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Production of unique metabolites by the marine dinoflagellate Prorocentrum minimum Trick, Charles Gordon 1982

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C J PRODUCTION OF UNIQUE METABOLITES BY THE MARINE DINOFLAGELLATE PROROCENTRUM MINIMUM by CHARLES GORDON TRICK B.Sc, University Of Manitoba, 1975 M.Sc, Acadia University, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department Of Oceanography We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1982 © Charles Gordon Trick, 1982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p ermission f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Oceanography The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: October 15, 1982 i i A b s t r a c t Marine phytoplankton produce e x t r a c e l l u l a r metabo l i t e s which may be important i n c o n t r o l l i n g the i n t e r a c t i o n s among spec ies or the compet i t i on for a l i m i t i n g n u t r i e n t . While the abso lute amount of these metabo l i t e s may be smal l compared to the pr imary organic s re leased by the phytoplankton c e l l , the c o n t r o l of the produc t ion of these unique metabo l i t e s may be an important f a c t o r i n the ecology of the producing s p e c i e s . These compounds have not been e x t e n s i v e l y s t u d i e d due to the d i f f i c u l t y i n i s o l a t i n g these minute q u a n t i t i e s from seawater. In t h i s t h e s i s , two e x t e r n a l l y produced metabo l i t e s have been i n v e s t i g a t e d . The c o n c e n t r a t i o n of 1 - ( 2 , 6 , 6 - t r i m e t h y l - 4 -h y d r o x y c y c l o h e x e n y l ) - 1,3-butanedione, a n o r - c a r o t e n i o d commonly r e f e r r e d to as the $ - d i k e t o n e , was q u a n t i t a t i v e l y determined dur ing the e x p o n e n t i a l and senescent stages of growth of Prorocentrum mi n imum i n P - , N - , and i r o n - d e f i c i e n t batch c u l t u r e s . The 3-diketone was re lea sed e x t r a c e l l u l a r l y i n a s i n g l e ' p u l s e ' dur ing the s t a t i o n a r y stage of growth. ' Severa l f a c t o r s such as temperature, i r r a d i a n c e , type of n u t r i e n t -d e f i c i e n c y (N, P, or F e ) , and the ambient n i t r a t e c o n c e n t r a t i o n were important in e s t a b l i s h i n g the amount of the 3- -d iketone produced. The environmental f a c t o r s d i d not i n f l u e n c e the temporal p a t t e r n of p r o d u c t i o n , only the absolute amount of the B-diketone produced. The l i m i t s of the range of p roduc t ion of t h e g • - d i k e t o n e were narrower than the range of maximum growth for any environmental i n f l u e n c e . The i n h i b i t i o n of growth and the h e t e r o t r o p h i c uptake of glucose by marine b a c t e r i a , demonstrated the a n t i b a c t e r i a l p r o p e r t i e s of t h e g -diketone. The second e x t r a c e l l u l a r organic examined was p r o r o c e n t r i n . P r o r o c e n t r i n i s the e x t r a c e l l u l a r siderophore produced by Prorocentrum minimum, P. m a r i a e - l e b o u r i a e , and P . g r a c i l e . F u n c t i o n a l l y s i m i l a r compounds are produced by T h a l a s s i o s i r a  pseudonana and D u n a l i e l l a t e r t i o l e c t a . T h i s study i s f i r s t to c h a r a c t e r i z e t h i s type of h i g h - a f f i n i t y i r o n ( 1 1 1 ) - t r a n s p o r t system in marine e u k a r y o t i c phytoplankton. The p a t t e r n of siderophore p r o d u c t i o n by a l l s p e c i e s i s the same, although the absolute amount of the m a t e r i a l produced i s s p e c i e s s p e c i f i c . There i s no i n t r a c e l l u l a r or e x t r a c e l l u l a r siderophore p r o d u c t i o n under i r o n - s u f f i c i e n t c u l t u r e c o n d i t i o n s . When i r o n was d e f i c i e n t there was a short p e r i o d of r a p i d e x t r a c e l l u l a r siderophore p r o d u c t i o n during the s t a t i o n a r y stage of growth. The i n t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n was very low which suggests that de novo s y n t h e s i s of the p r o r o c e n t r i n occurs j u s t p r i o r to e x t r a c e l l u l a r r e l e a s e . The p e r s i s t e n c e of the e x t r a c e l l u l a r siderophore i n the c u l t u r e medium was b r i e f . There was an increase i n the i_n v i v o f l u o r e s c e n c e f o l l o w i n g the l o s s of the siderophore from the medium. The increase in j_n v i v o f l u o r e s c e n c e was not accompanied by an increase i n c e l l c o n c e n t r a t i o n . An hypothesis concerning the mechanism of the iron-uptake system i s proposed. Procedures for the i s o l a t i o n and c h a r a c t e r i z a t i o n of p r o r o c e n t r i n are presented. P r o r o c e n t r i n appears to be a t r i -hydroxamate siderophore with a molecular weight between 560 and 590 d a l t o n s . The i r o n - p r o r o c e n t r i n complex i s s t a b l e over a ide pH range. V Table of Contents A b s t r a c t i i L i s t of Tables v i i L i s t of F i g u r e s v i i i Acknowledgement x I. INTRODUCTION 1 A. GENERAL INTRODUCTION 1 B. HISTORICAL BACKGROUND 2 C. TAXONOMIC CONSIDERATIONS 6 D. DISTRIBUTION OF PROROCENTRUM SPECIES 7 E. EXTRACELLULAR PRODUCTS FROM PROROCENTRUM SPECIES 8 F. PURPOSE 9 I I . A QUANTITATIVE EXAMINATION OF THE RELEASE OF AN EXTRACELLULAR METABOLITE BY THE MARINE DINOFLAGELLATE PROROCENTRUM MINIMUM 10 A. ABSTRACT 10 B. INTRODUCTION 11 C. MATERIALS AND METHODS 12 D. RESULTS AND DISCUSSION 15 I I I . THE INFLUENCE OF ENVIRONMENTAL FACTORS ON THE PRODUCTION OF AN ANTIBACTERIAL METABOLITE FROM PROROCENTRUM MINIMUM 22 A. ABSTRACT 22 B. INTRODUCTION 23 C. MATERIALS AND METHODS 26 D. RESULTS 30 E. DISCUSSION 45 IV. GENERAL INTRODUCTION TO SIDEROPHORES 55 A. ABSTRACT 55 B. INTRODUCTION 56 C. TRANSPORT OF IRON BY SIDEROPHORES 58 D. SIDEROPHORES IN HIGHER PLANTS 61 E. SIDEROPHORE PRODUCTION BY PHYTOPLANKTON 65 V. PROROCENTRIN:AN EXTRACELLULAR SIDEROPHORE PRODUCED BY THE MARINE DINOFLAGELLATE PROROCENTRUM MINIMUM 68 A. ABSTRACT 68 B. INTRODUCTION 69 C. MATERIALS AND METHODS 70 D. RESULTS AND DISCUSSION 72 VI. METHODOLOGY USED IN ISOLATING THE HYDROXAMATE SIDEROPHORE, PROROCENTRIN, FROM PROROCENTRUM MINIMUM 7 9 A. ABSTRACT 7 9 B. INTRODUCTION 80 C. MATERIALS 81 D. RESULTS 84 E. DISCUSSION 103 V I I . THE CONTROL OF THE PRODUCTION OF A SIDEROPHORE BY THE MARINE DINOFLAGELLATE, PROROCENTRUM MINIMUM 107 A. ABSTRACT 107 B. INTRODUCTION 108 C. MATERIALS AND METHODS 110 v i D. RESULTS 114 E. DISCUSSION 121 V I I I . EXTRACELLULAR HYDROXAMATE-SIDEROPHORE PRODUCTION BY NERITIC EUKARYOTIC MARINE PHYTOPLANKTON 128 A. ABSTRACT 128 B. INTRODUCTION 129 C. MATERIALS AND METHODS 131 D. RESULTS 132 E. DISCUSSION 138 BIBLIOGRAPHY 142 APPENDIX A - ANTIBACTERIAL PROPERTIES OF THE g-DIKETONE USING MARINE BIOASSAY SPECIES 158 v i i L i s t of Tables I. Recovery of the g-diketone using XAD-2 r e s i n 29 I I . Species examined f o r the pro d u c t i o n of the B-diketone. '. . .32 I I I . The i n f l u e n c e of i n c r e a s i n g ambient n i t r a t e c o n c e n t r a t i o n i n the medium on the pr o d u c t i o n of the B-diketone by P. minimum 41 IV. I n f l u e n c e of the a d d i t i o n of vanadium on the g-diketone p r o d u c t i o n i n i r o n - s t a r v e d batch c u l t u r e s of P. minimum 44 V. In f l u e n c e of c u l t u r e medium on growth rate of Prorocentrum minimum and pro d u c t i o n of Csaky p o s i t i v e compounds 73 VI. Comparison of f e r r i - p r o r o c e n t r i n with d e s f e r r i -p r o r o c e n t r in 75 VI I . The i n f l u e n c e of f i l t r a t e volume on the e f f i c i e n c y of e x t r a c t i o n and recovery of prorocentrum on XAD-2 r e s i n 87 V I I I . I n f l u e n c e of the volume of d e i o n i z e d , d i s t i l l e d water washes on the recovery of p r o r o c e n t r i n 88 IX. Removal of p r o r o c e n t r i n from XAD-2 r e s i n by su c c e s s i v e 100 ml methanol washes 89 X. Recovery of p r o r o c e n t r i n from XAD-2 r e s i n with i n c r e a s i n g c o n c e n t r a t i o n s of Csaky p o s i t i v e m a t e r i a l . 90 XI. P r o r o c e n t r i n p r o d u c t i o n by Prorocentrum s p e c i e s . .112 XI I . D e s c r i p t i o n and o r i g i n of b a c t e r i a l s p e c i e s examined. .1 60 v i i i L i s t of F igure s 1. The s t r u c t u r e of the e x t r a c e l l u l a r m e t a b o l i t e , g-d i ketone, (1 - (2 , 6, 6 - t r imethyl-4-hydr .oxycyclohexenyl ) -1,3-butanedione) from the marine d i n o f l a g e l l a t e , Prorocentrum minimum 13 2. P roduc t ion of the g-diketone i n batch c u l t u r e 16 3. Zeaxanthin and products of photooxygenation (from Isoe et a l . , . 1 972) 19 4. The i s o l a t i o n procedure for the g -d iketone from c u l t u r e s of P. minimum 28 5. P roduc t ion of the g-diketone i n batch c u l t u r e -i n f l u e n c e of i r r a d i a n c e 33 6. P r o d u c t i o n of the g-diketone i n batch c u l t u r e -i n f l u e n c e of a l i g h t : d a r k regime 34 7. P roduc t ion of the g-diketone i n batch c u l t u r e -i n f l u e n c e of s a l i n i t y 36 8. Produc t ion of the g-diketone i n batch c u l t u r e -i n f l u e n c e of temperature 37 9. Produc t ion of the g-diketone i n batch c u l t u r e -i n f l u e n c e of the i n i t i a l N:P atomic r a t i o i n the medi urn 38 10. Examinat ion of c e l l contents for the presence of the g - d i ketone 42 11. P roduc t ion of the g-diketone i n batch c u l t u r e -i n f l u e n c e of i r o n - d e f i c i e n c y 43 12. The s t r u c t u r e s of r e p r e s e n t a t i v e s iderophores 57 13. Representa t ive i r o n t r anspor t mechanisms 60 14. Representa t ive i r o n c h e l a t i n g agents from photosynthe t i c organisms 63 15. The U V - v i s i b l e absorp t ion spectra of d e s f e r r i -p r o r o c e n t r i n ( s o l i d l i n e ) and f e r r i - p r o r o c e n t r i n (broken l i n e ) 74 16. Summary of the procedure for the i s o l a t i o n of the aqueous f r a c t i o n c o n t a i n i n g the hydroxamate-type s iderophore , p r o r o c e n t r i n 86 17. Comparison of p r o f i l e s of the separa t ion of c o l l e c t e d metabo l i t e s based on molecular s i z e 95 18. Standard curve for the e s t i m a t i o n of the molecular weight of p r o c e n t r i n 96 19. The i n f l u e n c e of pH on the U V - v i s i b l e spectrum of p r o r o c e n t r i n 102 20. Procedure for the i s o l a t i o n of the aqueous f r a c t i o n c o n t a i n i n g p r o r o c e n t r i n 113 21. Comparison of the growth ra tes and p r o r o c e n t r i n produc t ion in batch c u l t u r e s of P. min imum 117 22. In f luence of the a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of EDTA on p r o r o c e n t r i n produc t ion 1 1 8 23. In f luence of i r o n on the growth of P. minimum 119 24. In f luence of i r o n on the growth of S. costatum 133 25. In f luence of i r o n on the growth of 0 . lu teus 134 26. In f luence of i r o n on the growth of T. pseudonana. . .135 ix 27. I n f l u e n c e of i r o n on the growth of D. t e r t i o l e c t a . .136 28. Growth c h a r a c t e r i s t i c s of Chromobacterium sp 161 29. The e f f e c t of the g-diketone on the h e t e r o t r o p h i c uptake of C-glucose by a n a t u r a l p o p u l a t i o n 162 X Acknowledgement I wish to s i n c e r e l y thank my s u p e r v i s o r , D r . Paul J . H a r r i s o n and D r . Raymond J . Andersen for t h e i r advice and guidance dur ing t h i s r e s e a r c h . The i r s t i m u l a t i n g guidance and l i m i t l e s s energy c rea ted for me a p o s i t i v e environment, without which t h i s work c o u l d not have been completed. The u n s e l f i s h g i f t of t h e i r time g r e a t l y broadened my a p p r e c i a t i o n of sc i e n c e . I would a l s o l i k e to g r e a t l y acknowledge the members of my research committee, D r s . T . R . Parsons and K. H a l l , for t h e i r p a t i e n t e f f o r t s i n reading the t h e s i s and t h e i r c o n s t r u c t i v e c r i t i c i s m in the development of the t h e s i s . Va luab le d i s c u s s i o n s w i t h a l l members of the Department of Oceanography c o n t r i b u t e d s i g n i f i c a n t l y to t h i s t h e s i s . I would e s p e c i a l l y l i k e to thank Dr . A. Lewis , D r . A. G i l l a m , and M. LeBlanc for t h e i r i n t e r e s t and a s s i s t a n c e in the c o l l e c t i o n of s iderophore da ta , N e i l P r i c e for c o n t r i b u t i n g to the f luorescence da ta , and Peter Thompson for h i s thought fu l encouragement in the l a b o r a t o r y . F i n a l l y , I wish to thank Janet for her p a t i e n c e , s a c r i f i c e s , and encouragement throughout these years of study and r e s e a r c h . 1 I . INTRODUCTION A. GENERAL INTRODUCTION The study of e x t r a c e l l u l a r o r g a n i c s produced by p h y t o p l a n k t o n i s of c o n s i d e r a b l e e c o l o g i c a l i n t e r e s t . These compounds a r e important i n the c y c l i n g of o r g a n i c matter by b a c t e r i a , the e s t a b l i s h m e n t and maintenance of s y m b i o t i c r e l a t i o n s h i p s and i n the c h e l a t i o n or b i n d i n g of n u t r i e n t s . E x t r a c e l l u l a r o r g a n i c s from p h y t o p l a n k t o n can be s e p a r a t e d i n t o a r t i f i c i a l g r o u p i n g s . 1) . The low m o l e c u l a r weight i n t e r m e d i a t e s of metabolism. These a r e compounds which are found i n t r a c e l l u l a r l y and are r e l e a s e d e x t r a c e l l u l a r l y as a c e l l u l a r response to s t r e s s or through d i f f u s i v e l eakage from the c e l l . Compounds of t h i s type i n c l u d e g l y c o l i c a c i d , g l y c e r o l , m a n n i t o l , and amino a c i d s and comprise the m a j o r i t y of the c e l l u l a r exudate ( H e l l e b u s t , 1965, 1974). 2) . High m o l e c u l a r weight compounds which a r e produced and r e l e a s e d as a c e l l u l a r response t o the environment (Fogg, 1975). T h i s second group of e x t r a c e l l u l a r m e t a b o l i t e s i s q u a n t i t a t i v e l y l e s s i m p o r t a n t than the low m o l e c u l a r weight i n t e r m e d i a t e s and l i t t l e r e s e a r c h has been done t o c h a r a c t e r i z e the components of t h i s f r a c t i o n . Compounds i n t h i s group may be i m p o r t a n t i n i n f l u e n c i n g the v i t a l a c t i v i t y of the p r o d u c i n g organism (e.g. p r o d u c t i o n of e x t r a c e l l u l a r a l k a l i n e phosphatase or v i t a m i n B>i 2 b i n d i n g f a c t o r ) or i n i n f l u e n c i n g the q u a l i t y of the water (e.g. p r o d u c t i o n of a n t i b a c t e r i a l or a n t i a l g a l 2 o r g a n i c s ) . To d i s p l a y an important r o l e in the ecology of the producing s p e c i e s , a q u a n t i t a t i v e l y l a r g e amount of m a t e r i a l i s not necessary. T h i s t h e s i s examines the c o n t r o l of production of organic compounds from t h i s second group. S p e c i f i c a l l y , two e x t r a c e l l u l a r b i o l o g i c a l l y a c t i v e m e t a b o l i t e s from Prorocentrum  minimum and r e l a t e d s p e c i e s w i l l be c o n s i d e r e d . The f i r s t m e t a b o l i t e , 1 - ( 6 , 2 , 6 - t r i m e t h y l - 4 - h y d r o x y c y c l o h e x e n y l ) - 1,3-butanedione, given the t r i v i a l name, the 3 -diketone, i s an a n t i b a c t e r i a l m e t a b o l i t e . The second, p r o r o c e n t r i n , i s an e x t r a c e l l u l a r i r o n - s p e c i f i c c h e l a t o r ( s i d e r o p h o r e ) . Both are r e p r e s e n t a t i v e of compounds i n the e x t r a c e l l u l a r , b i o l o g i c a l l y a c t i v e organic f r a c t i o n . B. HISTORICAL BACKGROUND The production of b i o l o g i c a l l y a c t i v e m e t a b o l i t e s by phytoplankton and the r o l e of these compounds in a l t e r i n g or d e l i m i t i n g the ecology of marine phytoplankton i s not a new concept. Quoting Johnson e_t a_l. (1924): "...we are p r e t t y sure that the plankton communities i n f l u e n c e each ot h e r . . . that there are what we can c a l l group symbiosis on the grand s c a l e so that the kind of plankton which we may expect to be present in a c e r t a i n sea area must depend to some extent, on the kind of plankton which was p r e v i o u s l y p r e s e n t . " (p.275, Lucas, 1947). Papers by Lucas (1944, 1947, 1955) have provided the p h i l o s o p h i c a l b a s i s for the c o n t r o l of marine r e l a t i o n s h i p s by the p r o d u c t i o n of organic compounds. 3 There are many reviews concerning various aspects of the production of b i o l o g i c a l l y active organics by phytoplankton (Wilson, 1951, 1954, 1981; Saunders, 1957; Lefevre, 1964, Sieburth, 1968; Hellebust, 1974). The production of b i o l o g i c a l l y active metabolites by phytoplankton, to a certain extent, is supported by f i e l d studies. The inh i b i t o r y nature of bloom water has been noted for a long time (Lefevre, 1964) and a l l e l o p a t h i c compounds have been implicated in establishing the sequence of species occurring within the bloom (Bentley, 1960; Pratt, 1966; Brockmann et a l . , 1977; Keating, 1978; Uchida, 1977) . Much of the supportive work for phytoplankton-phytoplankton relationships has been obtained through laboratory studies. These studies involved either measuring the growth response of species in multispecies cultures (Kayser, 1979; Sharp et a l . , 1979) or by examining the influence of c e l l - f r e e f i l t r a t e on the growth responses of the assay species ('conditioned' medium)(Uchida, 1977; Iwasaki, 1979). In two instances metabolite action has been characterized as; (1) e x t r a c e l l u l a r vitamin B i 2 binders (Kristensen, 1956; Pintner and Altmeyer, 1979), and (2) chelating compounds, especially e x t r a c e l l u l a r hydroxamic acids (Spencer e_t a l . , 1973; Murphy e_t a_l. , 1976). With regard to the production of a n t i b a c t e r i a l compounds by phytoplankton, there is well established a n t i b a c t e r i a l a c t i v i t y of seawater (Sieburth, 1968), often associated with declining phytoplankton blooms (Moebus, 1972). B e l l and Mitchell (1972), B e l l et a l . (1974) and Chan et a l . (1980) have noted the 4 p r o d u c t i o n of a n t i b a c t e r i a l compounds by phytoplankton grown under l a b o r a t o r y c o n d i t i o n s . The r e l a t i o n s h i p between phytoplankton and organisms i n higher t r o p h i c l e v e l s i s an important aspect in b i o l o g i c a l oceanography. I t i s thus s u r p r i s i n g that l i t t l e work has been performed in the area of phytoplankton-zooplankton chemical i n t e r a c t i o n s . However, recent work by Tomas (1980) has suggested that O l i s t h o d i s c u s l u t e u s out-competes Skeletonema  costatum by producing a chemical g r a z i n g d e t e r r e n t . Secondary m e t a b o l i t e s from higher p l a n t s growing in a s a l t marsh have been shown to deter d e t r i t u s feeders ( V a l i e l a e_t a_l. , 1979). While many papers d e s c r i b e the r o l e of e x t r a c e l l u l a r m e t a b o l i t e s in c o n t r o l l i n g the i n t e r a c t i o n s between s p e c i e s , the • approaches of many re s e a r c h e r s have f a i l e d to d i f f e r e n t i a t e between the a c t i v i t y of e x t r a c e l l u l a r m e t a b o l i t e s and other f a c t o r s : 1) In many s t u d i e s , there was a f a i l u r e to separate the c o m p etition f o r n u t r i e n t s from the i n h i b i t i o n by e x t r a c e l l u l a r m e t a b o l i t e s (Lam and S i l v e s t e r , 1979); 2) Other s t u d i e s f a i l e d to d i f f e r e n t i a t e between the e f f e c t s of pH and other non-metabolites from the e f f e c t s of i n h i b i t o r y substances, even though c o n s i d e r a b l e evidence i n d i c a t e s that f a c t o r s such as pH and l i g h t may be more important than e x t r a c e l l u l a r products in e x p l a i n i n g c e r t a i n i n t e r a c t i o n s (Kroes, 1973; E l b r a c h t e r , 1976; D ' E l i a et a l . , 1979; Nelson et a l . , 1979); 3) Many s t u d i e s have i n v o l v e d the i n h i b i t o r y nature of compounds 5 r e l e a s e d on c e l l l y s i s ( P r o c t o r , 1957; Duff et a l . , 1966; Bruce and Duff, 1967); 4) A l g a l c u l t u r e s are not always axenic and t h i s c o n d i t i o n i s necessary to e s t a b l i s h the source of the compound (Spencer et a l . , 1973); 5) Many experiments have used bioassay organisms from environments removed from that of the producing s p e c i e s . ' E c o l o g i c a l i m p l i c a t i o n s can not be drawn due to e n v i r o n m e n t a l l y c o n t r o l l e d p h y s i o l o g i c a l d i f f e r e n c e s between s t r a i n s ( F i s h e r et_ a l . , 1973; F i s h e r , 1977; Murphy and Belastock, 1980); 6) F i n a l l y , one can not be s a t i s f i e d with e s t a b l i s h i n g the b a s i c chemical c h a r a c t e r i s t i c s (molecular weight, heat s e n s i t i v i t y , e t c .) of the compound(s). T h i s p r o v i d e s l i t t l e i n f o r m a t i o n on e s s e n t i a l q u a n t i t a t i v e q u e s t i o n s r e l a t e d to the extent of p r o d u c t i o n , c r i t i c a l c o n c e n t r a t i o n s f o r e f f e c t i v e n e s s , p r e d i c t e d c o n c e n t r a t i o n s i n nature or the a c t i v i t y of s t r u c t u r a l l y s i m i l a r compounds. To f u l l y a p p r e c i a t e and understand the p o s s i b l e r o l e of e x t r a c e l l u l a r , b i o l o g i c a l l y a c t i v e m e t a b o l i t e s , the r e s p o n s i b l e e x t r a c e l l u l a r organic must be c h a r a c t e r i z e d and the p a t t e r n of p r o d u c t i o n of t h i s m e tabolite under the i n f l u e n c e of v a r i o u s environmental f a c t o r s be e s t a b l i s h e d . By understanding which o r g a n i c s are present, the extent of production and the mode of a c t i o n , one can s t a r t e n v i s i o n i n g p o s s i b l e s i t u a t i o n s where e x t r a c e l l u l a r , b i o l o g i c a l l y a c t i v e o r g a n i c s may occur. Research aimed at answering these q u e s t i o n s w i l l provide i n f o r m a t i o n necessary to assess the v a l i d i t y of e x t r a c e l l u l a r m e t a b o l i t e s as 6 important f a c t o r s in b i o l o g i c a l i n t e r a c t i o n s i n marine waters. C. TAXONOMIC CONSIDERATIONS The d i f f e r e n t i a t i n g f e a t u r e between Prorocentrum and other members of the Dinophyceae are the two a n t e r i o r l y i n s e r t e d f l a g e l l a and a theca c o n s i s t i n g of two l a r g e v a l v e s with a small f i e l d of p l a t e l e t s surrounding two, unequal f l a g e l l a pores ( T a y l o r , 1980). L o e b l i c h et a l . (1979) have examined the p o s i t i o n of the two f l a g e l l a and have suggested that both f l a g e l l a emerge from the l a r g e r of the two pores, rather than one f l a g e l l a per pore. The two v a l v e s are covered with minute spines (Dodge, 1975; Toriumi, 1980) and a l a r g e a p i c a l tooth may form near the a n t e r i o r pore. At l e a s t 21 sp e c i e s of Prorocentrum have been d e s c r i b e d . C r i t e r i a f o r the s e p a r a t i o n , of sp e c i e s i n c l u d e s i z e , shape, c o v e r i n g of t h e c a l p l a t e , and the extent of the a p i c a l tooth (Dodge, 1975). P. minimum can be d i s t i n g u i s h e d from the other s p e c i e s as the only s p e c i e s with spiny ornamentation on the v a l v e s , a n t e r i o r pores and an a p i c a l tooth (Toriumi, 1980). C l o s e l y r e l a t e d s p e c i e s i n c l u d e P^ b a i t i c u m (Lohmann) L o e b l i c h and P. mar ia e - l e b o u r iae (Parke et B a l l e n t i n e ) Hulburt. P. min imum can be separated from P^ b a i t icum based on s i z e and shape (P. minimum i s ovate and l a r g e r ) ( T o r i u m i , 1980). The d i s t i n c t i o n between P. minimum and P. mar i a e - l e b o u r iae i s based e n t i r e l y on the s i z e of the a p i c a l tooth near the f l a g e l l a r pore. Hulburt (1965) has suggested that a l l three are the same 7 s p e c i e s s i n c e each shows a l a r g e v a r i a t i o n in c e l l s i z e and the form of the a n t e r i o r tooth w i t h i n a s i n g l e sample. P.  b a i t i c u m and P. mar i a e - l e b o u r i a e r e t a i n v a r i e t a l s t a t u s . Synonymous species i n c l u d e E x u v i a e l l a minima P a v i l l a r d , E. mar i a e - l e b o u r i a e Parke & B a l l a n t i n e , P.triangulatum M a r t i n , and P . c o r d i f o r m i s Bursa (Dodge, 1975). D. DISTRIBUTION OF PROROCENTRUM SPECIES Members of the genus Prorocentrum are u b i q u i t o u s . The most s t u d i e d s p e c i e s i s P.micans , which i s a cosmopolitan n e r i t i c s p e c i e s (Dodge, 1975). H o l l i g a n et. a l . (1980) recognized P.  micans in over one-half of the samples c o l l e c t e d around the B r i t i s h I s l e s . P. minimum i s commonly found i n waters along the west coast of North America. T h i s s p e c i e s i s a common component of the phytoplankton of the Gulf of Mexico, E n g l i s h Channel, Caspian Sea, and the Mediterranean Sea (Dodge, 1975). T h i s s p e c i e s comprises about 20% of the f a l l bloom phytoplankton in Indian Arm and Port Moody Arm in B r i t i s h Columbian waters (Stockner and C l i f f , 1979). Large non-toxic blooms of P. minimum are a year-round component of the phytoplankton p o p u l a t i o n of Chesapeake Bay ( T y l e r and S e l i g e r , 1978) and in Japan ( O k a i c h i , 1975). Toxic P. minimum blooms have been reported but the nature of t h i s " t o x i c i t y " has not been c l a r i f i e d . Tangen (1980) recorded a t o x i c P. min imum bloom in the outer f j o r d of O s l o f j o r d i n the f a l l of 1979. The unique bloom was i n i t i a t e d 8 by the upwelling of deep water. The t o x i c nature was not d e s c r i b e d . Kat (1979) recorded an outbreak of g a s t r o i n t e s t i n a l i l l n e s s a s s o c i a t e d with a bloom of P. minimum in the, Netherlands. While P. minimum were not the d i r e c t cause of the outbreak, b a c t e r i a a s s o c i a t e d with P. minimum was thought to be the c a u s a t i v e agent. E. EXTRACELLULAR PRODUCTS FROM PROROCENTRUM SPECIES The e x t r a c e l l u l a r products of most marine phytoplankton have not been s t u d i e d thoroughly. T h i s i s e s p e c i a l l y true i n the case of Prorocentrum s p e c i e s . H e l l e b u s t (1965) examined the p h o t o e x c r e t i o n of organic carbon by P. min imum ( E x u v i a e l l a sp. P. c o r d i f o r m i s , Bursa, 1957) and found low amounts of organic carbon e x c r e t e d over a wide range of i r r i d i a n c e s . Less than 10% of the t o t a l carbon a s s i m i l a t e d was r e l e a s e d . Of the r e l e a s e d m a t e r i a l , n e a r l y o n e - t h i r d was g l y c o l i c a c i d . For comparison, O l i s t h o d i s c u s sp. and Skeletonema costatum r e l e a s e d 38 and 52% of the t o t a l a s s i m i l a t e d carbon, r e s p e c t i v e l y . While the absolute amount of e x t r a c e l l u l a r o r g a n i c s produced by Prorocentrum s p e c i e s may be s m a l l , the q u a l i t y of the exudate may be important. P.micans has been shown to produce s e v e r a l b i o l o g i c a l l y a c t i v e organic compounds. Uchida (1977) c h a r a c t e r i z e d an e x t r a c e l l u l a r d i a t o m - i n h i b i t i n g substance which was n o n - d i a l y z a b l e and was denatured on a u t o c l a v i n g . Aubert and Pesando (1971) and Gauthier et a l . 9 (1978) examined an e x t r a c e l l u l a r p r o t e i n which i n h i b i t e d the pro d u c t i o n of e x t r a c e l l u l a r a n t i b a c t e r i a l substances (a f a t t y a c i d and n u c l e o s i d e s ) by A s t e r i o n e l l a j aponica and Chaetoceros  l a u d e r i . I n h i b i t i o n of the p r o d u c t i o n of the a n t i b a c t e r i a l substances had no i n f l u e n c e on the growth of the diatom c u l t u r e s . S e v e r a l other r e s e a r c h e r s have attempted to show spe c i e s i n t e r a c t i o n s i n v o l v i n g micans, but i n t e r p r e t a t i o n i n each case was complicated by competition f o r n u t r i e n t s ( E l b r a c h t e r , 1976; Kayser, 1979). F. PURPOSE Th i s t h e s i s c o n t a i n s r e s u l t s of the examination of unique e x t r a c e l l u l a r m e t a b o l i t e s from P. minimum and r e l a t e d s p e c i e s . Emphasis has been p l a c e d on two compounds: 1 - ( 2 , 6 , 6 - t r i m e t h y l - 4 -hydroxycyclohexenyl)-1,3-butanedione (the g- d i k e t o n e ) , a unique e x t r a c e l l u l a r a n t i b a c t e r i a l compound (Chapters II and I I I ) and p r o r o c e n t r i n , an e x t r a c e l l u l a r i r o n - s p e c i f i c c h e l a t o r (Chapters IV to V I I I ) . 10 I I . A QUANTITATIVE EXAMINATION OF THE RELEASE OF AN  EXTRACELLULAR METABOLITE BY THE MARINE DINOFLAGELLATE PROROCENTRUM MINIMUM A. ABSTRACT Marine d i n o f l a g e l l a t e s produce e x t r a c e l l u l a r secondary m e t a b o l i t e s which may play a r o l e in the ecology of the producing s p e c i e s . The c o n c e n t r a t i o n of one such e x t e r n a l m e t a b o l i t e , 1 - ( 2 , 6 , 6 - t r i m e t h y l - 4 - h y d r o x y c y c l o h e x e n y l ) - 1,3 butanedione, produced by the marine d i n o f l a g e l l a t e , Prorocentrum  minimum, was determined q u a n t i t a t i v e l y during e x p o n e n t i a l growth and d u r i n g senescence in phosphate s t a r v e d batch c u l t u r e s . The p a t t e r n of production i s s i m i l a r to the production of many b a c t e r i a l t o x i n s . There was l i t t l e p r o d u c t i o n of the fr-diketone d u r i n g the ex p o n e n t i a l growth p e r i o d and highest production o c c u r r e d w i t h i n one week a f t e r c e s s a t i o n of c e l l d i v i s i o n . Approximately 50% of the t o t a l B-diketone produced, was ex c r e t e d on a s i n g l e day, f i v e days a f t e r phosphate became l i m i t i n g to growth. C e l l l y s i s or p h o t o d e s t r u c t i o n of c a r o t e n o i d s do not appear to be the cause of r e l e a s e of t h i s compound. 11 B. INTRODUCTION Marine phytoplankton excrete a wide v a r i e t y of organic compounds; however, the magnitude and the c o n d i t i o n s causing e x c r e t i o n are s t i l l matters of c o n t r o v e r s y (Smith and Wiebe, 1976; Sharp, 1977; Fogg, 1977; Mague et a l . , 1980). To-date most of the e x c r e t e d m a t e r i a l has been repo r t e d to be simple compounds such as organic a c i d s , amino a c i d s , and carbohydrates ( H e l l e b u s t , 1974). E x c r e t i o n of l a r g e molecular weight compounds has been reported and the i n h i b i t o r y or s t i m u l a t o r y e f f e c t s observed d u r i n g s p e c i e s i n t e r a c t i o n experiments have been a t t r i b u t e d to these compounds (Uchida, 1977; P i n t n e r and Altmeyer, 1979). Very few e x t r a c e l l u l a r secondary m e t a b o l i t e s from marine phytoplankton have been c h e m i c a l l y c h a r a c t e r i z e d . Most of the compounds i n v e s t i g a t e d p r i o r to t h i s work have been i n t r a c e l l u l a r t o x i n s and s t e r o l s from d i n o f l a g e l l a t e s (Shimizu et a l . , 1976; F i n e r et a l . , 1978; Alam et a l . , 1979; Schantz, 1979; Withers e_t a_l. , 1979)- and t o x i n s , a l k a l o i d s and novel l i p i d s from marine c y a n o b a c t e r i a (Moore, 1977; Mynderse and Moore, 1978; C a r d e l l i n a et a l . , 1978, 1979; C a r d e l l i n a and Moore, 1980). There i s i n c r e a s i n g i n t e r e s t i n i n t e r a c t i o n s among sp e c i e s in the f i e l d and i n c u l t u r e . E x t r a c e l l u l a r m e t a b o l i t e s are thought to play an important r o l e i n many of the observed c o m p e t i t i v e i n t e r a c t i o n s among sp e c i e s ( P r a t t , 1966). The c o n t r o l of production of i n d i v i d u a l e x c r e t e d compounds by environmental and p h y s i c a l f a c t o r s , has not been examined p r e v i o u s l y . 12 Recent work has provided c o n f l i c t i n g r e s u l t s on the a b i l i t y of Prorocentrum s p e c i e s to produce compounds i n h i b i t o r y to other a l g a l s p e c i e s ( E l b r a c h t e r , 1976; Uchida, 1977; Iwasaki, 1979; Kayser 1979). An e x t r a c e l l u l a r a n t i b i o t i c from f i l t r a t e s of Prorocentrum c u l t u r e has been i s o l a t e d and i t s chemical s t r u c t u r e shown to be the n o r - c a r o t e n o i d , 1 - ( 2 , 6 , 6 , - t r i m e t h y l - 4 -h y d r o x y c y c l o h e x e n y l ) - 1 , 3-butanedione ( F i g . 1; Andersen e_t a l . 1980). Compounds of t h i s type have a l s o been shown to be produced by Cyanidium (Rhodophyceae) i n mass c u l t u r e ( J u t t n e r 1979a). As part of a l a r g e r i n v e s t i g a t i o n to evaluate the b i o a c t i v e nature or e x t r a c e l l u l a r m e t a b o l i t e s from marine d i n o f l a g e l l a t e s , the k i n e t i c s of production of t h i s e x t e r n a l secondary m e t a b o l i t e by Prorocentrum minimum grown in the l a b o r a t o r y d u r i n g e x p o n e n t i a l growth and du r i n g d e f i n e d c o n d i t i o n s of phosphate s t a r v a t i o n were examined. C. MATERIALS AND METHODS Prorocentrum minimum S c h i l l e r was o r i g i n a l l y i s o l a t e d from E n g l i s h Bay, B.C. Canada and has been maintained i n the Northeast P a c i f i c C u l t u r e C o l l e c t i o n (NEPCC #96) s i n c e 1971. An axenic i s o l a t e was grown i n s t e r i l e e n r i c h e d a r t i f i c i a l seawater, m o d i f i e d by the replacement of Na glycerophosphate with Na 2HPO [ t and Fe (NH^ ) 2 SO^ with F e C l 3 (Harrison et a l . , 1980). The amount of phosphate in the enrichment s o l u t i o n was reduced to 6 yM (N:P = 27:1) to ensure that c u l t u r e s became P-starved d u r i n g senescence. Continuous l i g h t was provided by d a y l i g h t f l u o r e s c e n t bulbs at an i r r a d i a n c e of 160 U E n r 2 a n d a 13 F i g u r e 1 - The s t r u c t u r e of the e x t r a c e l l u l a r m e t a b o l i t e , 3-diketone, (1 -(2,6,6-trimethyl-4-hydroxycyclohexenyl)-1,3-butanedione) from the marine d i n o f l a g e l l a t e , Prorocentrum minimum. H O P - DI K E T O N E 14 temperature of 18 °C. C e l l s were grown axenically in 6-L batch cultures with constant s t i r r i n g (60 or 120 rpm). Cultures were tested frequently to determine i f they remained axenic throughout the experiment by plating on an organic-containing s t e r i l i t y medium. Disappearance of phosphate from the medium was monitored to ensure phosphate deficiency. Cultures were examined daily and c e l l s were counted using a Palmer-Maloney chamber. Several i d e n t i c a l cultures were set up and each culture was completely harvested at dif f e r e n t stages of growth. Production during the growth cycle was a compilation of three d i s t i n c t experimental repetitions. C e l l s were removed from the medium by passing the 6-L cultures through a continuous centrifuge (Sharpies, type TL). The c e l l - f r e e medium was membrane f i l t e r e d (0.45 y m) and adjusted to pH 2.0. A dissolved organic fraction was collected by passing the a c i d i f i e d f i l t r a t e through a 25 x 2 cm column of precleaned (Soxhlet extraction with methanol, 72 h) XAD-2 resin (Mallinckrodt). Remaining sal t s were removed by washing the column with 200-300 ml deionized, d i s t i l l e d water. The collected organic fraction was eluted with 150 ml methanol. The methanol extract was concentrated by rotary evaporation at 35 °C _in_ vacuo and the residue was partitioned between CHCL3 and H^O. Evaporation of the CHC13 _i_n vacuo at room temperature provided the crude f i l t r a t e extract. Pure & -diketone (Fig. 1) was used as an a n a l y t i c a l standard (Andersen et a l . , 1980). A l l crude extracts were quantitatively determined via HPLC equipped with a s i l i c a gel 15 column ( L i C h r o s o r b Si60) using i s o c r a t i c s e p a r a t i o n (2 ml-min ; hexane: i s o p r o p a n o l , 83:17) and UV d e t e c t i o n (287 nm). To determine the e f f i c i e n c y of the e x t r a c t i o n procedure, a known amount of standard compound was in t r o d u c e d i n t o two r e p l i c a t e f l a s k s with 6-L of seawater medium and, a f t e r e x t r a c t i o n , assayed by HPLC and by absorbance i n methanol (287 nm) . In order to estimate the importance of the g-diketone i n r e l a t i o n to the t o t a l organic e x c r e t i o n by the c u l t u r e , e x c r e t i o n was measured using two d i f f e r e n t methods. D i s s o l v e d organic carbon (DOC) in the c u l t u r e f i l t r a t e was determined by wet o x i d a t i o n with potassium p e r s u l f a t e , with c o r r e c t i o n for reagent and medium blanks. Rates of e x c r e t i o n were estimated 1 4 using the H 2 CCg technique of Mague e_t §_1. (1980). R a d i o a c t i v i t y was measured i n a Nuclear-Chicago Unilux III l i q u i d s c i n t i l l a t i o n counter. C e l l s were incubated f o r 4 h under experimental c o n d i t i o n s . D. RESULTS AND DISCUSSION Only two compounds were found in the e x t r a c t of the c u l t u r e f i l t r a t e from Prorocentrum minimum. The main compound was i d e n t i f i e d to be the 3-diketone ( F i g . 1; Andersen e_t a l . , 1980), while the second compound was only present in minor c o n c e n t r a t i o n s and was not i d e n t i f i e d or examined f u r t h e r . The p r o d u c t i o n of the g-diketone during c u l t u r e growth i s shown in F i g . 2. L i t t l e g-diketone was produced d u r i n g e x p o n e n t i a l growth. C e l l s became phosphate l i m i t e d a f t e r seven 16 F i g u r e 2 - Pr o d u c t i o n of the 3. -diketone i n batch c u l t u r e . Prorocentrum minimum. A: Changes i n g-diketone c o n c e n t r a t i o n and c e l l numbers d u r i n g e x p o n e n t i a l growth and du r i n g senescence i n phosphate-starved batch c u l t u r e s . Values are s i n g l e experiments u n l e s s noted. R e p l i c a t e compound c o n c e n t r a t i o n values are mean ± one SE, where n i s noted i n b r a c k e t s . B: Rates of compound p r o d u c t i o n . 50 r 40 30 o -t Q. cn E 5 ° c > ro 20 "•tJ -•-»' nj c IS 1 0 38 0 / (4)(3) (^ • ,o-o-(3V -J 1 I 10 Tim<2 ( D a y s ) 20 5.0 r-in O 4.5 UT 3 r~ 4.0 o 30 B c o •- -> 20 +-> X J CO "DO E. o u 10 0 -10 XJ 1 1 1 J J 10 T i m e ( D a y s ) 20 17 days of growth and 50% of the compound was produced i n a s i n g l e 'pulse' s i x days l a t e r . Decreasing amounts were produced dur i n g the l a t e r stages of senescence under phosphate s t a r v a t i o n . Based on ra t e s of e x c r e t i o n determined using the H^COg technique the B-di k e t o n e comprises approximately 2.8% of the t o t a l amount of carbon produced on Day 13 (the p e r i o d of maximum g-diketone p r o d u c t i o n ) . The average production over the e n t i r e p e r i o d of g-diketone p r o d u c t i o n (Day 11 through Day 19) was much lower, with the g-diketone accounting f o r an average of 0.4% of the carbon r e l e a s e d per day. T h i s was s i m i l a r to c a l c u l a t i o n s based on d i r e c t DOC d e t e r m i n a t i o n s where the B-diketone comprised 0.6% of the t o t a l DOC at the end of the experimental p e r i o d . The observed p a t t e r n of p r o d u c t i o n and the r e l a t i v e l y high l i g h t i n t e n s i t i e s u t i l i z e d , prompted the concern that the occurrence of t h i s compound i n the medium was a r e s u l t of e i t h e r c e l l l y s i s or the photochemical o x i d a t i o n of c a r o t e n o i d s . It i s u n l i k e l y that the compound was r e l e a s e d as a r e s u l t of c e l l l y s i s s i n c e l o s s of c e l l s based on c e l l count was only noted 18 days a f t e r most of the B - d i k e t o n e had been r e l e a s e d . In a d d i t i o n , i n t r a c e l l u l a r B-diketone i n the Prorocentrum i s o l a t e has never been found. Furthermore, the HPLC technique i n d i c a t e d the p r o d u c t i o n of only one major and one minor compound i n t h i s e x t r a c e l l u l a r f r a c t i o n throughout the e n t i r e growth p e r i o d except f o r Day 30, when a v a r i e t y of compounds were c o l l e c t e d , i n d i c a t i n g p o s s i b l e c e l l l y s i s at t h i s l a t e r date. A second c o n s i d e r a t i o n was whether t h i s compound was the 18 r e s u l t of ir\ v i t r o p h o t o o x i d a t i o n of c a r o t e n o i d s . The s t r u c t u r e of the g-diketone suggested a p o s s i b l e c a r o t e n o i d o r i g i n and p r e l i m i n a r y data i n d i c a t e d the compound was produced only at s a t u r a t i n g i r r a d i a n c e s I t has been shown that n o r - c a r o t e n o i d s of s i m i l a r carbon s k e l e t o n were produced by photooxygenation of c a r o t e n o i d s j_n v i t r o (Isoe et a l . 1969; Isoe e_t a l . 1972). T h i s suggests that the g-diketone i s a product of an enzyme c o n t r o l l e d metabolism, perhaps i n v o l v i n g a photobiochemical step, rather than a n o n - s p e c i f i c p h o t o o x i d a t i o n f o r the f o l l o w i n g reasons: a) Prorocentrum minimum produced only one compound of t h i s type. If non-enzymatic chemical breakdown of c a r o t e n o i d s was o c c u r r i n g , one would expect a l l c a r o t e n o i d s to undergo degradation and s e v e r a l compounds would be produced in d i r e c t p r o p o r t i o n to the c a r o t e n o i d composition. The p r e c i s e c a r o t e n o i d composition of P^ minimum i s not known; however, members of t h i s genus have been reported to c o n t a i n two major xan t h o p h y l l s ( p e r i d i n i n and d i a d i n o x a n t h i n ) , with g-carotene c o n s t i t u t i n g l e s s than 10% of the t o t a l c a r o t e n o i d content (Johansen e_t a_l. , 1974). Zeaxanthin and v i o l a x a n t h i n , two l i k e l y p r e c u r s o r s of the 3-diketone have not been repo r t e d i n members of the Dinophyceae (Goodwin, 1979), while on a s t r u c t u r a l b a s i s p e r i d i n i n does not appear to be a l i k e l y p r e c u r s o r . However, the g-diketone c o u l d have o r i g i n a t e d from d i a d i n o x a n t h i n by h y d r a t i o n of the a c e t y l e n e bond followed by o x i d a t i v e rupture of the c a r o t e n o i d chain at C - C ; b) Isoe et ^ 8 9 a l . (1972) have shown that the in_ v i t r o p h o t o o x i d a t i o n of zeaxanthin produces a complex mixture of products ( F i g . 3), 19 F i g u r e 3 - Zeaxanthin and products of photooxygenation (from Isoe et a l . , 1972). 20 while the i s o l a t i o n of a s i n g l e non-carotenoid argued a g a i n s t such n o n - s p e c i f i c p h o t o o x i d a t i o n s ; and c) the low r a t e of p r o d u c t i o n d u r i n g the i n i t i a l p e r i o d of phosphate s t a r v a t i o n , f o l l o w e d by a 'pulse' of e x c r e t i o n d u r i n g l a t e r stages, suggested some form of b i o l o g i c a l c o n t r o l . While t h i s i s the f i r s t r e p o rt on the p a t t e r n of production of an e x t r a c e l l u l a r secondary m e t a b o l i t e from phytoplankton, t h i s p a t t e r n was not unique among microorganisms. The p r o d u c t i o n p a t t e r n was very s i m i l a r to that observed f o r many b a c t e r i a l t o x i n s , where there i s no p r o d u c t i o n during e x p o n e n t i a l growth, low production as c e l l s undergo adjustment from e x p o n e n t i a l growth to the s t a t i o n a r y phase, high p r o d u c t i o n d u r i n g the e a r l y s t a t i o n a r y phase, and a r a p i d c e s s a t i o n of p r o d u c t i o n a f t e r a short p e r i o d of 'time (Weinberg 1970). U s u a l l y the length of p r o d u c t i o n was between one h a l f to two times the l e n g t h of the e x p o n e n t i a l growth p e r i o d i n b a c t e r i a l c u l t u r e s (Weinberg 1970). In t h i s study, 50% of the m a t e r i a l was produced in a s i n g l e pulse suggesting a r e a d i l y a v a i l a b l e p r e c u r s o r pool or a r a p i d exhaustion of a v a i l a b l e p r e c u r s o r s . In c o n c l u s i o n , the f i r s t p a t t e r n of p r o d u c t i o n of an i d e n t i f i a b l e e x t r a c e l l u l a r secondary m e t a b o l i t e produced by a marine d i n o f l a g e l l a t e has been e s t a b l i s h e d . Since the data show pro d u c t i o n only under n u t r i e n t s t a r v e d c o n d i t i o n s and a low y i e l d of compound when compared to t o t a l organic e x c r e t i o n determined by other techniques, the p r o d u c t i o n of t h i s compound would have n e g l i g i b l e i n f l u e n c e on organic carbon c y c l i n g under open ocean c o n d i t i o n s . However, the important i n f l u e n c e t h i s 21 compound may exert on s p e c i e s s u c c e s s i o n , e s p e c i a l l y under bloom c o n d i t i o n s i n which Prorocentrum minimum has o c c a s i o n a l l y been found to be abundant (Nakazima 1978; Kat 1979), cannot be ignored. 22 I I I . THE INFLUENCE OF ENVIRONMENTAL FACTORS ON THE PRODUCTION  OF AN ANTIBACTERIAL METABOLITE FROM PROROCENTRUM MINIMUM A. ABSTRACT Prorocentrum minimum , an u b i q u i t o u s non-toxic r e d - t i d e forming d i n o f l a g e l l a t e , produces b i o l o g i c a l l y a c t i v e e x t r a c e l l u l a r m e t a b o l i t e s . One such e x t r a c e l l u l a r organic compound i s 1 - ( 2 , 6 , 6 - t r i m e t h y l - 4 - h y d r o x y c y c l o h e x e n y l ) - 1,3-butanedione, a n o r - c a r o t e n o i d commonly r e f e r r e d to as the 3~ d i k e t o n e . The 3-diketone was r e l e a s e d e x t r a c e l l u l a r l y i n a s i n g l e 'pulse' d u r i n g the s t a t i o n a r y phase of growth. F a c t o r s such as i r r a d i a n c e and temperature are important environmental f a c t o r s c o n t r o l l i n g the p r o d u c t i o n of t h i s m e t a b o l i t e . D i f f e r e n t e x t r a c e l l u l a r c o n c e n t r a t i o n s were obtained depending on the type of l i m i t i n g n u t r i e n t . If c e l l s were P - d e f i c i e n t the production was twice that of the N - d e f i c i e n t c u l t u r e s . Iron-d e f i c i e n t growth f u r t h e r reduced the amount of the 3-diketone produced. The ambient n i t r a t e c o n c e n t r a t i o n i n the medium had a strong i n f l u e n c e on the amount of the 3-diketone produced. The p a t t e r n of pr o d u c t i o n and the c o n t r o l by environmental f a c t o r s suggest that the production of t h i s m e t a b o l i t e i s d i r e c t l y c o n t r o l l e d by the p h y s i o l o g i c a l s t a t e of the c e l l , and not merely a r e s u l t of the p h o t o d e s t r u c t i o n of c a r o t e n o i d s . The s p e c u l a t i v e r o l e of the 3 - d i k e t o n e in the n a t u r a l environment i s d i s c u s s e d . 23 B. INTRODUCTION Phytoplankton convert l a r g e amounts of the f i x e d carbon i n t o e x t r a c e l l u l a r m e t a b o l i t e s . The m a j o r i t y of these e x t r a c e l l u l a r m e t a b o l i t e s are primary organic compounds which p l a y an important r o l e i n the h e t e r o t r o p h i c c y c l e of a q u a t i c ecosystems (Aaronson, 1971; H e l l e b u s t , 1974). A smaller amount of l e s s w e l l d e f i n e d secondary, or unique, m e t a b o l i t e s are a l s o produced. Compounds i n t h i s l a t t e r group may be important i n c o n t r o l l i n g i n t e r a c t i o n s among phytoplankton and other organisms, or i n r e g u l a t i n g i n t r a c e l l u l a r metabolism. An example of these l e s s w e l l s t u d i e d , unique m e t a b o l i t e s i n c l u d e a c r y l i c a c i d , f i r s t noted by S i e b u r t h (1959). The p r e c u r s o r of a c r y l i c a c i d (DMPT: d i m e t h y l - B - p r o p r o t h e t i n ) , was found i n t e r n a l l y in c o l o n i a l forms of Phaeocyst i s pouchet i i (Haptophyceae). Evidence by G u i l l a r d and H e l l e b u s t (1971) suggested that a c r y l i c a c i d should e x i s t e x t r a c e l l u l a r l y (2% of t o t a l e x c r e t e d carbon) and they c a l c u l a t e d e x t e r n a l a c r y l i c a c i d c o n c e n t r a t i o n s in n a t u r a l blooms to reach 7 Vq-L l. Another unique e x t r a c e l l u l a r m e t a b o l i t e i n v o l v e d i n marine phytoplankton ecology i s 'aponin', a group of b a s e - l a b i l e o r g a n i c s produced by the cyanobacterium Gomphasphaeria aponina. At low c o n c e n t r a t i o n s t h i s compound causes l y s i s of the d i n o f l a g e l l a t e Gymnodinium breve (Martin and M a r t i n , 1976; Eng-Wilmt, et a l . , 1979). Of i n c r e a s i n g i n t e r e s t i s the production by phytoplankton of n o r - c a r o t e n o i d s , a group of o r g a n i c s r e l a t e d to c a r o t e n o i d s and some of t h e i r breakdown products. Nor-carotenoids have been 24 recognized as a n t i - f u n g a l and a n t i b a c t e r i a l agents ( Z a j i c - S e e r e t and Kuehn, 1961) and have been found in waters a s s o c i a t e d w i t h blooms of f r e s h water cyanobac ter i a (Tabachek and Yurkowsk i , 1976). N a t u r a l popu la t ions of M i c r o c y s t i s wesenberg, produce the n o r - c a r o t e n o i d , 3 - c y c l o c i t r a l , which g ives the bloom a t o b a c c o - l i k e odor ( J u t t n e r , 1979a). The f r a g r a n t , v o l a t i l e nature of nor -ca ro teno ids l e d J u t t n e r to i s o l a t e four nor-ca ro teno id s produced e x t r a c e l l u l a r l y by Cyan idium c a l d a r ium (Rhodophyceae) under large s ca l e c u l t u r i n g c o n d i t i o n s . These i n c l u d e d B - i o n o n e , geranylacetone , methylheptenone, and d i h y d r o t r i m e t h y l n a p h t h a l e n e ( J u t t n e r , 1979a). The a n t i b i o t i c nature of the nor-caro teno ids produced by C. c a l d a r ium has been e s t a b l i s h e d . Of the four n o r - c a r o t e n o i d s , B- ionone and d i h y d r o t r i m e t h y l n a p t h a l e n e were i n h i b i t o r y to cyanobac ter i a at 10 ppm. Geranylacetone reduced growth of cyanobac ter i a at 50 ppm. Methylheptenone was the l e a s t i n h i b i t o r y to the cyanobac ter i a species examined, s ince c o n c e n t r a t i o n greater then 100 ppm were requ i red to ' , reduce the growth of the assay species ( J u t t n e r , 1979b). The a n t i b a c t e r i a l nature of nor -ca ro teno ids i n aquat i c systems has a l s o been examined by Re ichardt (1981). Methylheptenone, i n h i b i t e d c o l o n i a l growth of a l l b a c t e r i a l species examined and was i n h i b i t o r y to glucose uptake at 2 ppm ( 18 P M ) . Converse ly 3-ionone and geranylacetone i n h i b i t e d only pigmented b a c t e r i a . The mechanism of i n h i b i t i o n i n v o l v e s the blockage of pigment produc t ion by the b a c t e r i a . Thi s i s a s i m i l i a r mechanism to the i n h i b i t i o n of c a r o t e n o i d s y n t h e s i s i n the cyanobacterium 25 Synechococcus by the a d d i t i o n of geranylacetone ( J i i t t n e r , 1979b). Recently, Andersen et a_l. (1980) c h a r a c t e r i z e d the e x t r a c e l l u l a r n o r - c a r o t e n o i d 1 1 - ( 2 , 6 , 6 , - t r i m e t h y l - 4 -h y d r o x y c y c l o h e x e n y l ) - 1,3-butanedione, from l a b o r a t o r y c u l t u r e s of the marine d i n o f l a g e l l a t e Prorocentrum minimum. T h i s compound, given the t r i v i a l name, t h e B -diketone, i s an example of an a n t i b a c t e r i a l , n o r - c a r o t e n o i d which imparts a t o b a c c o - l i k e odor to P^ minimum c u l t u r e s . The pro d u c t i o n of the g-diketone by P. minimum was examined i n Chapter 2. Production was dependent on the p h y s i o l o g i c a l s t a t e of the c e l l s and not simply a by-product of the photooxygenation of c a r o t e n o i d s . Since the g-diketone i s a n t i b a c t e r i a l under both l a b o r a t o r y (Andersen et a l . , 1980) and f i e l d c o n d i t i o n s (Appendix A), understanding environmental f a c t o r s which c o n t r o l the pro d u c t i o n and r e l e a s e of t h i s m e t a b o l i t e may provide i n s i g h t i n t o the p o s s i b l e r o l e of n o r - c a r o t e n o i d s in c o n t r o l l i n g the i n t e r a c t i o n s among microorganisms in a q u a t i c ecosystems. In t h i s chapter, the i n f l u e n c e of enviromental f a c t o r s on the amount of 3-diketone produced i s examined. F a c t o r s such as i r r a d i a n c e , temperature, and s a l i n i t y are con s i d e r e d f o r p h o s p h a t e - d e f i c i e n t batch c u l t u r e s . Since the g-diketone i s a p o t e n t i a l t r a c e metal c h e l a t o r (Andersen et a l . , 1980), pr o d u c t i o n under i r o n - l i m i t e d growth c o n d i t i o n s was a l s o i n v e s t i g a t e d . 1 The t r a d i t i o n a l d e f i n i t i o n , n o r c a t o t e n o i d s as p o s s i b l e c a r o t e n o i d breakdown products, i s employed. 26 C. MATERIALS AND METHODS A l l Prorocentrum s p e c i e s were grown in e n r i c h e d a r t i f i c i a l seawater medium (Har r i s o n e_t al. , 1980) , at 18 *C with the e x c e p t i o n of ' P. mar i a e - l e b o u r i a e which r e q u i r e d an e n r i c h e d n a t u r a l seawater-based medium of lower s a l i n i t y (15%.) and e l e v a t e d temperatures (22-24 °C) f o r growth. Both media were mo d i f i e d by the replacement of Na glycerophosphate with Na 2 HPO and Fe(NHi t) 2 SO^ with F e C l 3 . The amount of phosphate in the enrichment s o l u t i o n was reduced to 6vM-(N:P 27:1 by atoms) to ensure that the batch c u l t u r e s became phosphate-starved during senescence. To achieve i r o n - s t a r v e d growth, P. minimum was grown i n the f o l l o w i n g media: 1) c h a r c o a l - t r e a t e d n a t u r a l seawater followed by the a d d i t i o n of a l l r e q u i r e d n u t r i e n t s (ESNW) (Harrison et a l . , 1980); 2) C h a r c o a l - t r e a t e d n a t u r a l seawater f o l l o w e d by the removal of most of the a v a i l a b l e i r o n by the procedure of Lewin and Chen (1971), and then supplemented with a l l n u t r i e n t s except F e C l 3 and EDTA (ESNW-Fe); and 3) c h a r c o a l - and Chelex-100 r e s i n -t r e a t e d n a t u r a l seawater supplemented with AQUIL n u t r i e n t s minus F e C l 3 and EDTA (Morel et a l . , 1979). The f i r s t medium provided an i r o n - s u f f i c i e n t c o n t r o l while the other media pr o v i d e d i r o n -l i m i t e d growth c o n d i t i o n s . Vanadium was added as NHL+VCS to a l l e v i a t e the e f f e c t of i r o n - 1 i m i t i o n on growth in one set of experiments. Continuous l i g h t i n g was p r o v i d e d by d a y l i g h t f l u o r e s c e n t bulbs. I r r a d i a n c e was reduced to 120, 90, 60 and 30 y E - r 2 -s"1 by wrapping c u l t u r e s in n e u t r a l d e n s i t y s c r e e n i n g in c e r t a i n 27 experiments. C e l l s were grown a x e n i c a l l y i n 6 L or 10 L f l a t bottomed b o i l i n g f l a s k s with constant s t i r r i n g (60 rpm). The axenic nature of the c u l t u r e s was v e r i f i e d v i s u a l l y using the a c r i d i n e orange d i r e c t e p i f l u o r e s c e n t count technique (Hobbie et a l . , 1977), or by p l a t i n g on an organic s t e r i l i t y medium and examining f o r growth a f t e r 48 hours. I f b a c t e r i a l contamination was d i s c o v e r e d , the c u l t u r e s were d i s c a r d e d . C u l t u r e s were harvested and the 3-diketone c o l l e c t e d using the procedure s t a t e d p r e v i o u s l y (Chapter II) ( F i g . 4). B r i e f l y : c e l l s were removed from the e n t i r e 6 or 10 L c u l t u r e by continuous c e n t r i f u g a t i o n . The remaining c e l l s were removed by f i l t e r i n g the medium through a g l a s s f i b e r f i l t e r (GF/A), f o l l o w e d by a 0.45 pm membrane f i l t e r . The c e l l - f r e e f i l t r a t e was a c i d i f i d e d (pH 2.0) with HC1. Organics were removed from the a c i d i f i e d f i l t r a t e by passage over a 25 X 2 cm column of XAD-2 r e s i n ( M a l l i n c k r o d t ) . The XAD-2 r e s i n c o l l e c t e d a p o r t i o n of the e x t r a c e l l u l a r o r g a n i c s which i n c l u d e d the 3-diketone. The column was washed with 150 ml H-20 and the o r g a n i c s e l u t e d with 300 ml methanol. The methanol f r a c t i o n was reduced to dryness by r o t a t a r y evaporation and the residue p a r t i t i o n e d between CHCI3 and H 20. The CHC13 l a y e r was reduced to dryness and the amount of g-diketone in the crude f r a c t i o n determined using HPLC (Chapter I I ) . Using t h i s procedure recovery e f f i c i e n c y of the g-diketone was 80% (Table I ) . A l l reported values have been c o r r e c t e d a c c o r d i n g l y . 28 F i g u r e 4 - The i s o l a t i o n procedure f o r the g-diketone from c u l t u r e s of P. minimum. D e t a i l s are given i n the t e x t . C U L T U R E S C O N T I N U O U S C E N T R I F U G E F I L T E R ( 0 . 4 5 p m ) A C I D I F Y ( p H 2 . 0 ) X A D - 2 R E S I N H 2 0 W A S H M E T H A N O L E V A P O R A T E C H C L 3 : H 2 O E V A P O R A T E Q U A N T I F Y W I T H H P L C 29 Table I - Recovery of the g-diketone using XAD-2 resin. Six d i f f e r e n t amounts of the g-diketone were added separately to 5 L of ESNW medium. The a c i d i f i e d medium was passed over a column of XAD-2 resin and the col l e c t e d 3-diketone analysed by HPLC. The average recovery (tone SE) was 78.7 % ( 2.8)% (n = 6). I n i t i a l amount o f B-diketone added R e c o v e r e d amount R e c o v e r y t o medium ( yg) of g - d i k e t o n e ( yq)  10 7.4 74.3 20 16.2 81,0 30 24.0 80.0 40 30.9 77.2 60 46.7 77.9 100 82.0 82.0 30 C e l l c o n c e n t r a t i o n s were determined us ing an e l e c t r o n i c p a r t i c l e counter (Coul ter Counter Model T A - I I ) , and were used to determine growth r a t e s . D. RESULTS 1) Produc t ion under P - l i m i t e d growth c o n d i t i o n s Of the s i x species examined (Table I I ) , P. minimum v a r . b a i t i c u m , P. g r a c i l e and P_^  maximum showed no de tec tab le e x t r a c e l l u l a r g -d iketone produc t ion and were not examined f u r t h e r . P_;_ micans and P. mar i ae - l ebour i ae had lower amounts of 3-diketone p roduc t ion (10 y g - L - 1 ) compared to P. minimum ( 40 y g - L 1 ) (Table I I ) , the o r i g i n a l producer of the 3 -d iketone m a t e r i a l (Chapter I I ) . a) In f luence of ' i r r a d i a n c e The i n f l u e n c e of i r r a d i a n c e on the product ion of 3 - d ike tone by P. minimum and P. mar i ae - l ebour i ae i s shown i n F i g . 5. Smal l v a r i a t i o n s i n the i r r a d i a n c e had a s u b s t a n t i a l i n f l u e n c e on the amount of g-diketone produced by P. minimum ( F i g . 5A) . -2 -1 A r e d u c t i o n from 160 to 120 yE-m s reduced the amount of g-diketone produced by 50%, but had no i n f l u e n c e on the maximum growth rate or f i n a l c e l l y i e l d (not shown). Reducing the -2 -1 i r r a d i a n c e to 90 yE-m -s had no e f f e c t on the growth parameters but reduced the g-diketone produc t ion to only 3 y g - L " 1 . There was no 3-diketone produced at 60 pE-m'^s" 1 , a l e v e l at which l i g h t became l i m i t i n g to the growth of P. minimum. While the abso lute amount of 3-diketone produced by P. mar i ae - l ebour i ae was lower than that produced by P. minimum, 31 the i n f l u e n c e of i r r a d i a n c e on the temporal p a t t e r n of the 3-diketone p r o d u c t i o n was s i m i l a r ( F i g . 5B). In P. mariae-lebour iae , r e d u c t i o n of the i r r a d i a n c e from 160 to 120 yE-m'^s1 had no s i g n i f i c a n t i n f l u e n c e on the amount of -diketone produced. An i n c r e a s e or decrease i n i r r a d i a n c e to 270 or 90 yE m -s i n h i b i t e d 3-diketone p r o d u c t i o n . At each i r r a d i a n c e where 3-diketone was d e t e c t a b l e , the temporal p a t t e r n 'of pr o d u c t i o n was the same r e g a r d l e s s of the abs o l u t e amount of m a t e r i a l produced. At the onset of phosphate s t a r v a t i o n (Day 11 f o r P. minimum and Day 9 for P. mariae-l e b o u r i a e ) , the 3-diketone was produced r a p i d l y and maintained at a constant l e v e l in the medium. S i m i l a r p a t t e r n s of prod u c t i o n were seen under a l l other experimental c o n d i t i o n s . The p r o d u c t i o n of the 3-diketone under a l i g h t / d a r k c y c l e (18:6) suggests the pr o d u c t i o n of the 3-diketone i s l i g h t c o n t r o l l e d ( F i g . 6). While only one experiment was performed, there was no evidence of B-diketone p r o d u c t i o n during the dark p e r i o d . At the onset of the l i g h t , there was r a p i d production d u r i n g the f i r s t few hours, s h i f t i n g to a g r a d u a l l y slower r a t e of r e l e a s e . The r a p i d r e l e a s e of the 3 - d i k e t o n e may be due to the con v e r s i o n of an i n t r a c e l l u l a r p r e c u r s o r pool which forms d u r i n g the dark p e r i o d . While the k i n e t i c s of pr o d u c t i o n cannot be f u l l y a p p r e c i a t e d with a s i n g l e experiment, the l i n k of pr o d u c t i o n with the onset of the l i g h t p e r i o d suggests a p h o t o c o n t r o l mechanism. 32 Table II - Species examined for the produc t ion of the g-d i ketone. F i n a l c e l l y i e l d and the maximum recorded g -d ike tone c o n c e n t r a t i o n ( ± one SE, n = 3) are presented for c e l l s grown at 160 y E-m -s and 18 °C; except P . mar i a e - l e b o u r i a e which was grown at 22-23 °C . A l l c u l t u r e s were grown i n P - d e f i c i e n t batch c u l t u r e s . The e x t r a c e l l u l a r c o n c e n t r a t i o n of the g-diketone was monitored d a i l y . North East P a c i f i c C u l t u r e C o l l e c t i o n number i s g iven in brackets for the appropr i a t e s p e c i e s . Final Cell Yield Maximum -diketone Species (10-7cen.L-x) ( ug NH ? OH • IT1) P. minimum 3.3 39.6 + 7.2 TPavillard) Schiller (#96) P. minimum var. balticum 0.7 N.D. TPavillard) Schiller (#115) P_. micans 2.1 10.3± 2.7 Ehrenb. (#33) P. gracile 3.0 N.D. Schuett (#104) P. maximum 1.7 N,D. TGourret) Schiller (#250) P. mariae lebouriae ; 3.5 10.1+3.4. TParke et Ballentine) Hulburt* N.D. not detectable * Kindly supplied by Prof. H.H. Seliger, The Johns Hopkins University, Baltimore, Maryland 33 F i g u r e 5 - Production of the 3 - d i k e t o n e i n batch c u l t u r e -i n f l u e n c e of i r r a d i a n c e . The i n f l u e n c e of i r r a d i a n c e on the maximum growth r a t e (upper graph) and the temporal p r o d u c t i o n of the e x t r a c e l l u l a r g -diketone (lower graph) f o r : A) P. minimum, and B) P. mariae-l e b o u r i a e . Batch c u l t u r e s e v e n t u a l l y became P - d e f i c i e n t . R e p l i c a t e values are mean ± one SE where n i s noted i n b r a c k e t s . h f r j CO U Q E > I s X CD ra ~ ^ DC r 10-Q5H 6 0 . <5) (6) (6) <9> 3 0 6 0 9 0 120 160 uE-mV 6 0 9 0 120 160 uE ' r rv s " 1 <B ^ > CD * - C 03 O £ — ' 4 0 -20-160 o _ f o - o (6) 120 15i / ^  ^ ^ _ ^  y vj nn^n-o-y^n-nj-n—9 30/60 10 2 0 uE-m-s 1 e JV J 6 0 10 2 0 Days 34 F i g u r e 6 - Production of the g-diketone in batch c u l t u r e -i n f l u e n c e of a l i g h t : d a r k regime. The cumulative e x t r a c e l l u l a r g-diketone c o n c e n t r a t i o n f o r P. minimum grown under a 18:6 l i g h t : d a r k regime. Only the senescent phase of batch c u l t u r e growth i s shown. Days 35 b) I n f l u e n c e of s a l i n i t y S a l i n i t y had a s i g n i f i c a n t i n f l u e n c e on the f i n a l e x t r a c e l l u l a r g-diketone c o n c e n t r a t i o n . P. minimum had maximum growth r a t e s over a wide range of s a l i n i t i e s (from 1 5 % o to 30/») ( F i g . 7A). Maximum 3-diketone production had a r e s t r i c t e d s a l i n i t y range of 20-30 %.. At s a l i n i t i e s l e s s than 20%,, the amount of e x t r a c e l l u l a r g-diketone produced decreased r a p i d l y . P. mariae-lebour iae had a very d i f f e r e n t s a l i n i t y t o l e r a n c e range. S a l i n i t e s of 10 to 15^*° were r e q u i r e d to give maximum growth r a t e s ( F i g . 7B). Growth i n f u l l s t r e n g t h medium (27&,) reduced the growth rate by more than 50% and n e a r l y e l i m i n a t e d the 3-diketone p r o d u c t i o n . Growth ra t e s and g-diketone p r o d u c t i o n were a l s o reduced at s a l i n i t i e s l e s s than 10%,. Maximum e x t r a c e l l u l a r g-diketone c o n c e n t r a t i o n s were achieved at s a l i n i t i e s below those p r o v i d i n g maximum growth r a t e s . c) I n f l u e n c e of temperature Temperature a l s o had a profound e f f e c t on e x t r a c e l l u l a r g -diketone production ( F i g . 8). Temperatures l e s s then 15°C reduced the amount of g-diketone produced from 50 p g-L" 1 to l e s s then 3 ug-L 1 . There was no d e t e c t a b l e g-diketone when c e l l s were grown at 5 C. 2) Production under d i f f e r e n t N:P r a t i o s The i n f l u e n c e of the i n i t i a l nitrogen:phosphorus atomic r a t i o s on the production of the g-diketone i s shown i n F i g . 9. P. min imum was grown at N:P r a t i o s ranging from 2 to 100. The 36 Figure 7 - Product ion of the g-diketone in batch c u l t u r e -i n f l u e n c e of s a l i n i t y . The i n f l u e n c e of s a l i n i t y on the growth rate (upper graphs) and the maximum g -d iketone c o n c e n t r a t i o n (lower graphs) f o r : A) P. minimum and B) P. m a r i a e - l e b o u r i a e . R e p l i c a t e va lues are means (± one SE) , where n i s noted i n b r a c k e t s . A P. minimum 1.0-1 B P. mariae lebouriae t 3 ) (3. .3.? «L0l 10 20 30 10 20 30 Salinity (%») 37 F igure 8 - Produc t ion of the B -d ike tone i n batch c u l t u r e -i n f l u e n c e of temperature. The i n f l u e n c e of temperature on the growth ra te (upper graph) and the maximum 3 - d i k e t o n e c o n c e n t r a t i o n (lower graph) for P. minimum. R e p l i c a t e va lues are mean ± one SE where n i s noted i n b r a c k e t s . OS .Is ^ CL 1.0i > 0.5 19) -1— 5 (5 ) o — 4 -"lO 15 20 25 10 15 20 25 TemperatureCO 38 F igure 9 - Product ion of the 3-diketone i n batch c u l t u r e -i n f l u e n c e of the i n i t i a l N:P atomic r a t i o i n the medium. F i n a l c e l l y i e l d s for a l l experiments were e q u i v a l e n t . N:P. ATOMIC RATIO 39 atomic r a t i o of N:P = 25 was chosen as the t h e o r e t i c a l r a t i o where N and P were both l i m i t i n g to growth, based on the disappearance of n i t r a t e and phosphate from the batch c u l t u r e medium. To achieve these exper imenta l n u t r i e n t r a t i o s without decreas ing the f i n a l c e l l y i e l d of the c u l t u r e s , the amount of n o n - l i m i t i n g n u t r i e n t was increased i n the medium. For example, N:P r a t i o s greater than N:P = 25 were achieved by m a i n t a i n i n g the l i m i t i n g n u t r i e n t (P) at 6 yM and i n c r e a s i n g the amount of added N u n t i l the d e s i r e d r a t i o was a c h i e v e d . At very low N:P r a t i o s (N:P 2) a l a rge amount of inorgan ic p r e c i p i t a t e formed (C = 2.8%, H = 1.4%, N = 0.3%, P = 6.9%). Since autoc laved N:P = 2 medium, without c e l l s , f a i l e d to show any p r e c i p i t a t e , formation of t h i s p r e c i p i t a t e i s e i t h e r s t i m u l a t e d by the presence of the c e l l s or the a c t i v i t y of the c e l l s . The 3 - d i k e t o n e was not produced at t h i s r a t i o . At N:P r a t i o s from 5 to 22 ( N - d e f i c i e n t growth) , the l e v e l of 3 - d i k e t o n e produced was constant at 20 u g - L " 1 . As the c e l l s s h i f t e d to P - d e f i c i e n t growth c o n d i t i o n s (N:P = 27 to 35) , p roduc t ion doubled to about 40 y g - L " 1 . Phospha te -de f i c i en t c e l l s exposed to i n c r e a s i n g c o n c e n t r a t i o n s of ambient n i t r a t e , produced decreas ing amounts of g -d iketone . At N:P r a t i o s greater then 60, no g-diketone was produced. To t e s t whether the r e d u c t i o n in g-diketone product ion at N:P = 40 was a f u n c t i o n of the a c t u a l r a t i o or the amount of excess n i t r a t e in the c u l t u r e medium, a s e r i e s of phosphate-d e f i c i e n t c u l t u r e experiments was performed where the N:P atomic r a t i o was kept constant at 40, but the amount of phosphate added 40 was increased or decreased. As the amount of excess n i t r a t e in the c u l t u r e medium decreased, the amount of g - d i k e t o n e o normal ized on a per c e l l b a s i s , inc rea sed , reaching values s i m i l a r to the maximum amount of the g - d i k e t o n e produced under P - l i m i t a t i o n (Table I I I ) . Conver se ly , an increase i n the amount of excess n i t r a t e , at a constant N:P r a t i o of 40, complete ly i n h i b i t e d the g - d i k e t o n e p r o d u c t i o n . A s i m i l a r experiment to examine the i n f l u e n c e of excess phosphate at N:P = 2 f a i l e d to show an increase i n the g-diketone p r o d u c t i o n . In a l l experiments , a l a rge amount of p r e c i p i t a t e formed which may have i n t e r f e r r e d w i t h c e l l u l a r processes and reduced the amount of excess phosphate, s ince the p r e c i p i t a t e conta ined large amounts of phosphate. The amount of p r e c i p i t a t e c o u l d not be q u a n t i f i e d , s ince much of the m a t e r i a l adhered t e n a c i o u s l y to the g l a s s f l a s k . The methanol e x t r a c t s of P. minimum c e l l s grown at N:P = 5 were examined by TLC to determine i f the d i f f e r e n c e in e x t r a c e l l u l a r 3 - d i k e t o n e c o n c e n t r a t i o n was due to i n t r a c e l l u l a r storage when c e l l s were n i t r a t e - l i m i t e d . C e l l s from N:P = 5 and N:P = 27 were e x t r a c t e d w i t h methanol and run w i t h a g - d i k e t o n e standard i n a C H C l 3 : e t h y l aceta te (1:1) TLC system. Ne i ther of the c e l l e x t r a c t s conta ined the g - d i k e t o n e , a l though there were obvious d i f f e r e n c e s in the c e l l u l a r p r o f i l e s . C e l l s grown at N:P = 5 had l a rge amount of F e C l 3 p o s i t i v e m a t e r i a l w i t h Rf va lues greater than the -d ike tone (0.6 and 0.89 v s . 0.37 for the g - d i k e t o n e ) ( F i g . 10). N e i t h e r of the F e C l 3 p o s i t i v e spots gave the c h a r a c t e r i s t i c red-brown c o l o r of the g - d i k e t o n e . 41 Table III - The i n f l u e n c e of i n c r e a s i n g ambient n i t r a t e c o n c e n t r a t i o n i n the medium on the prod u c t i o n of the 3 - d i k e t o n e by P. minimum. C e l l s were grown i n N:P = 40 medium. Calculated * excess N Extracellular P added N added in the medium 3-diketone, ( u M) (yM) (yM) ( Pq • 10"7cells) 21.9 (control) 591.3 43. 8 6.3 2.7 109.8 36. 1 5.8 5.4 219.6 82. 4 3.7 10.9 439.2 164. 7 0.9 21.9 878.4 330. ,9 0.8 43.9 1756.8 657. 8 0.0 * Calculated based on a theoretical nutrient equilibrium ratio of N:P = 25. 42 Fi g u r e 10 - Examination of c e l l contents f o r the presence of the 3-diketone. Diagramatic r e p r e s e n t a t i o n of t h i n l a y e r chromatography s t r i p s (SIL G/UV 254) of the i n t r a c e l l u l a r , c h l o r o f o r m - s o l u b l e f r a c t i o n of P. minimum grown at two d i f f e r e n t N:P atomic r a t i o s . 1) c e l l s grown at N:P = 27; 2) 3-diketone standard; and 3) c e l l s grown at N:P = 5. The s o l v e n t system used was c h l o r o f o r m : e t h y l a c e t a t e (1:1). Shaded areas give a p o s i t i v e r e a c t i o n to FeCl (2% i n ethanol) spray. solvent front ight green yellow — 2 — r t o r i g i n 43 F i g u r e 11 - Production of the g-diketone i n batch c u l t u r e -i n f l u e n c e of i r o n - d e f i c i e n c y . Changes i n the g-diketone c o n c e n t r a t i o n and c e l l numbers during e x p o n e n t i a l growth and during senescence in i r o n - s t a r v e d batch c u l t u r e s . R e p l i c a t e values are mean + one SE where n i s noted in b r a c k e t s . 44 Table IV - Inf luence of the a d d i t i o n of vanadium on the B -diketone product ion i n i r o n - s t a r v e d batch c u l t u r e s of P. minimum. C u l t u r e s were harvested at Day 15 of batch c u l t u r e growth. Growth Conditions Maximum B-diketone produced (yg-L" 1) iron limited 10.9 (ESNW-Fe) iron 1imi ted with 4 X 10"8M NH ^  V0 3 0.0 iron 1imi ted with 8 X 10-8M NH 4 V03 0.0 iron sufficient (control) 38.3 45 3) Production under i r o n - d e f i c i e n t growth c o n d i t i o n s P. min imum c e l l s grown under i r o n - l i m i t e d c u l t u r e c o n d i t i o n s had a p a t t e r n of e x t r a c e l l u l a r g-diketone p r o d u c t i o n s i m i l a r to the p a t t e r n from p h o s p h a t e - d e f i c i e n t c u l t u r e s , but the a b s o l u t e amount of the g-diketone produced was s u b s t a n t i a l l y lower ( F i g . 11). Only c e l l s grown i n the ESNW-Fe medium produced the g-diketone. C e l l s grown i n the AQUIL-Fe medium f a i l e d to produce the e x t r a c e l l u l a r g-diketone. The a d d i t i o n of NH VO „ (a t r a c e metal used to a l l e v i a t e the k 3 i r o n l i m i t a t i o n ; Meisch and B i e l i g , 1975) had no e f f e c t on the growth parameters measured (growth rate and f i n a l c e l l y i e l d were e q u i v a l e n t to the i r o n - s u f f i c i e n t c o n t r o l s ) , but i t completely i n h i b i t e d the production of the g-diketone (Table IV). The a d d i t i o n of i r o n to the t r e a t e d medium r e e s t a b l i s h e d maximum e x t r a c e l l u l a r g-diketone c o n c e n t r a t i o n s . E. DISCUSSION The p r o d u c t i o n of the n o r - c a r o t e n o i d 1 - ( 2 , 6 , 6 - t r i m e t h y l - 4 -hydroxycyclohexeny1)- 1,3-butanedione by P. min imum i s under d i r e c t p h y s i o l o g i c a l c o n t r o l and cannot simply be co n s i d e r e d as a photooxygenation product of c a r o t e n o i d d e g r a d a t i o n . S e v e r a l l i n e s of evidence i n d i c a t e that the c o n t r o l of the g-diketone production i s under the same c o n t r o l processes as the t o t a l e x t r a c e l l u l a r carbon r e l e a s e (composed mostly of primary o r g a n i c s ) . Production of the g-diketone i s r e l a t e d to the p h y s i o l o g i c a l s t a t e of the c e l l . A l l p r o d u c t i o n i s l i m i t e d to 46 the s t a t i o n a r y phase of growth. T h i s i s the case f o r many e x t r a c e l l u l a r o r g a n i c s ( G u i l l a r d and Wangersky 1958; Marker 1965; Bolze and Soeder 1978; Melkonian 1979). However, i n the case of the g-diketone produced by P. minimum, the p r o d u c t i o n i n the s t a t i o n a r y phase i s t i g h t l y c o n t r o l l e d . Release of the g-diketone i s not gradual, suggesting a " l e a k i n g " of c e l l u l a r c o ntents, but r a t h e r , there i s a short p e r i o d of r a p i d p r o d u c t i o n followed by an e q u a l l y r a p i d c e s s a t i o n of 3-diketone p r o d u c t i o n a f t e r a short (1-2 days) p e r i o d of time. The r e s t r i c t e d p e r i o d of production suggests the exhaustion of c e l l u l a r p r e c u r s o r s a v a i l a b l e f o r 3 - d i k e t o n e p r o d u c t i o n . The de novo s y n t h e s i s of the -diketone i s supported by the p r o d u c t i o n under l i g h t / d a r k c y c l e s . Release of the g-diketone does not occur i n the dark. Since there was no evidence of accumulated i n t r a c e l l u l a r 3-diketone i n c e l l s c o l l e c t e d from the dark immediately p r i o r to the s t a r t of the l i g h t c y c l e , we must assume that p r e c u r s o r s have accumulated i n t r a c e l l u l a r l y and that the 3-diketone i s formed by photochemically mediated b i o s y n t h e s i s . I r r a d i a n c e was a strong i n f l u e n c e in c o n t r o l l i n g the 3-diketone p r o d u c t i o n . Increased i r r a d i a n c e s favor the r e l e a s e of DOC by marine phytoplankton (Fogg e_t a_l. , 1965; H e l l e b u s t , 1965; Nalewajko,1966) and the same can be seen for the p r o d u c t i o n of the 3 - d i k e t o n e . In the l a t t e r case, the extent of production i n c r e a s e d n o n - l i n e a r l y over the range of i r r a d i a n c e s . S i m i l a r l i g h t c o n t r o l of DOC from Skeletonema has a l s o been shown (Ignatiades and Fogg, 1973). The production of 3-diketone d i d 47 not occur i f l i g h t was l i m i t i n g to growth. T h i s i s not n e c e s s a r i l y the case i f one examines DOC r e l e a s e . Kuenzler (1970) found DOC r e l e a s e to be constant over a wide range of i r r a d i a n c e s , i n c l u d i n g l i g h t l e v e l s l i m i t i n g to growth r a t e s . The amount of the g-diketone p r o d u c t i o n was not c o n t r o l l e d e x t e n s i v e l y by temperature, although p r o d u c t i o n of the g-diketone ceased when temperatures reduced the growth rate of P.  minimum. The i n f l u e n c e of n u t r i e n t d e f i c i e n c y on the pr o d u c t i o n of the e x t r a c e l l u l a r g-diketone suggests a strong c o n t r o l by the p h y s i o l o g i c a l s t a t e of the c e l l . S h i f t i n g from N - d e f i c i e n t to P - d e f i c i e n t growth c o n d i t i o n s , s h i f t s the carbon metabolism of the c e l l . Enzymes (such as polyphosphokinase, polyphophatase, and a l k a l i n e phosphatase), storage products, and energy in t e r m e d i a t e s (such as adenylate charge and adenosine t r i p h o s p h a t e ) are a l l c o n t r o l l e d by the d e f i c i e n t n u t r i e n t (Healey, 1975). The i n f l u e n c e of t h e ' l i m i t i n g n u t r i e n t on the composition of the e x t r a c e l l u l a r exudate i s l e s s w e l l understood. Myklestad (1977) c l a r i f i e d the c o n t r o l of N:P r a t i o s on c e l l u l a r p rocesses. E x p o n e n t i a l l y growing c e l l s show l i t t l e v a r i a t i o n i n the c e l l u l a r compostion s i n c e n e i t h e r n u t r i e n t i s l i m i t i n g , but as the s t a t i o n a r y phase i s reached, the composition of the medium has profound e f f e c t s . The c e l l u l a r composition changes to r e f l e c t the composition of the medium. Examining the i n f l u e n c e of N:P r a t i o s on growth and carbohydrate metabolism, Myklestad (1977) noted l i t t l e d i f f e r e n c e i n the e x t r a c e l l u l a r p o l y s a c c h a r i d e s at 48 c o n c e n t r a t i o n s over the N:P range of 0.4 to 100 f o r Skeletonema  costatum. However, Myklestad d i d f i n d i n c r e a s e d l e v e l s of e x t r a c e l l u l a r p o l y s a c c h a r i d e d u r i n g the senescent stage of growth i f c e l l s were P - d e f i c i e n t i n s t e a d of N - d e f i c i e n t . P h o s p h a t e - d e f i c i e n t c e l l s produced two to three times the amount of e x t r a c e l l u l a r p o l y s a c c h a r i d e compared to n i t r a t e - d e f i c i e n t c e l l s . A s i m i l a r trend was shown f o r e x t r a c e l l u l a r p o l y s a c c h a r i d e production i n s t a t i o n a r y Chaetoceros af f i n i s c u l t u r e s (Myklestad, 1977). The i n f l u e n c e of the N:P r a t i o on the e x t r a c e l l u l a r p r o d u c t i o n of the g-diketone i s s i m i l a r in s e v e r a l s i g n i f i c a n t ways. When P.minimum was N - d e f i c i e n t , a constant amount of g-diketone was produced at 20 y g - L - 1 . When c e l l s s h i f t to P-d e f i c i e n c y there was an in c r e a s e d amount of the g-diketone produced (40 yg-L l ) . But, at N:P values g r e a t e r than N:P = 30, i t was the amount of excess n i t r a t e which i n h i b i t e d the amount of the g-diketone produced. The c o n t r o l of e x t r a c e l l u l a r m e t a b o l i t e s by excess n u t r i e n t s has been shown f o r many b a c t e r i a l and fungal m e t a b o l i t e s (Weinberg, 1970; Drew and Demain, 1977; Martin and Demain, 1980). The reduced amount of the g-diketone produced under i r o n -d e f i c i e n t growth c o n d i t i o n s compared to P - d e f i c i e n t c u l t u r e s suggests that the g-diketone was not d i r e c t l y l i n k e d to the high a f f i n i t y i r o n t r a n s p o r t system, even though the g-diketone forms an i r o n complex and has the p o t e n t i a l to c h e l a t e metals (Andersen et a l . , 1980). The a d d i t i o n of vanadium to i r o n - l i m i t e d c u l t u r e s of 49 P. minimum i s a c r i t i c a l experiment. Vanadium i s a t r a c e metal found commonly as a c o n s t i t u e n t of marine algae (O'Kelley, 1974) , but i t i s not n e c e s s a r i l y an e s s e n t i a l n u t r i e n t . The a d d i t i o n of vanadium to the c u l t u r e medium i n h i b i t s the formation of ^"-aminolevulinic a c i d ( *-ALA) , which i s a key step in the r e g u l a t i o n of c h l o r o p h y l l p r o d u c t i o n (Meisch and B i e l i g , 1975) . T h i s i s the same po i n t at which i r o n l i m i t a t i o n a f f e c t s the c e l l ' s p h y s i o l o g y (Jones, 1976). A second i n f l u e n c e of vanadium on the c e l l ' s p h y s i o l o g y was the s t i m u l a t i o n of the ph o t o i n d u c t i o n of c a r o t e n o i d g e n e s i s (Shropshire, 1980). The a d d i t i o n of vanadium to P. min imum i n h i b i t e d the 3-diketone pro d u c t i o n rather than s t i m u l a t e d the n o r - c a r o t e n o i d r e l e a s e , p r o v i d i n g f u r t h e r evidence a g a i n s t the 3-diketone as a non-s e l e c t i v e photooxygenation product of c a r o t e n o i d s . I t i s apparent from the l i m i t i n g n u t r i e n t experiments that the two processes c o n t r o l l i n g the B-diketone p r o d u c t i o n are re g u l a t e d by two n u t r i e n t - r e l a t e d c r i t e r i a : 1) the type of l i m i t i n g n u t r i e n t (P, N, or Fe, and 2) the amount of excess n i t r a t e a v a i l a b l e in the c u l t u r e medium. Se v e r a l p o i n t s can be s t r e s s e d . The pr o d u c t i o n of the e x t r a c e l l u l a r 3-diketone i s a normal p h y s i o l o g i c a l process r e l a t e d to the carbon flow w i t h i n the c e l l . The 3-diketone was formed immediately p r i o r to r e l e a s e and was never found i n t r a c e l l u l a r l y . The pr o d u c t i o n was a f u n c t i o n of the n u t r i e n t environment of the c e l l s and was mediated by photochemical c o n t r o l of compound formation. R e l a t i n g the production of the 3-diketone to the ecology of 50 P. min imum i s d i f f i c u l t . U n l i k e p r o r o c e n t r i n ( the siderophore which w i l l be d i s c u s s e d i n Chapters V and VII of t h i s t h e s i s ) , the r o l e of the g-diketone cannot be d i r e c t l y r e l a t e d to the p h y s i o l o g i c a l response of the c e l l . The l i n k between the p r o d u c t i o n of e x t r a c e l l u l a r g-diketone and the formation of P_;_ min imum blooms i s e s p e c i a l l y open to s p e c u l a t i o n . Researchers examining n a t u r a l phytoplankton blooms have n o t i c e d that even though c e l l s are s e v e r e l y s t r e s s e d or senescent, there i s no r e s u l t i n g i n c r e a s e i n the amount of e x t r a c e l l u l a r carbon r e l e a s e (Herbland and Dandonneau, 1975; Mague et a_l. , 1980). While the q u a n t i t y of e x t r a c e l l u l a r o r g a n i c s may not change s i g n i f i c a n t l y , the q u a l i t y might change. The g-diketone comprises only a small p r o p o r t i o n of the t o t a l r e l e a s e d organic and i s produced at l e v e l s which w i l l not r e s u l t in an i n c r e a s e i n DOC. But the g-diketone shows a n t i b a c t e r i a l a c t i v i t y when t e s t e d at the c o n c e n t r a t i o n s recorded in c u l t u r e . If the amount of the g-diketone produced in batch c u l t u r e s i s s i m i l a r to that i n the f i e l d , i t i s p r e d i c t e d that s u f f i c i e n t g -diketone w i l l be produced to i n h i b i t n a t u r a l p o p u l a t i o n s of b a c t e r i a (Appendix A). Part of the problem in e v a l u a t i n g the r o l e that the g-diketone may play in c o n t r o l l i n g b a c t e r i a l p o p u l a t i o n s i s our poor understanding of the l i n k between phytoplankton and b a c t e r i a . Researchers in the past have suggested that a n t i b a c t e r i a l m e t a b o l i t e s from algae are not important ( C a r l u c c i and Pramer i960), but recent work by Aubert and co-workers (Aubert et a l . 1968; 1970; Aubert and Pesando, 1971), has 51 p r o v i d e d s u b s t a n t i a l evidence of strong a l g a l - b a c t e r i a l i n t e r a c t i o n s . M e t a b o l i t e s which i n f l u e n c e a l g a l - b a c t e r i a l i n t e r a c t i o n s can be n o n - s p e c i f i c i n h i b i t o r s such as tannins or organic a c i d s (Lucas, 1955; N i g r e l l i , 1958), or s p e c i f i c , u n c h a r a c t e r i z e d m e t a b o l i t e s produced by l a b o r a t o r y c u l t u r e s ( B e l l et a l . , 1977; Kogure et a l . , 1979) or by n a t u r a l p o p u l a t i o n s ( S i e b u r t h , 1971; Moebus, 1972). C h a r a c t e r i z e d a n t i b a c t e r i a l agents produced by phytoplankton i n c l u d e c h l o r e l l u m from C h l o r e l l a ( P r a t t et. a l . , 1944), a c r y l i c a c i d from Phaeocyst i s pouchet i i ( G u i l l a r d and H e l l e b u s t , 1971) and Phaeodactylum t r i c o r n u t u m (Brown e_t a l . , 1977), and goniodomin from the marine d i n o f l a g e l l a t e Goniodoma (Sharma e_t al_. , 1 9 6 8 ) . There are s e v e r a l ways of viewing the organic carbon l i n k between phytoplankton and b a c t e r i a . The l i n k can be a one-way carbon exchange where the phytoplankton exudates are the source of organic m a t e r i a l f o r the b a c t e r i a (Smith, 1974; Smith and Higgins,1978; Larsson and Hagstrom, 1979; M e f f e r t and Overbeck, 1979). The extent of carbon t r a n s f e r to the b a c t e r i a can be s u b s t a n t i a l . Estimates i n d i c a t e that from 25% to 50% of the t o t a l f i x e d carbon i s i n c o r p o r a t e d i n t o the b a c t e r i a (Andrews and W i l l i a m s , 1971; Hagstrom et. a l . , 1979; Larsson and Hagstrom, 1979). These estimates suggest that a l a r g e part of the b a c t e r i a l carbon requirement i s met by phytoplankton exudates. To strengthen the l i n k between phytoplankton and b a c t e r i a , Larsson and Hagstrom (1982) have r e i n t r o d u c e d the concept of the "phycosphere". The phycosphere, as i t was o r i g i n a l l y d e s c r i b e d by B e l l and M i t c h e l l (1972), i s a zone surrounding each 52 phytoplankton c e l l . T h i s zone i s composed of a high c o n c e n t r a t i o n of a l g a l exudates. The zone c o n t a i n s a l l e x t r a c e l l u l a r o r g a n i c s i n c l u d i n g b a c t e r i a l a t t r a c t a n t s (Kogure et a l . , 1982) and a n t i b a c t e r i a l agents. Thus, the e p i p h y t i c b a c t e r i a l p o p u l a t i o n of a phytoplankton c e l l would be a f u n c t i o n of the c o n t r o l of e x t r a c e l l u l a r o rganic p r o d u c t i o n . T h i s r e l a t i o n s h i p has been p o o r l y c h a r a c t e r i z e d . B a c t e r i a gain by having a high c o n c e n t r a t i o n of r e a d i l y a v a i l a b l e o r g a n i c s , and a constant source of m a t e r i a l . The phytoplankton may gain r e q u i r e d o r g a n i c s such as vitamins or growth f a c t o r s from the b a c t e r i a but almost no evidence e x i s t s f o r the t r a n s f e r of o r g a n i c s from b a c t e r i a to phytoplankton (Haines and G u i l l a r d , 1 974) . U t i l i z i n g the concept of the phycosphere, e x p o n e n t i a l l y growing c e l l s would develop a m e t a b o l i c a l l y a c t i v e b a c t e r i a l p o p u l a t i o n , e s p e c i a l l y i f the b a c t e r i a show a chemotactic response to a l g a l exudates ( B e l l and M i t c h e l l , 1972; S j o b l a d and M i t c h e l l , 1979). As phytoplankton become n u t r i e n t d e f i c i e n t , the amount of o r g a n i c s r e l e a s e d a l s o decreases. The b a c t e r i a become o r g a n i c - s t r e s s e d and may s t i m u l a t e the degradation and r e m i n e r a l i z a t i o n of the a s s o c i a t e d phytoplankton by the p r o d u c t i o n of e x t r a c e l l u l a r enzymes. Using the concept of a phycosphere around the phytoplankton c e l l , the b a c t e r i a c o u l d produce s u f f i c i e n t c o n c e n t r a t i o n s of e x t r a c e l l u l a r enzymes to degrade the host. A phytoplankton c e l l which forms a r e s t i n g stage may a l t e r i t s s t r u c t u r e to r e s i s t b a c t e r i a l a t t a c k . However s p e c i e s such 53 as P^ minimum , for which no a l t e r n a t e stage i s known, c o u l d produce a n t i b a c t e r i a l metabo l i t e s such as the g-diketone to reduce the degradat ive a c t i v i t y of the a s s o c i a t e d b a c t e r i a l populat i o n . The p a t t e r n of p roduc t ion of the g-diketone lends i t s e l f to t h i s h y p o t h e s i s . P r i o r to the -d ike tone p r o d u c t i o n , b a c t e r i a a s s o c i a t e d w i t h P. min imum would have a favorable environment for h e t e r o t r o p h i c a c t i v i t y . With the h e t e r o t r o p h i c mechanism i n t a c t , the g - d i k e t o n e i s produced i n a s i n g l e r a p i d p u l s e , c r e a t i n g a t o x i c phycosphere. As soc i a t ed b a c t e r i a are i n h i b i t e d p r i o r to e x c r e t i o n of e x t r a c e l l u l a r degradat ive enzymes. The a c t i v i t y of the a s s o c i a t e d b a c t e r i a i s i n f l u e n c e d by the chemica l compostion of the substances re lea sed and the p a t t e r n of r e l e a s e . S t r i c t c o n t r o l of p roduc t ion i s necessary to ensure that the g-diketone i s re lea sed in s u f f i c i e n t q u a n i t i t i e s to e l i m i n a t e the a s s o c i a t e d b a c t e r i a l a c t i v i t y under p h y s i o l o g i c a l c o n d i t i o n s which are t empora l ly severe . I t has been argued that e x t r a c e l l u l a r organics serve no purpose to the producing spec ies and that a n t i b a c t e r i a l compounds, such as the g -d ike tone , are produced at c o n c e n t r a t i o n s too low to be a c t i v e in the marine environment. Under c e r t a i n c o n d i t i o n s , the produc t ion i s ha rd ly i n s i g n i f i c a n t and the abso lute c o n c e n t r a t i o n of the g -d ike tone may be much higher than i n i t i a l l y thought i f one examines the p a t t e r n of p r o d u c t i o n and the e x i s t e n c e of a physosphere. The study of the r o l e of h e t e r o t r o p h i c b a c t e r i a and m i c r o f l a g e l l a t e s i n degrading and r e m i n e r a l i z i n g the organics i n 54 marine waters i s of primary importance in the understanding of m i c r o b i a l i n t e r r e l a t i o n s h i p s ( S t r i c k l a n d , 1971). To understand the p h y t o p l a n k t o n - b a c t e r i a r e l a t i o n s h i p s not on ly must the q u a n t i t y of organic s b e , c o n s i d e r e d , but a l s o the q u a l i t y . The p r o d u c t i o n of the n o r - c a r o t e n o i d from P. min imum may be an example of the sever ing of organic l i n k s between t r o p h i c l e v e l s by e x t r a c e l l u l a r phytoplankton exudates . 55 IV. GENERAL INTRODUCTION TO SIDEROPHORES A. ABSTRACT A general review of siderophores ( f e r r i c i o n - s p e c i f i c c h e l a t o r s ) i s presented with emphasis on siderophore p r o d u c t i o n by higher p l a n t s and phytoplankton. Siderophores, by s t r i c t d e f i n i t i o n , have never been v e r i f i e d in higher p l a n t s . Higher p l a n t s may r e l y on the pro d u c t i o n of o r g a n i c s which i n c r e a s e the s o l u b l i z a t i o n of p a r t i c u l a t e i r o n , but these compounds have no r o l e in the t r a n s p o r t of i r o n i n t o the r o o t s . Cyanobacteria are we l l known producers of si d e r o p h o r e s , but the pro d u c t i o n of siderophores by e u k a r y o t i c phytoplankton i s c o n t r o v e r s i a l . Evidence, both, f o r and aga i n s t the siderophore p r o d u c t i o n by eu k a r y o t i c marine algae, i s reviewed. 56 B. INTRODUCTION Evidence for s iderophores as h i g h - a f f i n i t y i r o n - a q u i s i t i o n systems i s w e l l documented. Recent reviews have covered most genera l aspects of s iderophores such as c l a s s i f i c a t i o n and p r o p e r t i e s of s iderophores (Ne i l ands , 1981a), m i c r o b i a l i r o n uptake (Lankford , 1973; N e i l a n d s , 1980; N e i l a n d s , 1981b), s iderophore-mediated i r o n - t r a n s p o r t mechanisms (Raymond and Carrano, 1979; Bezkorova iny , 1980), and the e v o l u t i o n a r y development of s iderophore systems (Ne i l ands , 1966). S iderophores are low molecular weight o r g a n i c s , which coord ina te and s o l u b l i z e i r o n ( I I I ) by forming a h i g h l y s t ab le complex. Siderophore produc t ion i s s t i m u l a t e d by growth i n low i r o n c o n d i t i o n s ( G a r i b a l d i and N e i l a n d s , 1956). The mechanism of metabol ic c o n t r o l has only been examined i n a few systems but i t appears that the b i o s y n t h e t i c pathway for the produc t ion of s iderophores i s repressed by a v a i l a b l e i r o n . I t does not appear to be a system c o n t r o l l e d s imply by enzyme i n h i b i t i o n (Ne i l ands , 1973). S iderophores are of two chemical types . The c a t e c h o l - t y p e , of which e n t e r o b a c t i n i s an example, binds i r o n v i a three b identa te c a t e c h o l - f u n c t i o n a l i t i e s . The i r o n complex of the c a t e c h o l s iderophore i s red to purple i n c o l o r . The second group of s iderophores i s the hydroxamate-type s iderophore . Three hydroxamate groups are r e q u i r e d to f i l l the hexacoordinate f e r r i c complex (Ne i l ands , 1973). Ferr ichrome i s an example of a t r ihydroxamate s iderophore ( F i g . 12). As d i scus sed by Nei l ands (1973), t h i s dichotomy of 57 F i g u r e 12 - The s t r u c t u r e s of r e p r e s e n t a t i v e s i d e r o p h o r e s . A: E n t e r o b a c t i n ( t r i - c a t e c h o l s i d e r o p h o r e ) . B: Ferrichrome (tri-hydroxamate siderophore) (Adapted from Cooper e_t a l . , 1978). B. C O N H - C H p - C O N H - C H o — C O N H / /CH< C H — N H C O - C H — N H C O - C H - N H C O ' ( C H 2 ) 3 ( C H 2 ) 3 ( C H 2 ) 3 N-OH N-OH N-OH c=o oo c=o * . \ j C H 3 C H 3 C H ^ 58 s iderophore types i s m i s l e a d i n g . S iderophores are a h i g h l y d i v e r s e group of i r o n - c h e l a t o r s . Many s iderophores c o n t a i n a combinat ion of hydroxamate and c a t e c h o l f u n c t i o n a l i t i e s or c o n t a i n hydroxamate/catechol f u n c t i o n a l i t i e s w i t h another u n r e l a t e d group (eg. c i t r i c ac id ) forming the f i n a l c o o r d i n a t i o n s i t e . Given the present ev idence , the separa t ion of s iderophores i n t o the two groupings i s s u f f i c i e n t for the examination of s iderophores from e u k a r y o t i c phytop lankton . The only microorganisms known to produce c a t e c h o l - t y p e s iderophores are the t rue b a c t e r i a , such as Aerobacter aerogenes, E s c h e r i c h i a  c o l i , and Sa lmonel la typhimur ium (Ne i l ands , 1973). Species wi th mi tochondr ia produce s iderophores w i t h a hydroxamate-type l i g a n d , a l though hydroxamate-type s iderophores are produced by some b a c t e r i a . The s p e c i f i c i t y of the hydroxamate-type s iderophores in e u k a r y o t i c microorganisms may be a r e s u l t of a s p e c i f i c t r a n s p o r t mechanism r e q u i r e d to prov ide i r o n to the mi tochondr ia ( N e i l a n d s , 1972). C. TRANSPORT OF IRON BY SIDEROPHORES Produc t ion of s iderophores by microorganisms has been extremely w e l l documented in the l i t e r a t u r e . Less w e l l s t u d i e d i s the mechanism of s iderophore mediated i r o n t r a n s p o r t . While only a smal l number of organisms has been examined, c e r t a i n trends have begun to emerge. F i r s t , the uptake of the s i d e r o p h o r e - i r o n complex r e q u i r e s energy (Lankford , 1973). 59 Second, the i r o n - s i d e r o p h o r e t r a n s p o r t system i s h i g h l y s p e c i f i c for a g iven c h e l a t o r . For each s iderophore which can be t r a n s p o r t e d i n t o the c e l l there i s a s p e c i f i c , separate t r a n s p o r t system (Frost and Rosenberg, 1973; Hayden et a l . , 1973; A s w e l l e_t a_l. , 1977). Of equal importance i s the fac t that these s p e c i f i c t r a n s p o r t systems are i n d u c i b l e by the presence of the s p e c i f i c s i d e r o p h o r e - i r o n complex. These are important c r i t e r i a when one cons ider s the e c o l o g i c a l r o l e of s ide rophore s . The s e l e c t i v i t y of these t r a n s p o r t systems c o u l d prov ide the bas i s for compet i t ion between spec ies dur ing i r o n -l i m i t e d growth c o n d i t i o n s . The c e l l u l a r management of the s i d e r o p h o r e - i r o n complex has been s tud ied i n s evera l d i v e r s e s iderophore systems. G e n e r a l l y , hydroxamates are taken across the c e l l membrane as the f e r r i -hydroxamate complex. The ferr i -hydroxamate complex i s s t r o n g ; t h e r e f o r e to re lease the F e ( I I l ) , the c e l l must reduce the i r o n to F e ( I I ) which i s only weakly bound to the s iderophore . The i r o n ( I I ) i s re leased and the hydroxamate r e c y c l e d back i n t o the medium ( F i g . 13A). Thi s r e c y c l i n g has been r e f e r r e d to as the "European approach" (Bezkorovainy, 1980). Conver se ly , e n t e r o b a c t i n (a c a t e c h o l - t y p e s iderophore) t r a n s f e r s one i r o n atom i n t o the c e l l and i s de s t royed . There i s no r e c y c l i n g of the s iderophore . Thi s has been r e f e r r e d to as the "American approach" ( F i g . 13B). Thi s system i s o b v i o u s l y more expensive than the r e c y c l i n g hydroxamate-type s ide rophore . Cooper et. §_1. (1978) has suggested that the e n t e r o b a c t i n - i r o n complex has a r e d u c t i o n p o t e n t i a l which i s too 60 F i g u r e 13 - Representative t r a n s p o r t mechanisms. A: Ferrichrome (hydroxamate){Fer} - "European Approach". B: E n t e r o b a c t i n (phenolate){Ent} - "American Approach". C. Rh o d o t o r u l i c A c i d (di-hydroxamate){Rta}{'Sid' i s a membrane bound i r o n - t r a n s p o r t compound} (Adapted from Bezkorovainy, 1980; Cooper et a l . , 1978). extracellular cell membrane intracellular A ^ Fer <r-Fer Fe(lll) Fe(lll) uptake B Ent * Ent Fe(lll) Ent<j—Synthesis Fe(lll) uptake » Ent FeClll) lesterase byproduct^Fe(ll) 61 low for r e d u c t i v e i r o n r e l e a s e . These authors es t imated a redox p o t e n t i a l of the e n t e r o b a c t i n - i r o n complex of -750 mV. Thi s can be compared to the redox p o t e n t i a l of NADPH (-300 mV) . Thus, to re lease the i r o n , e n t e r o b a c t i n i s broken down i n t r a c e l l u l a r l y w i t h an e s t e r a s e . R e c e n t l y , Lodge et a l . (1980) have cha l l enged t h i s r a t i o n a l e and have shown that i r o n can be reduced of f e n t e r o b a c t i n and u t i l i z e d without the h y d r o l y s i s of the s iderophore . A t h i r d mechanism has been shown i n mycobacter ia and spec ies which produce r h o d o t o r u l i c a c i d ( F i g . 13C). Th i s system can be e a s i l y d i s t i n g u i s h e d from the prev ious systems s ince i t i n v o l v e s two s iderophores , one e x t r a c e l l u l a r and one membrane bound. In t h i s system, i r o n bound to the e x t r a c e l l u l a r s iderophore , i s t r a n s f e r r e d to the c e l l w a l l where the membrane-bound s iderophore accepts the i r o n and t r a n s p o r t s the i r o n to a reductant w i t h i n the c e l l (Bezkorova iny , 1980). While the system has not been f u l l y c h a r a c t e r i z e d , Rhodotorula pi lmanae, which produces r h o d o t o r u l i c a c i d ( A t k i n et a l . , 1970), a l s o appears to t r a n f e r i r o n in t h i s manner (Carrano and Raymond, 1978) . D. SIDEROPHORES IN HIGHER PLANTS There i s no evidence of s iderophores being produced by higher p l a n t s . Th i s i s not to suggest that i r o n does not become l i m i t i n g to these p l a n t s ( P r i c e , 1968), but that higher p l a n t s may depend on the product ion of s iderophores and i r o n -t r a n s p o r t i n g compounds by e p i p h y t i c b a c t e r i a . While h igher 62 p l a n t s have been l e s s f u l l y s t u d i e d than b a c t e r i a or f u n g i , there appears to be two processes to s o l u b l i z e i r o n . P l a n t s may depend on the humic content of s o i l , d e r i v e d from decaying p l a n t m a t e r i a l , to increase the a v a i l a b i l i t y of i r o n . The second mechanism i n v o l v e s the r e l e a s e of low molecular weight o r g a n i c s , such as c i t r i c a c i d , malic a c i d , or mugineic a c i d , from the r o o t s . Each of these compounds p l a y s an important r o l e i n i r o n a c q u i s i t i o n but none have the c h a r a c t e r i s t i c s of siderophores. C i t r i c a c i d , for example, i s a common c o n s t i t u e n t in p l a n t s which p o o r l y binds i r o n when compared to m i c r o b i a l siderophores. The f e r r i c i r o n i s thought to form a complex with the a l c o h o l and c a r b o x y l i c a c i d f u n c t i o n a l i t i e s of the c i t r a t e (Neilands, 1973). Under i r o n - l i m i t e d c o n d i t i o n s c i t r a t e accumulates in the root s of soybeans (Brown, 1966), but there was no reported s t i m u l a t i o n of e x t r a c e l l u l a r c i t r a t e . S i m i l a r l y , malic a c i d i s a common component of the TCA c y c l e and forms r e l a t i v e l y weak complexes with i r o n ( l l l ) . Ojima and Ohira (1980) report the r e l e a s e of malic a c i d and c i t r i c a c i d by c e l l suspension c u l t u r e s of r i c e . These compounds appeared to s t i m u l a t e the conver s i o n of i r o n ( I I l ) to i r o n ( I I ) . Release of these org a n i c s was not st i m u l a t e d by i r o n s t r e s s . The authors suggest that the r e l e a s e of these two o r g a n i c s was a c e l l u l a r response to adjust the i n t r a c e l l u l a r ion balance. As the pH dropped from 6.0 to 5.5 there was a s h i f t i n metabolic pathways r e s u l t i n g i n the r e l e a s e of these two organic a c i d s . Again, the production of the or g a n i c s and a need f o r i r o n c o u l d not be r e l a t e d . 63 F i g u r e 14 - Rep r e s e n t a t i v e i r o n c h e l a t i n g agents from p h o t o s y n t h e t i c organisms. A: Mugineic a c i d , a i r o n c h e l a t i n g agent from higher p l a n t s (from Sugiura et a_l. , 1981). B: S c h i z o k i n e n , a dihydroxamate siderophore from c y a n o b a c t e r i a (from Simpson and N e i l a n d s , 1976). A •B H O O Ii I I I H C H ^ > h K C H 2 ) 3 N H C O C H 2 C O N C I - ^ C O N H - ( C H 2 ^ - N - C C H ; 64 Takemota et a l . (1978) and Sugiura et a l . (1981) have suggested that mugineic a c i d i s a p o s s i b l e phytosiderophore. While o r i g i n a l l y i s o l a t e d from b a r l e y , s i m i l a r compounds have been found i n r i c e and oats (Takagi, 1976; Fushiya e_t a l . , 1980). Examination of the s t r u c t u r e of t h i s compound ( F i g . 14A) shows that mugineic a c i d i s not a hydroxamate nor a phenolate. Sugiura e_t a_l. (1981) pr o v i d e d evidence of mugineic a c i d forming a 1:1 complex with i r o n ( I I I ) with the l i g a n d s c o n s i s t i n g of the c a r b o x y l , amine, and hydroxyl groups, The formation constant i s very low ( l o g K =18.1) for i r o n ( I I I ) , but the formation constant f o r i r o n ( I I ) was very high ( l o g K =8.1) compared to m i c r o b i a l s i d e r o p h o r e s . There was no evidence of mugineic a c i d being p o s i t i v e l y l i n k e d with i r o n - s t r e s s ; although unpublished work by Takagi ( c i t e d by Sugiura e_t a_l. , 1981) has shown mugineic a c i d to s t i m u l a t e iron-uptake i n b a r l e y . In c o n c l u s i o n , while there i s no evidence f o r the prod u c t i o n of siderophores ( s t r i c t l y defined) by higher p l a n t s , there i s evidence to suggest that i r o n - c h e l a t i n g agents are produced by higher p l a n t s . These o r g a n i c s occur e i t h e r i n t r a c e l l u l a r l y (accumulated i n the roots) or e x t r a c e l l u l a r l y , but there i s no evidence of the s t i m u l a t i o n of production under i r o n - s t r e s s e d c o n d i t i o n s . Higher p l a n t s may r e l y on these lower a f f i n i t y iron-complexing reagents as i r o n - s o l u b i l i z i n g f a c t o r s which a i d i n i r o n removal from humic m a t e r i a l s and from m i c r o b i a l siderophores (Lindsay, 1972; Lankford,'1973). 65 E. SIDEROPHORE PRODUCTION BY PHYTOPLANKTON Siderophore p r o d u c t i o n by phytoplankton has r e c e i v e d i n c r e a s i n g a t t e n t i o n i n recent years. Lange (1974) found n a t u r a l c h e l a t o r production from 6 out of 10 p l a n k t o n i c freshwater c y a n o b a c t e r i a . C u l t u r e s produced s u f f i c i e n t c h e l a t i n g m a t e r i a l to grow without the a d d i t i o n of a r t i f i c i a l c h e l a t o r s . These n a t u r a l c h e l a t o r s were thought to be l a r g e molecular weight p e p t i d e s or p o l y s a c c h a r i d e s which c h e l a t e t r a c e metals (Fogg and Westlake, 1955; Fogg, 1966). Murphy e_t a_l. (1976) found hydroxamate-type siderophores in c u l t u r e s of Anabaena flos-aquae and in f i e l d samples from blooms of Anabaena sp. Hydroxamate-type siderophores have a l s o been rep o r t e d from Anabaena sp. by B a i l e y and Taub (1980). Simpson and Neilands (1976) have i s o l a t e d the dihydroxamate siderophore ( s c h i z o k i n e n , F i g . 14B) from the c u l t u r e supernatant of i r o n - s t a r v e d Anabaena sp. Sev e r a l authors have provided evidence to support the prod u c t i o n of hydroxamate-type siderophores by marine c y a n o b a c t e r i a . Estep et a l . (1975) found c y a n o b a c t e r i a c o l l e c t e d from two d i f f e r e n t h a b i t a t s ( c y a n o b a c t e r i a l mats and seagrass beds) to c o n t a i n e x t r a c e l l u l a r compounds which promoted the growth of the siderophore auxotroph, A r t h r o b a c t e r JG-9. Using the same siderophore auxotroph bioassay, Armstrong and Van Baalen (1979) i s o l a t e d a siderophore from the marine cyanobacteriurn, Agmenellum quadruplicayurn. The siderophore was p o s i t i v e to the Csaky t e s t (a c o l o r i m e t r i c t e s t f o r secondary hydroxamate f u n c t i o n a l i t i e s ) and was found i n t r a c e l l u l a r l y when 66 c e l l s were c u l t u r e d under low i r o n c o n d i t i o n s . While there i s some supporting evidence f o r the p r o d u c t i o n of siderophores by p r o k a r y o t i c phtoplankton, p r o d u c t i o n of s i m i l a r i r o n - s p e c i f i c c h e l a t o r s by e u k a r y o t i c phytoplankton i s c o n t r o v e r s i a l . Murphy e_t a l . (1976) hypothesize that c y a n o b a c t e r i a can outcompete e u k a r y o t i c phytoplankton d u r i n g i r o n - l i m i t e d growth c o n d i t i o n s s i n c e the l a t t e r group cannot produce s i d e r o p h o r e s . T h i s i n a b i l i t y was f u r t h e r supported by work of McKnight and Morel (1979), who c o u l d not induce siderophore p r o d u c t i o n in any of the 13 e u k a r y o t i c phytoplankton i n v e s t i g a t e d . However, a l l seven p r o k a r y o t i c p h y t o p l a n k t e r s that were examined produced hydroxamates. Evidence in support of siderophore production by e u k a r y o t i c phytoplankton i n c l u d e s Spencer et a_l. (1973), who i s o l a t e d a trihydroxamate-type siderophore from a b a c t e r i z e d Chaetoceros  s o c i a l i s c u l t u r e . The i s o l a t e d compound reac t e d with F e C l 3 , had a broad a b s o r p t i o n band between 425 and 525 nm f o r the f e r r i -siderophore complex, and contained secondary hydroxamate groups based on the i n f r a - r e d spectrum. These are a l l c r i t e r i a f o r a hydroxamate-type siderophore ( N e i l a n d s , 1981a). Researchers have been c r i t i c a l of t h i s work s i n c e the c u l t u r e s c ontained b a c t e r i a , and b a c t e r i a are e x c e l l e n t sources of hydroxamate-type siderophores (Goyne and Carpenter, 1974). Key papers d e a l i n g with siderophore production i n phytoplankton have chosen not to c i t e t h i s work, due, most l i k e l y , to the ambiguous o r i g i n of the siderophore (Murphy et_ a_l. , 1976; McKnight and Morel, 1979; Anderson and Morel, 1980; Huntsman and 67 Sunda, 1980). F u r t h e r evidence for the p r o d u c t i o n of siderophores from e u k a r y o t i c phytoplankton i s presented by Armstrong and Van Baalen (1979). The authors found that i n t r a c e l l u l a r and e x t r a c e l l u l a r e x t r a c t s of the marine diatom C y l i n d r o t h e c a sp. s t i m u l a t e d the growth of the siderophore auxotroph, A r t h r o b a c t e r JG-9. The e x t r a c e l l u l a r supernatent was not r e a c t i v e to the Csaky t e s t . Thus, i t does not f i t the c r i t e r i o n f o r a hydroxamate-type siderophore. The e x i s t e n c e of t h i s siderophore w i l l have to be confirmed, s i n c e the f r a c t i o n s which produced s t i m u l a t i o n i n the bioassay t e s t were concentrated 1000 f o l d and the bioassay w i l l respond to non-hydroxamate siderophores present in high c o n c e n t r a t i o n s (Morrison' e_t a l . , 1965). The f o l l o w i n g chapters d e s c r i b e p r o r o c e n t r i n , a hydroxamate-type siderophore i s o l a t e d from the marine din'of l a g e l l a t e , Prorocentrum minimum. Production of hydroxamate-type siderophores i n other s p e c i e s i s a l s o e s t a b l i shed. 68 V. PROROCENTRIN:AN EXTRACELLULAR SIDEROPHORE PRODUCED BY THE  MARINE DINOFLAGELLATE PROROCENTRUM MINIMUM A. ABSTRACT P r o r o c e n t r i n i s a Csaky p o s i t i v e m e t a b o l i t e that can be e x t r a c t e d from the f i l t r a t e s of Prorocentrum minimum c u l t u r e s by XAD-2 r e s i n . Production of p r o r o c e n t r i n can be s t i m u l a t e d by c u l t u r i n g P. minimum under c o n d i t i o n s of i r o n d e f i c i e n c y . The i r o n ( I I I ) complex of p r o r o c e n t r i n has a U V - v i s i b l e a b s o r p t i o n spectrum t y p i c a l of hydroxamate si d e r o p h o r e s . 69 B. INTRODUCTION Marine phytop lankton , l i k e most other l i v i n g organisms, have a n u t r i t i o n a l requirement for i r o n ( P r i c e , 1968; O ' K e l l y , 1974). I ron i n seawater i s e i t h e r a s s o c i a t e d w i t h organic c h e l a t o r s (Sugimura e_t a_l. , 1978) or present as aggregates of — 3 8 the almost t o t a l l y i n s o l u b l e i r o n ( I I I ) hydroxide (Ksp=lO ) (Ne i l ands , 1972). A p o r t i o n of the c h e l a t e d i r o n ( I I I ) may be u t i l i z e d by phytop lankton , however, the l a rge pool of i n s o l u b l e i r o n ( I I I ) hydroxide i s presumed to be u n a v a i l a b l e as a n u t r i t i o n a l source . Aquat ic microorganisms, for example b a c t e r i a (Gonye and Carpenter , 1974) and cyanobacter i a (Murphy et. a l . , 1976; Simpson and N e i l a n d s , 1976; Armstrong and Van Baa len , 1979), have been shown to produce e x t r a c e l l u l a r i r o n ( I I I ) c h e l a t i n g agents ( s iderophores ) that enable .them to s o l u b l i z e and there fore acqu i re the i r o n present i n the i r o n ( I I I ) hydroxide aggregates . I t i s not c l e a r whether e u k a r y o t i c phytoplankton acquire i r o n ( I I I ) i n a s i m i l a r manner. Spencer et a l . (1973) c h a r a c t e r i z e d a p o s s i b l e s iderophore from a non-axenic marine d iatom, Chaetoceros soc i a l i s. The i s o l a t e d substance had chemical p r o p e r t i e s s i m i l a r to a hydroxamate-type s iderophore but d e f i n i t i v e evidence for the o r i g i n of the compound (from the diatom and not the b a c t e r i a ) was not presented . More r e c e n t l y , McKnight and Morel (1979) have concluded that a number of axenic e u k a r y o t i c phytoplankton are unable to produce i r o n ( I I I ) s p e c i f i c c h e l a t o r s . They have suggested that the diatom T h a l a s s i o s i r a w e i s s f l o g i i u t i l i z e s i r o n ( I I ) ra ther than i r o n 70 ( I I I ) (Anderson and Morel, 1980), because f e r r o u s i r o n i s more s o l u b l e i n seawater and t h e r e f o r e i t should be more n u t r i t i o n a l l y a c c e s s i b l e . Since i r o n (II) i s extremely l a b i l e in the marine environment, t r a n s f o r m a t i o n of i r o n ( I I I ) to i r o n (I I ) at the c e l l membrane may be r e q u i r e d f o r e f f i c i e n t uptake. Armstrong and Van Baalen (1979) showed that a concentrated c h l o r o f o r m e x t r a c t of the marine diatom C y l i n d r o t h e c a was Csaky p o s i t i v e (Csaky, 1948; G i l l a m et a l . , '1981) and that i t s t i m u l a t e d the growth of the siderophore auxotroph A r t h r o b a c t e r  f i a v e s c e n s JG-9 (Burnhard and Ne i l a n d s , 1961). C i r c u m s t a n c i a l evidence f o r the u t i l i z a t i o n of siderophores by phytoplankton comes from numerous o b s e r v a t i o n s that e i t h e r s y n t h e t i c or n a t u r a l c h e l a t o r s s t i m u l a t e phytoplankton growth i n c u l t u r e and i n the f i e l d , p o s s i b l y by enhancing the a v a i l a b i l i t y of i r o n (Johnston, 1964; Barber and Ryther, 1969; Barber et a l . , 1971; Barber, 1973). C. MATERIALS AND METHODS In an attempt to c l a r i f y whether' marine eukaryotes do produce siderophores, a survey was conducted of marine phytoplankton s p e c i e s i n the North East P a c i f i c C u l t u r e C o l l e c t i o n (NEPCC) f o r the pr o d u c t i o n of e i t h e r i n t r a c e l l u l a r or e x t r a c e l l u l a r m e t a b o l i t e s which were Csaky p o s i t i v e (a q u a n t i t a t i v e t e s t for hydroxamate siderophores) ( G i l l a m e_t a l . , 1981) and which would s a t i s f y the requirements of the siderophore auxotroph A^ fIavescens JG-9 (Burnhard and Ne i l a n d s , 1961). The survey ( G i l l a m et a l . , i n prep.) re v e a l e d 71 that the c e l l and f i l t r a t e e x t r a c t s of the non-toxic, red t i d e d i n o f l a g e l l a t e Prorocentrum min imum S c h i l l e r (NEPCC #96) showed c o n s i d e r a b l e promise as a source of hydroxamate s i d e r o p h o r e s . It i s w e l l documented that the m i c r o b i a l p r o d u c t i o n of siderophores i s s t i m u l a t e d by growth in i r o n - l i m i t e d medium ( G a r i b a l d i and Neilands, 1956). Six L axenic c u l t u r e s (Chapter 2) of P. minimum were grown in the f o l l o w i n g media: 1) c h a r c o a l - t r e a t e d n a t u r a l seawater followed by a d d i t i o n of a l l r e q u i r e d n u t r i e n t s (ESNW) (Harr i s o n e_t al.,1980); 2) c h a r c o a l - t r e a t e d n a t u r a l seawater followed by the removal of most of the a v a i l a b l e i r o n by the procedure of Lewin and Chen (1971), and then supplemented with a l l n u t r i e n t s , except F e C l 3 and EDTA (ESNW-Fe); and 3) c h a r c o a l - and Chelex-100 r e s i n - t r e a t e d n a t u r a l seawater supplemented with AQUIL n u t r i e n t s (Morel e_t a l . , 1979), minus F e C l 3 and EDTA (AQUIL-Fe). The f i r s t medium provided an i r o n s u f f i c i e n t c o n t r o l while the other media provided i r o n - s t r e s s e d c o n d i t i o n s . A l l c u l t u r e s were harvested when e x t r a c e l l u l a r siderophore c o n c e n t r a t i o n was maximal. T h i s occurred approximately three days a f t e r the c u l t u r e reached senescence. C e l l s were harvested by continuous c e n t r i f u g a t i o n and membrane f i l t r a t i o n (0.45 ^m). The c e l l - f r e e f i l t r a t e s were a c i d i f i e d to pH 2 and passed over XAD-2 r e s i n (Sugimura e_t a_l. , 1978) to e x t r a c t the Csaky p o s i t i v e m e t a b o l i t e s . Methanol e l u t i o n removed the o r g a n i c s from the XAD-2 r e s i n . In vacuo evaporation of methanol followed by p a r t i t i o n i n g the r e s u l t i n g r e s i d u e between chl o r o f o r m and 72 water generated an aqueous f r a c t i o n which c o n t a i n e d the Csaky a c t i v i t y . D. RESULTS AND DISCUSSION The i n f l u e n c e of the media on growth parameters and Csaky p o s i t i v e m e t a b o l i t e p r o d u c t i o n i s shown i n Table V. The red u c t i o n in growth rate and f i n a l c e l l y i e l d i s a t t r i b u t e d to the i r o n - d e f i c i e n t c u l t u r e medium. C e l l s grown under i r o n -s t r e s s e d c o n d i t i o n s produced more Csaky p o s i t i v e compound. The a d d i t i o n of f r e s h l y prepared F e C l 3 to a l l e v i a t e the i r o n d e f i c i e n c y i n ENSW-Fe and AQUIL-Fe r e e s t a b l i s h e d maximum growth ra t e s (1.0V±0.07, 0.94± 0.01 d i v i s i o n s / d a y , r e s p e c t i v e l y ) and reduced the pro d u c t i o n of Csaky p o s i t i v e compound to l e s s than 5 u g NH2OH • 10 ~ 8 c e l l s . A 40 L c u l t u r e of P. minimum grown in ESNW-Fe medium and harvested s h o r t l y a f t e r the c e s s a t i o n of growth, provided a s u f f i c i e n t amount of the e x t r a c e l l u l a r Csaky p o s i t i v e compound to enable p u r i f i c a t i o n and p r e l i m i n a r y chemical c h a r a c t e r i z a t i o n ; t h i s compound has been named p r o r o c e n t r i n . C u l t u r e f i l t r a t e s were e x t r a c t e d as d e s c r i b e d above. F e r r i - p r o r o c e n t r i n was formed by adding f r e s h l y prepared i r o n ( I I I ) hydroxide (Neilands, 1966) to the r e s u l t i n g aqueous f r a c t i o n and heating the suspension at 80°C f o r 2 h. Excess i r o n ( I I I ) hydroxide was removed by f i l t r a t i o n and the deep red f i l t r a t e was reduced i n volume by r o t a r y e v a p o r a t i o n _in_ vacuo. A white p r e c i p i t a t e that formed in the concentrated f i l t r a t e was 73 Table V - Influ e n c e of c u l t u r e medium on growth r a t e of Prorocentrum minimum and production of Csaky p o s i t i v e compounds. Medium and Final cell Csaky test nutrient Growth rate yield ( yg NH2 OH-108 status (divisions • day"1) (107 cells • L'1) cells"1)  ESNW, iron sufficient 1.04 + 0.08 6.27 5.9 AQUIL-Fe, iron deficient 0.42±0.01 4.01 127.2 ESNW-Fe, iron deficient 0.75 ± 0.02 3.95 133.0 74 F i g u r e 15 - The U V - v i s i b l e a b s o r p t i o n s p e c t r a of d e s f e r r i -p r o r o c e n t r i n ( s o l i d l i n e ) and f e r r i - p r o r o c e n t r i n (broken l i n e ) . 6 111 5 ^ W A V E L E N G T H (nm) 75 Table VI - Comparison of ferri-prorocentrin with desferri-prorocentrin. Test or ' parameter Ferri-prorocentrin Desferri-prorocentrin Rf (But:AcOH:H 2 0) 0.60 Reaction to I 2 vapor Reaction to FeCl3 spray positive none 0.18 - 0.20 positive light pink color Reaction to perchlorate test Reaction to Csaky test Estimated molecular weight (daltons) positive positive positive positive not determined 560-590 76 removed and the mother l i q u o r was a p p l i e d to a r o t a r y t h i n l a y e r chromatography p l a t e (Chromatotron-Harrison Research, HF S i l i c a Gel with a s t a r c h b i n d e r ) . F r a c t i o n a t i o n was achieved by e l u t i o n with a s e r i e s of s o l v e n t s (MeOH, 1:24 H20:MeOH, 3:7 H20:MeOH, 60:25:15 Bu t a n o l : H 2 0 : A c e t i c a c i d ) . S e v e r a l red-orange bands were c o l l e c t e d in each s o l v e n t pass. F o l l o w i n g c o n c e n t r a t i o n by r o t a r y evaporation each f r a c t i o n was assayed by the Csaky t e s t . A s i n g l e orange red f r a c t i o n from the Butanol:H 0 : A c e t i c a c i d pass was p o s i t i v e . A n a l y t i c a l t h i n l a y e r chromatography of the Csaky p o s i t i v e f r a c t i o n on s i l i c a g e l (SIL G/UV254 - But a n o l : H 2 0 : A c e t i c a c i d -60:25:15) showed a s i n g l e i o d i n e p o s i t i v e spot at Rf=0.6 corresponding to f e r r i - p r o r o c e n t r i n . The i r o n c o u l d be removed from the complex by treatment with 1N NaOH. Thin l a y e r chromatography of the d e s f e r r i - p r o r o c e n t r i n showed a s i n g l e spot at Rf=0.2 which took up i o d i n e and gave a pink spot with f e r r i c c h l o r i d e spray (2% i n EtOH). P r o r o c e n t r i n was d e s a l t e d on a Bio Gel P-2 column to give 7 mg of a white s o l i d . The Bio Gel column was c a l i b r a t e d with s e v e r a l low molecular weight standards. The estimated molecular weight f o r p r o r o c e n t r i n i s 560--590 d a l t o n s . The molecular weight f o r f e r r i - p r o r o c e n t r i n c o u l d not estimated because i t streaked on the column. P r o r o c e n t r i n shows only strong end a b s o r p t i o n i n i t s UV-v i s i b l e spectrum ( F i g . 15). Formation of the ir o n ( I I I ) complex at n e u t r a l pH, generates a new a b s o r p t i o n band with a = 440 nm (H 20), which s h i f t s to a = 450 nm upon a c i d i f i c a t i o n to pH 2. The UV- v i s i b l e a b s o r p t i o n spectrum of f e r r i - p r o r o c e n t r i n 77 i s t y p i c a l of tri-hydroxamate siderophore such as ferr i c h r o m e (Neilands, 1966). S e v e r a l o b s e r v a t i o n s support the c l a i m that p r o r o c e n t r i n i s a hydroxamate c o n t a i n i n g siderophore produced by the d i n o f l a g e l l a t e P. minimum. F i r s t , the o r i g i n of the me t a b o l i t e cannot be questio n e d . The axenic c o n d i t i o n of our c u l t u r e s and the i s o l a t i o n of the same compound ( i d e n t i c a l TLC) from c e l l e x t r a c t s e l i m i n a t e s the p o s s i b i l i t y of a b a c t e r i a l o r i g i n . Secondly, the p o s i t i v e Csaky t e s t shown by pure p r o r o c e n t r i n (Table VI) and the U V - v i s i b l e a b s o r p t i o n spectrum of f e r r i - p r o r o c e n t r i n are c h a r a c t e r i s t i c of hydroxamate si d e r o p h o r e s . T h i r d l y , i n c r e a s e d p r o d u c t i o n of p r o r o c e n t r i n in i r o n - l i m i t e d c u l t u r e medium i s the r e q u i r e d p h y s i o l o g i c a l m a n i f e s t a t i o n of a siderophore-based i r o n a c q u i s i t i o n mechanism. F i n a l l y , i t i s a r o u t i n e matter to i n t e r c o n v e r t the d e s f e r r i and f e r r i forms of p r o r o c e n t r i n . Success i n i s o l a t i o n of the siderophore can be a t t r i b u t e d to the use of i r o n - d e f i c i e n t medium and the c o n c e n t r a t i o n of p r o r o c e n t r i n on XAD-2 r e s i n . While the recovery using XAD-2 i s l e s s than i d e a l ( 50%), i t i s a necessary step in i s o l a t i n g and q u a n t i f y i n g e x t r a c e l l u l a r m e t a b o l i t e s from t h i s marine dinof l a g e l l a t e (Andersen et al.. , 1980; Chapter I I ) . Without c o n c e n t r a t i o n , i r o n ( I I I ) s p e c i f i c c h e l a t o r s from marine eukaryotes may remain undetected, whereas these compounds from marine c y a n o b a c t e r i a can be measured (Armstrong and Van Baalen, 1979). Whether or not marine e u k a r y o t i c phytoplankton produce q u a n t i t a t i v e l y l e s s siderophore than marine c y a n o b a c t e r i a i s 78 u n c l e a r . I t i s w e l l documented that c y a n o b a c t e r i a have a higher s p e c i f i c requirement f o r i r o n ( e s p e c i a l l y when f i x i n g n i t r o g e n ) (Stewart, 1980) than marine e u k a r y o t i c phytoplankton, however, q u a n t i t a t i v e comparisons between the groups cannot be made u n t i l f u r t h e r i n f o r m a t i o n i s a v a i l a b l e on the p r o d u c t i o n , e x c r e t i o n and c e l l u l a r uptake of si d e r o p h o r e s . P r o r o c e n t r i n i s the f i r s t siderophore to be i s o l a t e d from a eu k a r y o t i c marine phytoplankton. The d i s c o v e r y suggests that the i r o n uptake mechanism of d i n o f l a g e l l a t e s may c l o s e l y p a r a l l e l that of p r o k a r y o t i c siderophore producing organisms. The production of a strong t r a c e metal c h e l a t o r by a red t i d e organism has many important e c o l o g i c a l i m p l i c a t i o n s . Blue-green algae can e f f e c t i v e l y e l i m i n a t e competing a l g a l s p e c i e s in f r e s h water lakes by seq u e s t e r i n g a l l the a v a i l a b l e i r o n as a siderophore complex (Murphy et. a_l. , 1976) and there i s some i n d i c a t i o n that exogenous siderophores can both p o s i t i v e l y and n e g a t i v e l y a f f e c t the growth of marine phytoplankton ( B a i l e y and Taub, 1980). Strong t r a c e metal c h e l a t o r s have a l s o been shown to reduce the t o x i c i t y of c u p r i c ions to s e n s i t i v e marine phytoplankton s p e c i e s (Sunda and G u i l l a r d , 1976). It w i l l be i n t e r e s t i n g to t e s t whether t h i s d i n o f l a g e l l a t e siderophore p r o v i d e s i r o n to p r o k a r y o t i c organisms. 79 VI. METHODOLOGY USED IN ISOLATING THE HYDROXAMATE SIDEROPHORE,  PROROCENTRIN, FROM PROROCENTRUM MINIMUM A. ABSTRACT The experimental procedure f o r the i s o l a t i o n of p r o r o c e n t r i n , a hydroxamate-type siderophore, from c e l l - f r e e Prorocentrum minimum c u l t u r e i s c r i t i c a l l y examined. The siderophore adsorbs p o o r l y to the Amberlite XAD-2 r e s i n but with a r i g i d l y d e f i n e d experimental procedure a constant recovery of approximately 60% can be achieved. T h i s recovery allows f o r an accu r a t e q u a n t i t a t i v e estimate of the t o t a l amount of p r o r o c e n t r i n produced. The i s o l a t e d p r o r o c e n t r i n i s a trihydroxamate with a molecular weight of 560-590 d a l t o n s . The p r o r o c e n t r i n - i r o n complex i s s t a b l e over a wide pH range. 80 B. INTRODUCTION The i s o l a t i o n of the e x t r a c e l l u l a r hydroxamate-type siderophore, p r o r o c e n t r i n , from the marine e u k a r y o t i c marine phytoplankton, Prorocentrum minimum, has r e c e n t l y been undertaken (Chapter V ) . S i m i l a r compounds are produced by marine b a c t e r i a (Gonye and Carpenter, 1974), marine cy a n o b a c t e r i a (Estep, et a l . , 1978; Armstrong and Van Baalen, 1979), and other e u k a r y o t i c phytoplankton such as T h a l a s s i o s i r a  pseudonana (Chapter VIII) and Chaetoceros s o c i a l i s (Spencer e_t a l . , 1973). In an i n i t i a l paper d e s c r i b i n g p r o r o c e n t r i n (Chapter V) i t was s t r e s s e d that the success of i s o l a t i o n was p r i m a r i l y due to the c o n c e n t r a t i o n technique u t i l i z e d . T h i s technique i s based on the c o n c e n t r a t i o n of the hydroxamate-type siderophore on XAD-2 r e s i n , p r i o r to a n a l y s i s f o r hydroxamate-containing compounds (Csaky t e s t ) . T h i s chapter c r i t i c a l l y examines the i s o l a t i o n steps of p r o r o c e n t r i n . Since p r o r o c e n t r i n does not bind s t r o n g l y to the XAD-2 r e s i n , c a r e f u l l y d e f i n e d procedures must be f o l l o w e d to achieve constant recovery e f f i c i e n c i e s f o r q u a n t i t a t i v e s t u d i e s . T h i s chapter a l s o r e p o r t s the attempted p u r i f i c a t i o n of p r o r o c e n t r i n . Various s e p a r a t i o n techniques were u t i l i z e d and p r o r o c e n t r i n was c h a r a c t e r i z e d with respect to molecular s i z e and chromatographic behavior. 81 C. MATERIALS Prorocentrum min imum was obtained from the North East P a c i f i c C u l t u r e C o l l e c t i o n (NEPCC #96). Axenic c u l t u r e s were grown i n 20 L batch c u l t u r e s , using e n r i c h e d n a t u r a l seawater. medium which had been t r e a t e d to remove r e s i d u a l i r o n (ESNW-Fe; Chapter V). C u l t u r e c o n d i t i o n s , were as f o l l o w s : constant d a y l i g h t f l u o r e s c e n t l i g h t i n g at ^ O y E - m ^ s 1 , 18"C, continuous s t i r r i n g at 60 rpm. Since maximum i n t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n s occur p r i o r to maximum e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n s (Chapter V I I ) , c u l t u r e s were harvested 8 to 10 days a f t e r i n o c u l a t i o n to o b t a i n i n t r a c e l l u l a r p r o r o c e n t r i n and 10 to 12 days a f t e r i n o c u l a t i o n i f e x t r a c e l l u l a r p r o r o c e n t r i n was r e q u i r e d . The h a r v e s t i n g procedure has been d e s c r i b e d p r e v i o u s l y (Andersen, e_t §_1. , 1979; Chapter I I ) . C e l l s were harvested using a Sharpies continuous flow c e n t r i f u g e . The c u l t u r e medium was then f i l t e r e d through a g l a s s f i b e r f i l t e r (GF/A) followed by a 0.45 -urn membrane f i l t e r . The c e l l - f r e e medium was a d j u s t e d to pH 2.0 with HC1. The d i s s o l v e d organic f r a c t i o n was c o l l e c t e d by p a s s i n g the a c i d i f i e d f i l t r a t e through a column of precleaned (Soxhlet e x t r a c t i o n with methanol, 72 h ) XAD-2 r e s i n ( M a l l i n c k r o d t ) . Remaining s a l t s were removed by washing the column with 150 ml d e i o n i z e d , d i s t i l l e d water The adsorbed organic f r a c t i o n was removed using 300 ml of methanol. The organic f r a c t i o n was d i s s o l v e d in water and repeatedly washed with c h l o r o f o r m u n t i l the water f r a c t i o n was c o l o r l e s s . To q u a n t i f y the amount of p r o r o c e n t r i n , the m o d i f i e d Csaky 82 t e s t of G i l l a m et a l . (1981) was employed. The Csaky t e s t i s a c o l o r i m e t r i c t e s t s p e c i f i c f o r secondary hydroxamates. The t e s t r e q u i r e s a long a c i d h y d r o l y s i s step (4 h, 121 *C, 15 p s i ) . The hydroxy-amino compound r e l e a s e d i s o x i d i z e d with I 2 to n i t r i t e which i s measured c o l o r i m e t r i c a l l y a f t e r r e a c t i o n with s u l f a n i l i c a c i d and a-naphthylamine. For each 5 ml sample, a 2 ml a l i q u o t was used f o r the t e s t and a second, 2 ml sample was used f o r the c o l o r and reagent blank. A l l c o n c e n t r a t i o n s are presented as the amount of product produced by the Csaky t e s t , c o r r e c t e d f o r subsampling and the i n i t i a l volume of seawater con c e n t r a t e d (e.g. N^OH-L - 1 ). C e r t a i n procedures such as g e l permeation chromatography, r e q u i r e d the examination of a l a r g e number of samples. Since t h i s was t e c h n i c a l l y d i f f i c u l t to do by the Csaky'test, i n i t i a l s c r e e n i n g of samples by the a d d i t i o n of f e r r i c p e r c h l o r a t e served to dete c t the presence of o r g a n i c s which formed c o l o r e d f e r r i c complexes (Atk i n e_t a_l. , 1970). The procedure was m o d i f i e d s l i g h t l y to f a c i l i t a t e a n a l y s i s of small sample volumes (2.5 ml reagent, 0.5 ml sample). F r a c t i o n s p o s i t i v e to the f e r r i c p e r c h l o r a t e t e s t were f u r t h e r examined using the Csaky t e s t to confirm the presence of p r o r o c e n t r i n . The t h i n l a y e r chromatography solvent systems employed to i s o l a t e p r o r o c e n t r i n were b u t a n o l : a c e t i c acid:water (60:15:25) (BAW), methanol:water (70:30), isopropanol:water (70:30) and methanol:water (98:2). Samples were run on s i l i c a s t r i p s with a f l u o r e s c e c n c e i n d i c a t o r (SIL G/UV 254) or using a r o t a r y chromatography system with a s i l i c a - s t a r c h p l a t e (Chromatotron, 83 H a r r i s o n Research). Organics were v i s u a l i z e d by f l u o r e s c e n c e or by r e a c t i o n to any of the f o l l o w i n g : F e C l 3 spray (2% i n e t h a n o l ) , F e C l 3 spray with monochloroacetic a c i d (Bernhard et a l . , 1964), n i n h y d r i n spray, or I 2 vapor. The a c i d i f i e d ammonium vanadate t e s t , a chemical t e s t f o r hydroxamates, was a l s o employed f o r aqueous samples (Abbasi, 1976). P u r i f i c a t i o n and e s t i m a t i o n of molecular s i z e were performed by g e l chromatography using a 55 X 1'. 3 cm column s l u r r y - p a c k e d with BioGel P-2 (200-400 mesh). The g e l was swollen and washed w i t h d e i o n i z e d d i s t i l l e d water. Bio Gel P-2 has a molecular s i z e e x c l u s i o n l i m i t of 1800 d a l t o n s . Samples of 1 to 2 ml were a p p l i e d to the top of the column and chromatographed with d e i o n i z e d , d i s t i l l e d water. The flow r a t e was maintained at 15 ml*h 1 using a p e r i s t a l t i c pump. F r a c t i o n s were c o l l e c t e d every 5 ml. Standards f o r molecular s i z e c a l i b r a t i o n (blue dextran, vitamin B 1 2 , r i b o f l a v i n , and lumichrome) were detected by absorbance at 260 nm. Desferrioxamine B (Ciba-Geigy) and p r o r o c e n t r i n were det e c t e d using the f e r r i c p e r c h l o r a t e t e s t f o r i r o n - b i n d i n g o r g a n i c s . Absorption s p e c t r a i n the v i s i b l e and UV ranges were obt a i n e d with a Model 2200 Bausch and Lomb double-beam spectrophotometer. The formation of a f e r r i - p r o r o c e n t r i n complex was performed by a d d i t i o n of f r e s h l y p r e c i p i t a t e d f e r r i c hydroxide (Carrano and Raymond, 1978), or by a d d i t i o n of f r e s h l y prepared F e C l 3 . The a d d i t i o n of i r o n using f e r r i c a c e t y l a c e t o n a t e as a source was a l s o attempted. 84 To convert f e r r i - p r o r o c e n t r i n to d e s f e r r i - p r o r o c e n t r i n the i r o n was removed using a strong base f o l l o w i n g the methods of Emery and Neilands ( i 9 6 0 ) . An equal volume of 1.0 N KOH was added to the s o l u t i o n c o n t a i n i n g f e r r i - p r o r o c e n t r i n . The r e a c t i o n mixture was c h i l l e d f o r 30 minutes, then c e n t r i f u g e d f o r 20 minutes. The supernatant c o n t a i n i n g i r o n - f r e e p r o r o c e n t r i n was decanted from the f e r r i c hydroxide p r e c i p i t a t e and n e u t r a l i z e d with 1.0 N HC1. D. RESULTS 1. Recovery of P r o r o c e n t r i n Using XAD-2 Resin The procedure f o r the i s o l a t i o n and c o n c e n t r a t i o n of d i s s o l v e d o r g a n i c s from the c e l l - f r e e c u l t u r e medium i s summarized in F i g . 16. To st a n d a r d i z e a l l experiments, the bed-volume of the XAD-2 r e s i n was constant (25 X 2 cm). A flow r a t e of 1 L-h"1 was employed. To determine the maximum e f f i c i e n c y of p r o r o c e n t r i n c o l l e c t i o n using XAD-2 r e s i n , four v a r i a b l e s were t e s t e d as f o l l o w s : 1) the volume of the c e l l -f r e e , a c i d i f i e d f i l t r a t e passed through the column; 2) the volume of the d e i o n i z e d , d i s t i l l e d water used to d e s a l t (wash) the column; 3) the amount of methanol used to removed the c o l l e c t e d o r g a n i c s from the column; and 4) the i n i t i a l c o n c e n t r a t i o n of the siderophore i n the c u l t u r e medium. a) In f l u e n c e of F i l t r a t e Volume Table VII shows the i n f l u e n c e of i n c r e a s i n g volumes of 85 a c i d i f i e d f i l t r a t e on the e f f i c i e n c y of recovery. Csaky-p o s i t i v e m a t e r i a l was c o l l e c t e d from 50 L of c e l l - f r e e f i l t r a t e u sing the procedure from F i g . 16. A s u b t r a c t i o n of the XAD-2 c o l l e c t e d o r g a n i c s was taken to determine the c o n c e n t r a t i o n of c o l l e c t e d Csaky p o s i t i v e m a t e r i a l . The remaining XAD-2 c o l l e c t e d o r g a n i c s were added to ESNW-Fe medium to give a f i n a l r e a c t i v e Csaky c o n c e n t r a t i o n of 6.0 ug NH OH*!.""1. Volumes ranging from 1 to 20 L were passed through the standard XAD-2 column. The column was washed with 150 ml water and the adsorbed organic e l u t e d with 300 ml methanol. Recovery i s presented as a percentage of the recovered r e a c t i v e m a t e r i a l (based on the Csaky t e s t ) compared to the c o n c e n t r a t i o n i n the i n i t i a l sample before i t was passed through the column. The maximum e f f i c i e n c y of recovery was in the range of 60%. Using the standard column, o r g a n i c s from 6 L c o u l d be scavenged without f u r t h e r l o s s i n recovery. Passage of more than 6 L of medium per column, provided l i t t l e f u r t h e r b i n d i n g of r e a c t i v e m a t e r i a l and the e f f i c i e n c y of recovery d e c l i n e d r a p i d l y . b) Washing or D e s a l t i n g the Column The next step in the a n a l y s i s i n v o l v e s a d j u s t i n g the volume of the water f o r the column wash. The c o l l e c t e d w a t e r - s o l u b l e organic f r a c t i o n was added back to 40 L of a c i d i f i e d ESNW-Fe medium. I n i t i a l c o n c e n t r a t i o n of hydroxamate-type siderophore was e q u i v a l e n t to 3.6 y g NH2OH•L 1 . The 40 L was s p l i t i n t o e i g h t , 5 L f r a c t i o n s . Each f r a c t i o n was passed over a standard XAD-2 column which was then washed with e i t h e r 50, 150, 1000, or 86 igure 16 - Summary of the procedure f o r the i s o l a t i o n of the aqueous f r a c t i o n c o n t a i n i n g the hydroxamate-type siderophore, p r o r o c e n t r i n . culture continuous centrifuge I cells extract with MeOH evaporate medium I 0-45 urn membrane filtration CHCI3 soluble H 20 soluble acidify to pH 20 I XAD-2 column 150 ml H 20 wash I 300 ml MeOH evaporate CHCU soluble cell- ree filtrate " L Hp soluble Csak^ test 87 Table VII - The i n f l u e n c e of f i l t r a t e volume on the e f f i c i e n c y of e x t r a c t i o n and recovery of prorocentrum on XAD-2 r e s i n . The c o n c e n t r a t i o n of the hydroxamate-type siderophore i n the i n i t i a l seawater was 6.0 yg NH2OH•L 1 (Csaky t e s t e q u i v a l e n t s ) . Csaky Prorocentrin Recovery Volume (L) Absorbance ( yg NH2 OH) (%) 1 0.068 3.6 59.4 3 0.197 11.2 61.9 5 0.331 18.8 62.5 6 0.383 21.8 60.3 10 0.420 23.9 39.6 20 0.443 25.2 20.8 88 Table VIII - Influence of the volume of deionized, d i s t i l l e d water washes on the recovery of prorocentrin. Five L of seawater containing 3.6 yg NH2OH equivalents of Csaky posit i v e material was passed through each column. Values are e f f i c i e n c y of recovery of the o r i g i n a l material (expressed as a % ). Experiments were performed in duplicate. (*-These samples contained too much salt to enable a Csaky test to be performed.) Wash Volume (L ) 0.050 0.050 Csaky A b s o r b a n c e P r o r o c e n t r i n C o l l e c t e d ( yg NH2OH. L) R e c o v e r y 0.150 0.150 0.105 0.098 2.3 2.1 63 59 1.0 1.0 0.0.70* 0.069 1. 5V 1.4 41 40 2.0 2.0 0.048 0.038 1.0: 0.8 27 21 89 Table IX - Removal of p r o r o c e n t r i n from XAD-2 r e s i n by suc c e s s i v e 100 ml methanol washes. N.D.-none de t e c t e d . Methanol Csaky Prorocentrin Wash (mL) Absorbance Collected ( yg NH2OH) 000-100 0.188 10.4 100-200 0.197 11.2 200-300 0.037 2.1 300-400 <0.010 ND: 400-500 <0.010 ND 500-600 <0.010 ND 600-700 <0.010 ND 90 Table X - Recovery of prorocentrin from XAD-2 resin with increasing concentrations of Csaky positive material. Recovery (%) i s based on the o r i g i n a l concentration of prorocentrin added to 5 L of a c i d i f i e d ESNW-Fe medium. Average recovery (and one standard deviation) was 62.2 (±4.2) %. I n i t i a l Csaky Material ( u g NH2 OH Csaky Absorbance Prorocentrin Collected (yg NH 2 QH) Recovery (%) 5 10 20 30 50 0.057 0.117 0.215 0.328 1.524 2.9 6.3 11.9 18.3 34.5 58 63 60 61 69 91 2000 ml d e i o n i z e d , d i s t i l l e d water. The adsorbed o r g a n i c s were removed from the column with 300 ml methanol. Each experiment was run i n d u p l i c a t e . The recovery of Csaky p o s i t i v e m a t e r i a l i s presented i n Table V I I I . Washing the column with 50 ml was u n s a t i s f a c t o r y as the l a r g e amount of s a l t remaining i n t e r f e r r e d with the c o n c e n t r a t i o n step p r i o r to performance of the Csaky t e s t . Recovery a f t e r washing the column with 1000 ml or 2000 ml of water was very low, i n d i c a t i n g p r o r o c e n t r i n d i d not bind s t r o n g l y to the XAD-2 column. c) Volume of Methanol to E l u t e the Column Organics from 10 L of c e l l - f r e e f i l t r a t e were c o l l e c t e d on a standard XAD-2 column. The column was washed with 150 ml of d e i o n i z e d , d i s t i l l e d water. Organics were e l u t e d from the column i n seven s u c c e s s i v e 100 ml methanol passes. The amount of Csaky r e a c t i v e m a t e r i a l was measured in each f r a c t i o n . A l l of the Csaky r e a c t i v e m a t e r i a l was e l u t e d i n the f i r s t 300 ml (Table IX). d) I n i t i a l C o n c e n t r a t i o n of Siderophore Based on the previous experiments, the f o l l o w i n g standard procedure was adopted for t h i s t e s t . Six l i t e r s or l e s s of sample was passed through a 25 X 2 cm column of Soxh l e t - c l e a n e d XAD-2 r e s i n . The column was washed with 150 ml d e i o n i z e d , d i s t i l l e d water and the adsorbed o r g a n i c s e l u t e d with 300 ml methanol. Siderophore was c o l l e c t e d from 100 L of P. minimum 92 c u l t u r e . The e l u t e d aqueous f r a c t i o n from the XAD-2 c o n c e n t r a t i o n procedure was added back to f i v e , 5 L a l i q u o t s of a c i d i f i e d ESNW-Fe medium. Each f l a s k was i n o c u l a t e d at one of the f o l l o w i n g Csaky e q u i v a l e n t c o n c e n t r a t i o n s : 5, 10, 20, 30, or 50 ug NH2OH-L 1 . Recovery was constant over the range of 5 to 50 yg NH2OH-L"" 1 (Table X ). 2. I s o l a t i o n and C h a r a c t e r i z a t i o n of P r o r o c e n t r i n From XAD-2 C o l l e c t e d F r a c t i o n s a) D e t e c t i o n of P r o r o c e n t r i n in C o l l e c t e d Aqueous F r a c t i o n s To f o l l o w the i s o l a t i o n and p u r i f i c a t i o n of p r o r o c e n t r i n , two methods were r o u t i n e l y employed. For accurate a n a l y s i s the p r e v i o u s l y d e s c r i b e d Csaky t e s t was used. The a d d i t i o n of f e r r i c p e r c h l o r a t e to the aqueous f r a c t i o n s ( c o l l e c t e d from the g e l permeation chromatography column) was used to detect the presence of or g a n i c s which form c o l o r e d f e r r i c complexes ( A t k i n , et a l . , 1970). A third- procedure, to c h a r a c t e r i z e p r o r o c e n t r i n in aqueous samples was attempted but was u n s u c c e s s f u l . The r e a c t i o n with ammonium metavanadate i s a s e n s i t i v e t e s t f o r microgram amounts of some hydroxamates (Abbasi, 1976). T h i s technique i n v o l v e s forming the vanadium-hydroxamic a c i d complex and the e x t r a c t i o n of the v i o l e t - c o l o r e d complex i n t o a water-immiscible o r g a n i c . The d e t e c t i o n of p r o r o c e n t r i n by t h i s procedure was u n s u i t a b l e s i n c e a c o l o r e d v a n a d a t e - p r o r o c e n t r i n complex co u l d not be det e c t e d . E i t h e r p r o r o c e n t r i n d i d not form a p p r e c i a b l e amounts of the vanadium-complex or the 93 vanadium-prorocentrin complex was not s o l u b l e in the chloroform, b u t a n o l , or e t h y l a c e t a t e organic l a y e r s t e s t e d . b) D e t e c t i o n of P r o r o c e n t r i n by Thin Layer Chromatography Of the t h i n l a y e r chromatography s o l v e n t systems examined only b u t a n o l : a c e t i c acid:water (60:15:25) {BAW} s u i t a b l y chromatographed p r o r o c e n t r i n from the o r g i n (Rf= 0.18-0.20 f o r the d e s f e r r i - p r o r o c e n t r i n ) . P r o r o c e n t r i n remained a t the o r i g i n in a l l the other t h i n l a y e r chromatography so l v e n t systems. The other s o l v e n t systems separated other o r g a n i c s away from the o r i g i n (and, thus, away from p r o r o c e n t r i n ) . To d e t e c t the presence of p r o r o c e n t r i n on t h i n l a y e r chromatography p l a t e s , a F e C l 3 spray (2% i n ethanol) was u t i l i z e d . P r o r o c e n t r i n was v i s i b l e immediately a f t e r s p r a y i n g as a l i g h t pink spot which q u i c k l y faded. To reduce the speed at which the i r o n - p r o r o c e n t r i n l o s t i t s c o l o r , the mod i f i e d FeCl spray with c h l o r o a c e t i c a c i d was used. T h i s spray i s co n s i d e r e d to form a more s t a b l e complex (Bernhard et a l . , 1964), but i t was u n s u i t a b l e because a c o l o r e d p r o r o c e n t r i n -product f a i l e d to form. P r o r o c e n t r i n on t h i n l a y e r chromatography p l a t e s c o u l d be a l s o v i s u a l i z e d by r e a c t i o n with n i n h y d r i n spray (brown, yellow c o l o r ) or by I vapor. These r e a c t i o n s c o u l d only be used i n co n j u n c t i o n with the F e C l 3 spray, as n e i t h e r i s s p e c i f i c f o r i r o n - b i n d i n g o r g a n i c s . 94 c) F r a c t i o n a t i o n by Gel Permeation Chromatography I n i t i a l s e p a r a t i o n of p r o r o c e n t r i n from other XAD-2 c o l l e c t e d w a t e r - s o l u b l e o r g a n i c s , was attempted on a g e l permeation chromatography column. The column was packed with BioGel P-2, a polyacrylamide g e l with a f u n c t i o n a l molecular s i z e s e p a r a t i o n range between 100 and 1800 d a l t o n s . P r o f i l e s of the c o l l e c t e d f r a c t i o n s are presented i n F i g . 17. In each case s e v e r a l compounds or groups of compounds were r e a c t i v e to the f e r r i c p e r c h l o r a t e . Four major f e r r i c p e r c h l o r a t e peaks c o u l d be d i s t i n g u i s h e d , but only one of the peaks co n t a i n e d Csaky-p o s i t i v e compounds (peak III i n F i g . 17C). Fi g u r e 17 a l s o serves as a comparison of the organic p r o f i l e s from v a r i o u s experimental treatments. Based on these p r o f i l e s , c e l l s grown in l a r g e - s c a l e p o l y e t h y l e n e b a r r e l s c o n t a i n e d the same type of i n t r a c e l l u l a r o r g a n i c s as d i d axenic c e l l s grown in g l a s s carbouys. In c o n t r a s t , the p r o f i l e s of the e x t r a c e l l u l a r XAD-2 c o l l e c t e d o r g a n i c s d i f f e r e d s i g n i f i c a n t l y . The e x t r a c e l l u l a r compounds of c e l l s grown in the b a r r e l s always had a strong peak of high molecular weight and p e r c h l o r a t e -p o s i t i v e compounds at the v o i d volume f r a c t i o n s (peak I i n F i g . 17C and 17D). T h i s was evident r e g a r d l e s s of whether n a t u r a l or a r t i f i c i a l seawater was employed (data not shown). T h i s peak was v i r t u a l l y e l i m i n a t e d from the 95 F i g u r e 17 - Comparison of p r o f i l e s of the s e p a r a t i o n of c o l l e c t e d m e t a b o l i t e s based on molecular s i z e . C o l l e c t e d o r g a n i c s were i s o l a t e d from: a) c e l l s grown i n p o l y e t h y l e n e b a r r e l s ; b) c e l l s grown in g l a s s carbouys; c) c e l l -f r e e c u l t u r e medium from p o l y e t h y l e n e b a r r e l s ; and d) c e l l - f r e e c u l t u r e medium from g l a s s carbouys. 96 F i g u r e 18 - Standard curve f o r the e s t i m a t i o n of the molecular weight of p r o c e n t r i n . Standards i n c l u d e : dextran blue (1) , v i t a m i n B 1 2 (2 ) , d e s f e r r i o x a m i n e B (3) , r i b o f l a v i n (4) , and lumichrome (5 ) . The shaded area represents the best estimate of p r o r o c e n t r i n based on the c o l l e c t i o n of a f r a c t i o n p o s i t i v e to the p e r c h l o r a t e t e s t and the Csaky t e s t . Ve/Vo i s the r a t i o of the c o l l e c t e d volume c o n t a i n i n g the standard compound compared with the v o i d volume of the column. 3 04 : / / / / / / / / I 2.5 O JCD O ° . 2,0 2 3? 97 e x t r a c e l l u l a r compounds of the glass-grown c e l l s ( F i g . 17C), suggesting that these compounds o r i g i n a t e d e i t h e r from the b a r r e l or as a c e l l u l a r response to some component of the b a r r e l . There was no evidence of peak I in e i t h e r of the i n t r a c e l l u l a r p r o f i l e s ( F i g . 17A and 17D). The only organic f r a c t i o n that was c o n s i s t e n t l y observed was the C s a k y - p o s i t i v e , peak I I I . Examination of a l l f r a c t i o n s by the BAW t h i n l a y e r chromatography s o l v e n t system and F e C l 3 spray, v e r i f i e d peak III as the only f r a c t i o n with a p p r e c i a b l e amounts of p r o r o c e n t r i n . d) Molecular S i z e Determination To determine the molecular s i z e of p r o r o c e n t r i n the p r e v i o u s l y d i s c u s s e d BioGel P-2 column was employed. The column was c a l i b r a t e d using a s e r i e s of compounds (molecular weights, d a l t o n s ) : blue dextran (>1800), v i t a m i n B 12 (1350), r i b o f l a v i n (376), desferrioxamine B (560), and lumichrome (242). P r o r o c e n t r i n was run s e p a r a t e l y on the same column immediately p r i o r to running the standards. While the standards gave d i s c r e t e estimates of molecular weight, p r o r o c e n t r i n spread somewhat while descending the column. The best estimate of molecular weight i s between 560 and 590 d a l t o n s ( F i g . 18). e) F r a c t i o n a t i o n by Rotary Thin Layer Chromatography The p r o r o c e n t r i n - c o n t a i n i n g f r a c t i o n from the g e l permeation chromatography column (peak III ) was f u r t h e r f r a c t i o n a t e d using the r o t a r y t h i n l a y e r chromatography system. 98 Rotary t h i n l a y e r chromatography i s f u n c t i o n a l l y d i f f e r e n t from t r a d i t i o n a l t h i n l a y e r chromatography in that there i s no l i m i t to the s o l v e n t f r o n t . Thus, s e p a r a t i o n of p r o r o c e n t r i n i s s u i t e d to t h i s system s i n c e p r o r o c e n t r i n has a low Rf i n BAW and remains at the o r i g i n with the other s o l v e n t systems examined. M a t e r i a l from peak III was d i s s o l v e d i n a small volume of methanol:water (50:50). T h i s sample was a p p l i e d to the o r i g i n of the r o t a r y t h i n l a y e r chromatography p l a t e and allowed to dry o v e r n i g h t . The p l a t e was washed with methanol (100%), followed by water:methanol (2:98) to remove unwanted o r g a n i c s away from the o r i g i n . P r o r o c e n t r i n was then recovered from the p l a t e with the BAW s o l v e n t system. Any o r g a n i c s remaining on the p l a t e were removed with 100 ml wash with hexane. Examination of each f r a c t i o n by t r a d i t i o n a l t h i n l a y e r chromatography and FeClg spray confirmed p r o r o c e n t r i n only i n the BAW f r a c t i o n . F r a c t i o n a t i o n by r o t a r y chromatography was the best way to i s o l a t e p r o r o c e n t r i n , but i t was not without c e r t a i n problems. The high amount of water in the BAW s o l v e n t system weakened the support. A small amount of m a t e r i a l (probably s i l i c a ) was c o l l e c t e d along with p r o r o c e n t r i n . While t h i s contaminating m a t e r i a l c o u l d be removed by passing the c o l l e c t e d f r a c t i o n back over the g e l permeation chromatography column, the a d d i t i o n a l pass r e s u l t e d i n a very reduced y i e l d of p r o r o c e n t r i n . f) A d d i t i o n of Iron Since the i r o n - p r o r o c e n t r i n complex i s c o l o r e d (as shown by the r e a c t i o n with f e r r i c p e r c h l o r a t e ) , i t was thought that the 99 formation of the complex p r i o r to the a p p l i c a t i o n on the r o t a r y chromatography p l a t e might r e s u l t i n a more e f f i c i e n t s e p a r a t i o n technique. S e v e r a l procedures to add i r o n were attempted. While p r o r o c e n t r i n d i d r e a d i l y form the i r o n - p r o r o c e n t r i n complex, the s e p a r a t i o n of t h i s complex from the non-complexed i r o n was a more d i f f i c u l t task. The simplest technique of adding i r o n as F e C l 3 was u n s u c c e s s f u l . The f e r r i - p r o r o c e n t r i n c o u l d not be separated from the r e s i d u a l c o l l o i d a l f e r r i c hydroxide which formed at the experimental pH (7 - 9). A p p l i c a t i o n of the iron-complex and i r o n hydroxide system to e i t h e r of the t h i n l a y e r chromatography systems r e s u l t e d i n the i r o n adsorbing to the p l a t e . None of the s o l v e n t systems was s u c c e s s f u l i n r e l e a s i n g the f e r r i - p r o r o c e n t r i n complex from the plate-bound i r o n hydroxide. A d d i t i o n of the complex to a g e l permeation chromatography column was u n s u c c e s s f u l s i n c e the i r o n and the f e r r i - p r o r o c e n t r i n bound i r r e v e r s i b l y to the column. A d d i t i o n of i r o n using f r e s h l y p r e c i p i t a t e d i r o n hydroxide was more s u c c e s s f u l , but i t c o u l d not be r o u t i n e l y performed. F o l l o w i n g the procedure of Carrano and Raymond (1978), f r e s h l y p r e c i p i t a t e d and washed f e r r i c hydroxide was added to the aqueous s o l u t i o n c o n t a i n i n g p r o r o c e n t r i n . The mixture was s t i r r e d f o r two hours at 80°C. The non-bound c o l l o i d a l i r o n was removed by f i l t r a t i o n {glass f i b e r (GF/A), followed by f i l t r a t i o n through a 1 pm membrane f i l t e r } . The f e r r i - p r o r o c e n t r i n complex remained i n s o l u t i o n . The aqueous sample was a p p l i e d to the r o t a r y chromatography 100 p l a t e and allowed to dry o v e r n i g h t . The same s e r i e s of s o l v e n t s were employed to remove o r g a n i c s a s s o c i a t e d with the p r o r o c e n t r i n . During the BAW wash a red-orange band chromatographed away from the o r i g i n . T h i s band was c o l l e c t e d and i t s UV/VIS a b s o r p t i o n spectrum obtained. The m a t e r i a l had the c h a r a c t e r i s t i c a b s o r p t i o n spectrum of a ferri-hydroxamate. The spectrum has been d e s c r i b e d elsewhere in the t h e s i s (Chapter V) . To v e r i f y that t h i s compound was the iron-complex of p r o r o c e n t r i n , a sample was run on the t r a d i t i o n a l t h i n l a y e r chromatography s t r i p . Using the BAW so l v e n t system, the sample showed a s i n g l e spot with an Rf of 0.6. Based on s h o r t -wavelength f l u o r e s c e n c e and r e a c t i o n to I 2 vapor and F^C1 3 spray, there was no evidence of contaminating compounds. To remove the i r o n , a small subsample was b a s i f i e d with 1 ml 1N KOH. The reagent mixture was c h i l l e d and the f e r r i c hydroxide removed by c e n t r i f u g a t i o n . Chromatography of the c o l o r l e s s supernatant showed a s i n g l e , F e C l 3 p o s i t i v e spot, corresponding to the o r i g i n a l d e s f e r r i - p r o r o c e n t r i n . Thus, the i n t e r c o n v e r s i o n of the f e r r i - f o r m and d e s f e r r i - f o r m of p r o r o c e n t r i n was v e r i f i e d . The a d d i t i o n of i r o n by f e r r i c hydroxide was not always s u c c e s s f u l . Of the f i v e attempts, only two provided s u f f i c i e n t t r a n s f e r of i r o n to p r o r o c e n t r i n . It seems the p r e p a r a t i o n of the f r e s h l y p r e c i p i t a t e d and washed f e r r i c hydroxide i s a c r i t i c a l step which i s not e n t i r e l y r e p r o d u c i b l e . 101 g) A n a l y s i s of I s o l a t e d P r o r o c e n t r i n S e v e r a l a n a l y s e s were performed on the s m a l l amount of f e r r i - p r o r o c e n t r i n . The i s o l a t e d f e r r i - p r o r o c e n t r i n gave a p o s i t i v e Csaky t e s t and d i d not r e a c t to the a d d i t i o n of f e r r i c p e r c h l o r a t e . The i s o l a t e d f e r r i - p r o r o c e n t r i n had a Rf = 0.6 i n the BAW s o l v e n t system. However, when f e r r i - p r o r o c e n t r i n was c o - s p o t t e d with e i t h e r f e r r i c c h l o r i d e or f e r r i c hydroxide, the i r o n - p r o r o c e n t r i n complex remained at the o r i g i n . T h i s confirms the p r e v i o u s suggestion that d i f f i c u l t i e s i n i s o l a t i n g the i r o n complex may be due to our i n a b i l i t y to separate the complex away from the o r i g i n , rather than our i n a b i l i t y to form the i r o n complex. The unbound i r o n must be removed from the sample before f e r r i - p r o r o c e n t r i n w i l l chromatograph. A s u b t r a c t i o n of the c o l l e c t e d f e r r i - p r o r o c e n t r i n was examined s p e c t r o s c o p i c a l l y to determine the i n f l u e n c e of pH on the iron-complex. The i n f l u e n c e of a wide range of pH v a l u e s i s documented in F i g . 19. For each t r i a l , the same amount of f e r r i - p r o r o c e n t r i n was d i s s o l v e d in pH-adjusted water. The s h i f t in the a b s o r p t i o n was recorded immediately and a f t e r 24 hours. While the absorbance s h i f t e d and s t a b i l i z e d immediately, some siderophores r e q u i r e longer i n c u b a t i o n s before a s t a b l e absorbance i s obtained (Ong e_t a l . , 1979). In the case of f e r r i - p r o r o c e n t r i n , a c i d i f i c a t i o n of the sample had l i t t l e • e f f e c t on e i t h e r the i n t e n s i t y of absorbance or in the absorbancy maximum. B a s i f y i n g the sample to pH 11 d i d d i m i n i s h the i n t e n s i t y of the absorbance but an i r o n - p r o r o c e n t r i n complex s t i l l e x i s t e d with an absorbance maximum at 440 nm. 1 02 F i g u r e 19 - The i n f l u e n c e of pH on the U V - v i s i b l e spectrum of p r o r o c e n t r i n . wavelength (nm) 1 03 The remaining m a t e r i a l was submitted for C:N:Fe a n a l y s i s . Based on t h i n l a y e r chromatography, the f e r r i - p r o r o c e n t r i n sample appeared to be pure, however, the a c t u a l percentage of organic m a t e r i a l was low (10%). The sample contained a l a r g e amount of contaminating i n o r g a n i c m a t e r i a l (probably s i l i c a from the r o t a r y t h i n l a y e r chromatography p l a t e ) . The low amount of f e r r i - p r o r o c e n t r i n d i d not allow f o r an accurate measurement of the i r o n conte'nt, although i r o n was v e r i f i e d as a component of the sample. The measured atomic r a t i o of C:N was 2.4:1. T h i s i s lower than the 3:1 r a t i o f o r the trihydroxamate siderophore, f e r r i c h r o m e , and much lower than the 3.6:1 r a t i o f o r r h o d o t o r u l i c a c i d (a dihydroxamate siderophore) (Neilands, 1981). E. DISCUSSION Examination of the t o t a l i s o l a t i o n procedure c l a r i f i e s the d i f f i c u l t i e s i n i s o l a t i n g the h i g h l y water-soluble hydroxamate siderophore. S p e c i a l care must be taken d u r i n g the XAD-2 e x t r a c t i o n of p r o r o c e n t r i n from the c u l t u r e supernatant. Under the most r i g o r o u s procedure, up to 40% of the p r o r o c e n t r i n was unrecoverable. However, for the q u a n t i t a t i v e a n a l y s i s of the p r o d u c t i o n of p r o r o c e n t r i n , the standard i s o l a t i o n procedure c o n s i s t e n t l y recovered 60-65% of the e x t r a c e l l u l a r siderophore. T h i s recovery was . maintained over the e n t i r e range of siderophore c o n c e n t r a t i o n s recorded. Thus, as a q u a n t i t a t i v e a n a l y s i s , the XAD-2 technique as o u t l i n e d in F i g . 16, p r o v i d e s 1 04 a good approximation of the a c t u a l amount of p r o r o c e n t r i n produced. The procedure for i s o l a t i n g pure p r o r o c e n t r i n was not s a t i s f a c t o r y . Each step d i d separate p r o r o c e n t r i n from other o r g a n i c s , but the f i n a l product contained a l a r g e amount of c o l o r l e s s i n o r g a n i c m a t e r i a l . While t h i s was only v e r i f i e d f o r the f e r r i - p r o r o c e n t r i n sample (by elemental a n a l y s i s ) , one must suspect an i n o r g a n i c contaminant in the d e s f e r r i - p r o r o c e n t r i n samples a l s o . The i n o r g a n i c contaminant i s of l i t t l e consequence s i n c e i t s presence d i d not i n t e r f e r e with the a n a l y s i s of the i s o l a t e d m a t e r i a l . In Chapters V and VII, the r o l e of p r o r o c e n t r i n as a f u n c t i o n a l hydroxamate-type siderophore was e s t a b l i s h e d . In review, the r a t i o n a l e was that p r o r o c e n t r i n gave a p o s i t i v e Csaky t e s t , formed an iron-complex with an a b s o r p t i o n maximum near 440 nm, and p r o d u c t i o n was s t i m u l a t e d by l o w - i r o n medium. Neilands (1981a) has c h a r a c t e r i z e d hydroxamate-type siderophores as having the f o l l o w i n g p r o p e r t i e s : formation of a c o l o r e d complex by a d d i t i o n of f e r r i c c h l o r i d e or f e r r i c p e r c h l o r a t e , p o s i t i v e Csaky t e s t , formation of a c o l o r e d complex with a c i d i f i e d ammonium vanadate, and a c o l o r e d iron-complex with maximum absorbancy w i t h i n 425-450 nm. In c o n t r a s t , a c a t e c h o l - t y p e of siderophore forms an iron-complex in an absorbancy range c l o s e r to 500 nm and does not react to the Csaky t e s t . Of the four c r i t e r i a , three were e x h i b i t e d by p r o r o c e n t r i n . The f o u r t h , the formation of a c o l o r e d complex 105 with vanadium, was not met. However, not a l l hydroxamate-sideophores give a p o s i t i v e ammonium vanadate t e s t (Abbasi, 1976). The a b s o r p t i o n s p e c t r a presented in t h i s paper provide evidence f o r p r o r o c e n t r i n being s p e c i f i c a l l y a tri-hydroxamic s i d e r o p h o r e . Tri-hydroxamic siderophores are c h a r a c t e r i z e d by a strong, broad a b s o r p t i o n band between 425 and 440 nm. The band i s not s t r o n g l y s h i f t e d by a c i d i f i c a t i o n from pH 7 to pH 2 and t h i s agrees with e a r l i e r o b s e r v a t i o n s by Warren and Neilands (1964). There i s only a s l i g h t l o s s i n absorbancy which has been d e s c r i b e d in other tri-hydroxamates (Anderegg, et a l . , 1963). S i m i l a r l y the r e s i s t a n c e to decomposition of the i r o n -complex under a l k a l i n e c o n d i t i o n s i s a c h a r a c t e r i s t i c of some tri-hydroxamates (Neilands, 1966). While some tri-hydroxamates l o s e i r o n e a s i l y at e l e v a t e d pH values (eg. f e r r i c h r o m e ; Emery and N e i l a n d s , 1970), the s t a b i l i t y of the i r o n - p r o r o c e n t r i n complex at b a s i c pH values would be e s s e n t i a l for t h i s compound to f u n c t i o n as a siderophore i n the marine environment. In c o n c l u s i o n , the w a t e r - s o l u b l e nature of p r o r o c e n t r i n makes i s o l a t i o n a c h a l l e n g i n g problem. With care, XAD-2 c o u l d be used as a s u i t a b l e method of c o n c e n t r a t i o n of p r o r o c e n t r i n . However, the l i m i t s are e a s i l y exceeded. The procedure used to i s o l a t e p r o r o c e n t r i n would not be s u i t a b l e i f l a r g e amounts of m a t e r i a l were necessary. The l a r g e number of steps r e s u l t e d in s i g n i f i c a n t l o s s of m a t e r i a l . The presence of the i n o r g a n i c contaminant would hamper any f u r t h e r c h a r a c t e r i z a t i o n . The absorbance s p e c t r a of the f e r r i - p r o r o c e n t r i n , p r o v i d e s evidence 1 06 of p r o r o c e n t r i n being a t r i - h y d r o x a m i c siderophore. The s t a b i l i t y of the iron-complex at e l e v a t e d pH values may be an important c r i t e r i o n for e x t r a c e l l u l a r siderophores produced i n marine waters. 1 07 V I I . THE CONTROL OF THE PRODUCTION OF A SIDEROPHORE BY THE  MARINE DINOFLAGELLATE, PROROCENTRUM MINIMUM. A. ABSTRACT Fa c t o r s which i n f l u e n c e the pr o d u c t i o n of p r o r o c e n t r i n by sp e c i e s of Prorocentrum were i n v e s t i g a t e d . P r o r o c e n t r i n , the e x t r a c e l l u l a r siderophore i s o l a t e d from P. minimum, was a l s o produced by P.mariae-lebouriae and P . g r a c i l e . P . g r a c i l e was s u s c e p t i b l e to i r o n - s t r e s s and was not c o n s i d e r e d f u r t h e r . Under i r o n - s a t u r a t e d c o n d i t i o n s there was no measurable siderophore found e i t h e r i n t r a c e l l u l a r l y or e x t r a c e l l u l a r l y . Under i r o n - d e f i c i e n t c u l t u r e c o n d i t i o n s , p r o r o c e n t r i n was produced 1 to 2 days a f t e r the c e s s a t i o n of growth i n the s t a t i o n a r y phase. Production was over a short p e r i o d of time {1 to 2 days) and the p r o r o c e n t r i n d i d not remain i n the medium. The rate of p r o r o c e n t r i n disappearance from the medium was s i m i l a r to the rate of p r o d u c t i o n . Immediately f o l l o w i n g the removal of p r o r o c e n t r i n from the medium, there was a r a p i d i n c r e a s e in i_n v i v o f l u o r e s c e n c e . There was no i n c r e a s e i n c e l l numbers and the inc r e a s e was not seen i n i r o n - s u f f i c i e n t c u l t u r e s . An hypothesis on the i r o n uptake mechanism i s proposed. 108 B. INTRODUCTION The r e l a t i o n s h i p between t r a c e metals and c h e l a t o r s i n c o n t r o l l i n g primary p r o d u c t i v i t y i n the ocean has r e c e i v e d an i n c r e a s i n g amount of a t t e n t i o n i n recent y e a r s . Of p a r t i c u l a r importance i s the r o l e of i r o n . In a s e r i e s of e a r l y experiments, Johnston (1964) found that the growth of n a t u r a l phytoplankton was o f t e n c o n t r o l l e d by the a v a i l a b i l i t y of c h e l a t i n g agents. Barber and Ryther (1969) suggested that p r o d u c t i o n of n a t u r a l c h e l a t o r s s t i m u l a t e d phytoplankton growth r a t e s i n upwelled seawater. Since the s t i m u l a t i o n of growth c o u l d be mimicked by the a d d i t i o n of F e C l 3 , the i n i t i a l i n t e r p r e t a t i o n was that i r o n became a v a i l a b l e f o r growth by forming i r o n - o r g a n i c complexes (Barber et. a_l. ,1971; Barber, 1973). Most of the i r o n i n seawater appears to be u n a v a i l a b l e for the d i r e c t u t i l i z a t i o n by phytoplankton because i t i s a s s o c i a t e d with l a r g e organic p a r t i c l e s (Sugimura, e_t §_1. , 1978) or i t forms i n s o l u b l e , Fe(OH)3 aggregates (Lewin and Chen, 1971). The a d d i t i o n of' a c h e l a t o r i s thought to enhance the a v a i l a b l i t y of i r o n ( I I I ) by s o l u b l i z i n g the f e r r i c hydroxide. Thus, a v a i l a b l e i r o n i n marine waters may be low enough to l i m i t growth (Menzel and Ryther, 1961; Menzel e_t a_l. , 1963; Tranter and Newell, 1963; Glover, 1978). In g e n e r a l , microorganisms can scavenge i r o n from a low i r o n environment by producing e x t r a c e l l u l a r low molecular weight i r o n ( I I I ) s p e c i f i c c h e l a t o r s ( s i d e r o p h o r e s ) . Siderophores are of two ge n e r a l types. Those c o n t a i n i n g secondary hydroxamate groups and those with c a t e c h o l f u n c t i o n a l i t i e s (Neilands, 1980). 109 High a f f i n i t y i r o n a c q u i s i t i o n systems have been demonstrated f o r marine b a c t e r i a (Gonye and Carpenter, 1974) and cy a n o b a c t e r i a (Simpson and N e i l a n d s , 1976; Armstrong and Van Baalen, 1979). U n t i l r e c e n t l y , siderophore p r o d u c t i o n by e u k a r y o t i c phytoplankton was unknown. Barber and co-workers (Spencer e_t a_l. , 1973) c h a r a c t e r i z e d a p o s s i b l e siderophore from a non-axenic c u l t u r e of the marine diatom Chaetoceros s o c i a l i s The i s o l a t e d substance had chemical p r o p e r t i e s s i m i l a r to a hydroxamate-type siderophore but the p o s s i b i l i t y of a b a c t e r i a l o r i g i n f o r t h i s compound was not r i g o r o u s l y excluded. Recent work aimed at s t i m u l a t i n g the p r o d u c t i o n of siderophores in axenic e u k a r y o t i c phytoplankton has demonstrated they are unable to produce i r o n ( 1 1 1 ) - s p e c i f i c c h e l a t o r s (Swallow et a l . , 1978; McKnight and Morel, 1979). This has l e d to the suggestion that e u k a r y o t i c phytoplankton may not u t i l i z e i r o n ( I I I ) , but rather i r o n ( I I ) , which i s more r e a d i l y t r a n s p o r t e d a c r o s s the c e l l membrane, but i s extremely l a b i l e in marine waters (Anderson and Morel, 1980). Recently the i s o l a t i o n of p r o r o c e n t r i n , a low molecular weight e x t r a c e l l u l a r hydroxamate-type siderophore produced by the marine d i n o f l a g e l l a t e Prorocentrum minimum was d e s c r i b e d (Chapter V ) . P r o r o c e n t r i n represents the f i r s t example of an e x t r a c e l l u l a r siderophore produced by a marine e u k a r y o t i c p h y t o p l a n k t e r . T h i s paper presents the r e s u l t s of an experimental i n v e s t i g a t i o n of the f a c t o r s c o n t r o l l i n g the pr o d u c t i o n of p r o r o c e n t r i n in c u l t u r e . 1 10 C. MATERIALS AND METHODS The f i v e Prorocentrum s p e c i e s examined and the source of each c u l t u r e are presented i n Table 11. C e l l s were grown in s t e r i l e e n r i c h e d n a t u r a l seawater, m o d i f i e d by the replacement of Na glycerophosphate with NagHPO^ and Fe (NH^ )2 SO^ with FeClg (ESNW) (H a r r i s o n et al.,1980) Experiments were conducted i n 6 L or 10 L f l a t bottomed b o i l i n g f l a s k s in i r o n s u f f i c i e n t or i r o n d e f i c i e n t media. To reduce i r o n contamination from c u l t u r e glassware, a l l c u l t u r e f l a s k s were repeatedly r i n s e d with 3N NaOH, fo l l o w e d by 3N HC1 and f i n a l l y , d e i o n i z e d , d i s t i l l e d water. Seawater was c o l l e c t e d at a depth of 60 m and i n i t i a l l y f i l t e r e d through a g l a s s f i b e r p r e - f i l t e r f o l l o w ed by another g l a s s f i b e r f i l t e r (GF/A). The seawater was t r e a t e d with a c t i v a t e d c h a r c o a l (24 h, constant s t i r r i n g ) to remove d i s s o l v e d o r g a n i c s . Charcoal was removed by passage through a g l a s s f i b e r f i l t e r f o l l o w e d by a 0.45 urn membrane f i l t e r . Two methods for removal of r e s i d u a l i r o n i n c h a r c o a l t r e a t e d seawater were compared: 1) seawater was heated and f i l t e r e d by the procedure of Lewin and Chen (1971), and then the medium was supplemented with ESNW n u t r i e n t s , minus E e C l 3 and EDTA (ESNW-Fe); and 2) seawater was passed through a column of Chelex-100 r e s i n to remove t r a c e metals (procedure of Morel e_t a l . , 1979), and then supplemented with AQUIL n u t r i e n t s , minus EDTA. A small known amount of i r o n was added back ( 1 u M F e C l 3 ) in order to provide some growth under t h i s i r o n - l i m i t e d c o n d i t i o n (AQUIL-Fe). Both the ESNW-Fe and AQUIL-Fe media 111 p r o v i d e d i r o n - s t r e s s e d growth c o n d i t i o n s . Iron s u f f i c i e n t c o n t r o l s were the above media supplemented with f r e s h l y prepared FeCl (ESNW + Fe and AQUIL + Fe, r e s p e c t i v e l y ) . Experimental f l a s k s were incubated at 18 C using continuous l i g h t p r o v i d e d by d a y l i g h t f l u o r e s c e n t bulbs at an i r r a d i a n c e of 160 yE-m -s . C u l t u r e s were s t i r r e d c o n t i n u o u s l y (60 rpm). Growth was monitored by d a i l y c e l l counts using an e l e c t r o n i c p a r t i c l e counter (Coulter Counter T A - I I ) . The axenic nature of a l l c u l t u r e s was t e s t e d f r e q u e n t l y by i n o c u l a t i n g onto an o r g a n i c - c o n t a i n i n g s t e r i l i t y - m e d i u m p l a t e and examining the p l a t e f o r growth of c o l o n i e s a f t e r 48 h of i n c u b a t i o n . The axenic nature was a l s o v e r i f i e d by a c r i d i n e orange d i r e c t s t a i n i n g (Hobbie et al.,1977). C e l l s were harvested using a Sharpies continuous c e n t r i f u g e f o l l o w e d by passage of the supernatant through g l a s s f i b e r f i l t e r s (GF/C) ( F i g . 20). Remaining c e l l s were removed from the f i l t r a t e by passage through a 0.45 um membrane f i l t e r under a g e n t l e vacuum (<200 mm Hg). Both the c e l l s and the f i l t r a t e were t e s t e d f o r p r o r o c e n t r i n . A crude f r a c t i o n c o n t a i n i n g p r o r o c e n t r i n was obtained in the f o l l o w i n g manner. The c e l l s were e x t r a c t e d by s t i r r i n g with MeOH (2 X 100 ml, 16 h, 8 h), foll o w e d by f i l t r a t i o n through a g l a s s f i b e r f i l t e r (GF/A). The MeOH f i l t r a t e s were evaporated to dryness i_n vacuo and the resi d u e was p a r t i t i o n e d between 50 ml H 20 and 100 ml CHC1 3 The aqueous l a y e r was repeatedly e x t r a c t e d with CHC1 3 u n t i l a n e a r l y c o l o r l e s s l a y e r was achieved. The H 20 l a y e r was evaporated ir\ vacuo to a volume of 5 ml. E x t r a c e l l u l a r 112 Table XI - P r o r o c e n t r i n p r o d u c t i o n by Prorocentrum s p e c i e s . Maximum e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n s and f i n a l c e l l y i e l d of s i x Prorocentrum s p e c i e s grown i n n a t u r a l , e n r i c h e d , i r o n - d e f i c i e n t medium (ESNW-Fe). Species and NEPCC Identification No. Final Cell Yield (10 7-l-i) Maximum Extracellular Prorocentrin ( yg NH 2 OH • L"1;) P. minimum #96 TPavillard) Schiller P. mariae lebouriae * TParke et Ballantine) Loeblich P. gracile #104 (a) Schuett P. maximum #250 TGourret)Schiller IP. micans #33 Ehrenb. 3.2 3.0 3.0 1.7 1.9 37.5 41.0 44.9 5.7 3.4 * Kindly supplied by Prof. H.H. Seliger, The Johns Hopkins University, Baltimore, Maryland. 1 1 3 F i g u r e 20 - Procedure f o r the i s o l a t i o n of the aqueous f r a c t i o n c o n t a i n i n g p r o r o c e n t r i n . r cells extract with MeOH culture . I continuous centnfuge : i V mediurri I 0-45 ym membrane filtration evaporate i C H C i 3 soluble H 2 0 soluble L f acidify to pH 2 0 I XAD-2 column 150 ml H 2 0 wrash I 300 ml MeOH I , evaporate cell-free fi ltrate I r C K C ! solubfe ?e so lub le Csaky* test 1 1 4 p r o r o c e n t r i n was c o l l e c t e d by a d j u s t i n g the c e l l - f r e e f i l t r a t e to pH 2.0 and passing i t over a 25 X 2 cm column of precleaned (Soxhlet e x t r a c t i o n with methanol,72 h) XAD-2 r e s i n ( M a l l i n c k r o d t ) . The column was subsequently washed with 150 ml of d e i o n i z e d and d i s t i l l e d H 2 0 (pH 2.0) and the adsorbed o r g a n i c s were e l u t e d with 300 ml MeOH. The MeOH was reduced to dryness by r o t a r y evaporation and the residue was p a r t i t i o n e d between water and CHC13. The aqueous f r a c t i o n was evaporated i n  vacuo to a f i n a l volume of 5 ml. To q u a n t i f y the amount of p r o r o c e n t r i n , a modified Csaky t e s t which determines the c o n c e n t r a t i o n of hydroxamate f u n c t i o n a l i t i e s c o l o r i m e t r i c a l l y ( G i l l a m e_t a_l. , 1981) was used. In the i n i t i a l s t u d i e s where a l l f r a c t i o n s were examined, only f r a c t i o n s c o n t a i n i n g p r o r o c e n t r i n gave p o s i t i v e Csaky r e s u l t s . The presence of p r o r o c e n t r i n was v e r i f i e d by t h i n l a y e r chromatography of each f r a c t i o n (Chapter V). For each sample, a 2 ml a l i q u o t was used as a t e s t and a second, 2 ml sample was used as a c o l o r and reagent blank. D. RESULTS Three of the f i v e Prorocentrum s p e c i e s showed high amounts of e x t r a c e l l u l a r Csaky p o s i t i v e compound when grown i n i r o n -d e f i c i e n t medium. Prorocentrum g r a c i l e produced the l a r g e s t amount of Csaky p o s i t i v e m a t e r i a l but was i n t o l e r a n t of r e p e t i t i v e t r a n s f e r s and was not s t u d i e d f u r t h e r (Table 10). A l l subsequent experiments were performed with a l o c a l i s o l a t e of P. minimum. This i s o l a t e i s the o r i g i n a l source of 1 15 p r o r o c e n t r i n (Chapter V), although t h i n l a y e r chromatography i n d i c a t e d that p r o r o c e n t r i n was present in the other two s p e c i e s that gave strong p o s i t i v e Csaky t e s t s ( m a r i a e - l e b o u r i a e and P. q r a c i l e ). In order to provide accurate a b s o l u t e values of the amount of p r o r o c e n t r i n produced, the e f f i c i e n c y of i s o l a t i o n of t h i s m a t e r i a l from c u l t u r e f i l t r a t e s was q u a n t i f i e d . F i g u r e 20 shows a schematic of the i s o l a t i o n procedure. The c r i t i c a l step i s the d e s a l t i n g of the column with a d i s t i l l e d water wash. If the column was washed with a minimum amount of d i s t i l l e d , d e i o n i z e d water (150 ml) recovery based on s u c c e s s i v e t r a n s f e r s was 60%. Using t h i s wash volume a small amount of s a l t remained, but i t had no i n f l u e n c e on the Csaky t e s t . D e s a l t i n g the column with l a r g e r volumes of water* (1 L and 2 L) e l i m i n a t e d r e s i d u a l s a l t but a l s o s t r i p p e d the r e s i n of p r o r o c e n t r i n (45% and 23% recovery, r e s p e c t i v e l y ) . Columns washed with l e s s than 50 mL of d i s t i l l e d water r e t a i n e d l a r g e q u a n t i t i e s of s a l t which i n t e r f e r r e d with the evaporation s t e p . As a r e s u l t of the above o b s e r v a t i o n s , a l l columns were washed with 150 ml H 20 and values have been c o r r e c t e d f o r a 40% l o s s . The production of p r o r o c e n t r i n under i r o n - s u f f i c i e n t and i r o n - d e f i c i e n t c o n d i t i o n s i s presented in F i g . 21. There was l i t t l e d i f f e r e n c e in p r o r o c e n t r i n p r o d u c t i o n between the ESNW-Fe and AQUIL-Fe media, although growth r a t e s were s i g n i f i c a n t l y d i f f e r e n t (75% and 40% of the c o n t r o l , r e s p e c t i v e l y ) . The p a t t e r n of pr o d u c t i o n of p r o r o c e n t r i n i s of i n t e r e s t . Under i r o n - s u f f i c i e n t c o n d i t i o n s a small amount of p r o r o c e n t r i n 116 was produced both e x t r a c e l l u l a r l y and i n t r a c e l l u l a r l y at the onset of n u t r i e n t d e f i c i e n c y . A s i m i l a r small pulse i n i n t r a c e l l u l a r p r o r o c e n t r i n p r o d u c t i o n occurred under i r o n -d e f i c i e n c y (ESNW-Fe ). I r o n - d e f i c i e n t c e l l s produced most of the e x t r a c e l l u l a r p r o r o c e n t r i n w i t h i n three days of the c e s s a t i o n of growth. There was no corresponding e x t r a c e l l u l a r p r o d u c t i o n of p r o r o c e n t r i n i n medium f u l l y supplemented with i r o n . P r o r o c e n t r i n e x i s t e d e x t r a c e l l u l a r l y f o r only a short p e r i o d of time before i t r a p i d l y disappeared from the medium. The l o s s of e x t r a c e l l u l a r p r o r o c e n t r i n was not fol l o w e d by an inc r e a s e i n i n t r a c e l l u l a r p r o r o c e n t r i n . Within two to three days a f t e r maximum e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n s were observed, n e i t h e r the c e l l s nor the medium c o n t a i n e d p r o r o c e n t r i n . The e f f e c t of i r o n - d e f i c i e n c y can be c l e a r l y seen by examining the growth r a t e s of the c u l t u r e s . I r o n - s u f f i c i e n t c e l l s grew at maximum growth r a t e s whereas c e l l s grown i n the medium without.added i r o n had s i g n i f i c a n t l y reduced growth r a t e s and s l i g h t l y reduced c e l l y i e l d s . Since the f i n a l c e l l y i e l d s of ESNW-Fe and AQUIL-Fe approached maximum y i e l d s , i t appeared that there was s u f f i c i e n t r e s i d u a l i r o n in the two c u l t u r e s unsupplemented with i r o n to pro v i d e growth, but the form of the i r o n c o u l d only be slowly u t i l i z e d by the c e l l s . To t e s t t h i s h ypothesis, v a r y i n g amounts of EDTA were added to the i r o n - d e f i c i e n t medium (ESNW-Fe) and maximum p r o r o c e n t r i n c o n c e n t r a t i o n and c e l l y i e l d s were measured. If i r o n was i n the medium, but not i n an a v a i l a b l e s t a t e , then the a d d i t i o n of an 1 1 7 Fi g u r e 21 - Comparison of the growth r a t e s and p r o r o c e n t r i n p r o d u c t i o n i n batch c u l t u r e s of P. minimum. P r o r o c e n t r i n values have been c o r r e c t e d f o r l o s s i n the recovery procedure. Medium used was: a) ESNW+Fe; b) ESNW-Fe; c) AQUIL+Fe; and d) AQUIL-Fe. 7.8-1 o o o / a. E S N W + Fe b. E S N W - F e Days 1 18 Fi g u r e 22 - In f l u e n c e of the a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of EDTA on p r o r o c e n t r i n p r o d u c t i o n . A) F i n a l c e l l y i e l d . B) Maximum i n t r a c e l l u l a r or e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n produced by P. minimum. R e p l i c a t e values are mean (± one SE), f o r n = 3. 0.05 15 150 450 EDTA (pM) 1 19 F i g u r e 23 - Influence of i r o n on the growth of P. minimum. Comparison of growth, measured by _in v i v o f l u o r e s c e n c e (log s c a l e ) f o r i r o n - s u f f i c i e n t and i r o n - d e f i c i e n t batch c u l t u r e s of P. minimum. Temporal changes i n e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n f o r the i r o n - d e f i c i e n t c u l t u r e are a l s o i n c l u d e d . 2oJ 1 20 a r t i f i c i a l c h e l a t o r such as EDTA should a l l e v i a t e the i r o n s t r e s s . I n c r e a s i n g the a v a i l a b l e i r o n should i n h i b i t siderophore p r o d u c t i o n and r e - e s t a b l i s h a maximum growth r a t e and f i n a l c e l l y i e l d . The a d d i t i o n of a low amount of EDTA (0.05 y M) had no e f f e c t on these parameters ( F i g . 22). As the EDTA c o n c e n t r a t i o n was i n c r e a s e d a l l parameters a t t a i n e d values s i m i l a r to those of the i r o n - s u f f i c i e n t c u l t u r e s . Using p r o r o c e n t r i n p r o d u c t i o n as an i n d i c a t o r of i r o n s t r e s s the a d d i t i o n of 15 yM EDTA was s u f f i c i e n t to remove i r o n - l i m i t a t i o n , even though i r o n was not added to the medium. Further i n c r e a s e s in EDTA c o n c e n t r a t i o n (450 yM) reduced c e l l y i e l d s and growth r a t e s to values s i m i l a r to the i r o n - d e f i c i e n t v a l u e s . T h i s was accompanied by an i n c r e a s e d p r o d u c t i o n of p r o r o c e n t r i n suggesting that i r o n a v a i l a b i l i t y was reduced due to ex c e s s i v e c h e l a t i o n . The i n f l u e n c e of i r o n d e f i c i e n c y on growth was measured by f l u o r e s c e n c e and i s presented i n F i g u r e 23. Fluoresence values provide a s i g n i f i c a n t l y d i f f e r e n t p i c t u r e than growth monitored by c e l l numbers ( F i g . 21). C e l l counts provided the t r a d i t i o n a l growth curve (exponential growth, followed by a p l a t e a u due to n u t r i e n t l i m i t a t i o n ) f o r both i r o n - s u f f i c i e n t and i r o n - d e f i c i e n t c u l t u r e s . The same p a t t e r n was seen f o r growth i n i r o n - s u f f i c i e n t medium monitored by _in v i v o f l u o r e s c e n c e . However, growth i n medium without added i r o n p r ovided a bimodal f l u o r e s c e n c e p a t t e r n . There was an i n i t i a l i n c r e a s e in f l u o r e s c e n c e as c e l l numbers i n c r e a s e d . As c e l l s became i r o n - l i m i t e d the f l u o r e s c e n c e values plateaued. 121 F l u o r e s c e n c e increased s i g n i f i c a n t l y again, c o i n c i d e n t a l with the disappearance of e x t r a c e l l u l a r p r o r o c e n t r i n . C e l l numbers showed no corresponding change. E. DISCUSSION S u c c e s s f u l examination of siderophore p r o d u c t i o n by marine e u k a r y o t i c phytoplankton i s dependent upon two processes: the s u c c e s s f u l c r e a t i o n of i r o n - l i m i t e d growth medium and the c o n c e n t r a t i o n of the siderophore p r i o r to a n a l y s i s . Both of the procedures used to reduce or e l i m i n a t e a v a i l a b l e i r o n from seawater were s u c c e s s f u l , a l b e i t to d i f f e r e n t degrees. The procedure of Lewin and Chen (1971) reduced the amount of a v a i l a b l e i r o n to c o n c e n t r a t i o n s l i m i t i n g to growth rate and f i n a l c e l l y i e l d . The f i n a l c e l l y i e l d was , however, high enough f o r adequate accumulation of e x t r a c e l l u l a r products. . R e p e t i t i o n of the treatment f a i l e d to remove enough a d d i t i o n a l i r o n to s i g n i f i c a n t l y reduce e i t h e r c e l l y i e l d or growth rate or to f u r t h e r s t i m u l a t e p r o r o c e n t r i n p r o d u c t i o n . The p r e p a r a t i o n of i r o n - s u f f i c i e n t c o n t r o l s was e q u a l l y important. To achieve maximum growth r a t e s and y i e l d s and a minimum production of p r o r o c e n t r i n , i t was e s s e n t i a l to use FeCl which had been f r e s h l y prepared. Iron stocks which were st o r e d i n po l y e t h y l e n e b o t t l e s f o r as short as 2 to 3 days formed enough u n a v a i l a b l e i r o n upon a u t o c l a v i n g to s t i m u l a t e p r o r o c e n t r i n p r o d u c t i o n ; however, f i n a l p r o r o c e n t r i n c o n c e n t r a t i o n s were f a r l e s s than the values obtained using ESNW-Fe or AQUIL-Fe. No n o t i c e a b l e change i n growth rate or 1 22 f i n a l c e l l y i e l d was recorded. The re d u c t i o n i n a v a i l a b l e i r o n in an aged i r o n stock s o l u t i o n has been shown p r e v i o u s l y (Lewin and Chen 1973). A c o n c e n t r a t i o n procedure was a l s o necessary to de t e c t and q u a n t i f y the production of p r o r o c e n t r i n . As d i s c u s s e d p r e v i o u s l y (Chapter V), past r e s e a r c h e r s have concluded that marine e u k a r y o t i c phytoplankton are unable to produce si d e r o p h o r e s . An a l t e r n a t i v e hypothesis i s that p r o d u c t i o n i s at a l e v e l which r e q u i r e s c o n c e n t r a t i o n f o r d e t e c t i o n using present assays. XAD-2 r e s i n has been used p r e v i o u s l y i n marine systems to i s o l a t e humic substances (Stuermer and Harvey, 1974; Mantoura and R i l e y , 1975; Lyons et. a_l. , 1979) and novel e x t r a c e l l u l a r m e t a b o l i t e s (Andersen et a l . , 1980; T r i c k et a_l. , 1981). Sugimura e_t a l . (1978) used XAD-2 to i s o l a t e i r o n - o r g a n i c complexes from seawater. T h i s r e s i n has a l s o been used i n the i s o l a t i o n of siderophores from other organisms (Horowitz et a l . , 1976). While XAD-2 r e s i n has been used i n many r e l a t e d a p p l i c a t i o n s , the recovery of p r o r o c e n t r i n was l e s s than i d e a l . The h i g h l y water s o l u b l e p r o r o c e n t r i n binds i n e f f e c t i v e l y to the r e s i n and c o u l d e a s i l y be l o s t through e x c e s s i v e d e s a l t i n g of the XAD-2 column. Using the d e f i n e d c o n d i t i o n s of sample p r e p a r a t i o n (150 mL H 20 wash) recovery was c o n s i s t e n t and t h e r e f o r e the t o t a l p r o r o c e n t r i n c o n c e n t r a t i o n c o u l d be estimated with c o n f i d e n c e . The s t i m u l a t i o n of p r o r o c e n t r i n production under low i r o n growth c o n d i t i o n s s a t i s f i e s the t r a d i t i o n a l t e s t f o r a high 1 23 a f f i n i t y i r o n t r anspor t system ( G a r i b a l d i and N e i l a n d s , 1956). Growth of P. minimum in e i t h e r of the two low i r o n media s t i m u l a t e d e x t r a c e l l u l a r p r o r o c e n t r i n p roduc t ion and the e f f e c t c o u l d be reversed by the a d d i t i o n of f r e s h l y prepared FeCl While t h i s r e s u l t p o i n t s to p r o r o c e n t r i n being a s iderophore , the p a t t e r n of p roduc t ion i s e q u a l l y important . A l l of the p r o r o c e n t r i n was produced i n the e a r l y s t a t i o n a r y phase of batch c u l t u r e . S t i m u l a t i o n of e x t r a c e l l u l a r organic p roduc t ion dur ing s t a t i o n a r y phase i s common for both pr imary organic s ( H e l l e b u s t , 1974) and unique e x t r a c e l l u l a r m e t a b o l i t e s . ( T r i c k e_t a l . , 1981). A s i m i l a r p e r i o d of p roduct ion has been reported for the copper complexing agents from marine and freshwater phytoplankton (McKnight and M o r e l , 1979). Produc t ion of p r o r o c e n t r i n in the s t a t i o n a r y phase d i f f e r s from the p a t t e r n of s iderophore product ion by the marine cyanobacter ium, Agmenellum  quadruplicaturn , grown i n batch c u l t u r e (Armstrong and Van Baa len , 1979). E x t r a c e l l u l a r s iderophore from the blue-green a lga accumulated dur ing e x p o n e n t i a l growth and was removed from the medium as c e l l s approached s t a t i o n a r y phase. Whether t h i s d i f f e r e n c e i n produc t ion i s a c h a r a c t e r i s t i c d ivergence between the e u k a r y o t i c and p r o k a r y o t i c phytoplankton remains to be ver i f i e d . Our a n a l y s i s of p r o r o c e n t r i n produc t ion in batch c u l t u r e prov ides f u r t h e r i n s i g h t i n t o the unsucces s fu l attempts of other researchers to v e r i f y the p roduc t ion of e x t r a c e l l u l a r s iderophore by e u k a r y o t i c phytop lankton . Under i r o n l i m i t a t i o n , the p e r i o d of h igh e x t r a c e l l u l a r p r o r o c e n t r i n c o n c e n t r a t i o n i s 124 v e r y s h o r t and c o u l d e a s i l y be missed i n a r i g i d , s i n g l e p o i n t s a m p l i n g s c h e d u l e . T h i s i s a l s o t r u e f o r i n t r a c e l l u l a r p r o r o c e n t r i n , a l t h o u g h the d i f f e r e n c e s are l e s s d r a m a t i c due t o a lower a b s o l u t e c o n c e n t r a t i o n f o r c e l l u l a r p r o r o c e n t r i n . The low i n t e r n a l p r o r o c e n t r i n c o n c e n t r a t i o n compared t o the l a r g e amount of e x t r a c e l l u l a r s i d e r o p h o r e produced, i s e v i d e n c e f o r the de novo s y n t h e s i s of p r o r o c e n t r i n i m m e d i a t e l y p r i o r t o r e l e a s e . T h i s r a p i d f o r m a t i o n of p r o r o c e n t r i n a f t e r the onset of i r o n s t r e s s i n b a t c h c u l t u r e i s s i m i l a r t o the de novo s y n t h e s i s of the 3 - d i k e t o n e , 1 - ( 2 , 6 , 6 - t r i m e t h y 1 - 4 -h y d r o x y c y c l o h e x e n y 1 ) - 1,3-butanedione, a unique e x t r a c e l l u l a r m e t a b o l i t e e x c r e t e d by P. min imum ( T r i c k e_t a_l. , 1981). In the case of the g - d i k e t o n e , p r o d u c t i o n o c c u r r e d i n a narrow time p e r i o d i n the s t a t i o n a r y growth phase. Maximum g-diketone c o n c e n t r a t i o n s were r a p i d l y a c h i e v e d but they were m a i n t a i n e d a t maximal l e v e l s . There was no i n d i c a t i o n of r e a b s o r p t i o n or breakdown. In the case of p r o r o c e n t r i n , maximum e x t r a c e l l u l a r c o n c e n t r a t i o n was e x h i b i t e d f o r o n l y a s h o r t p e r i o d of time ( l e s s than one d a y ) . The r e d u c t i o n i n e x t r a c e l l u l a r c o n c e n t r a t i o n was as r a p i d as the i n i t i a l p r o d u c t i o n , w i t h almost no Csaky p o s i t i v e m a t e r i a l r e m a i n i n g i n the medium or i n the c e l l s two days a f t e r maximum e x t r a c e l l u l a r c o n c e n t r a t i o n . The l o s s of e x t r a c e l l u l a r p r o r o c e n t r i n w i t h o u t a s i m u l t a n e o u s i n c r e a s e i n c e l l u l a r c o n c e n t r a t i o n s u g gests a p o s s i b l e mechanism of i r o n a c q u i s i t i o n by P. minimum. I t i s h y p o t h e s i z e d t h a t the h i g h , a f f i n i t y i r o n t r a n s p o r t system i n P. minimum f u n c t i o n s as f o l l o w s . The e x t r a c e l l u l a r p r o r o c e n t r i n 125 forms the i r o n - p r o r o c e n t r i n complex which i s then taken up by the c e l l s . The i r o n i s removed and a modified p r o r o c e n t r i n by-product, not r e a c t i v e to the Csaky t e s t , i s produced. The by-product i s e i t h e r maintained i n t r a c e l l u l a r l y or r e l e a s e d back i n t o the medium. The f r e e i r o n i s u t i l i z e d i n the b i o s y n t h e s i s of c h l o r o p h y l l r e s u l t i n g in the corresponding i n c r e a s e i n f l u o r e s c e n c e . A s i m i l a r mechanism of i r o n a c q u i s i t i o n (termed the "American approach") has been shown in organisms producing the c a t e c h o l - t y p e siderophores (Bezkorovainy, 1980). The mechanism i s unique to e u k a r y o t i c microorganisms. F u r t h e r s t u d i e s of the mechanism of i r o n a q u i s i t i o n are necessary. I t i s c l e a r , however, that the mechanisms f o r hydroxamate-type i r o n t r a n s p o r t f o r fungi and molds (termed the "European approach") which i n v o l v e s the r e l e a s e of the i n t a c t , Csaky p o s i t i v e siderophore back i n t o the medium (Raymond and Carrano, 1979; Bezkoravainy,1980) c o u l d not account f o r the r a p i d disappearance of p r o r o c e n t r i n . The s t i m u l a t i o n of c e l l f l u o r e s c e n c e a f t e r the disappearance of e x t r a c e l l u l a r p r o r o c e n t r i n i s an i n t e r e s t i n g f e a t u r e . There have been many r e p o r t s s u g g e s t i n g that c h o l o r o p h y l l a s y n t h e s i s i s governed by the supply of i r o n (Myers 1947; Hayward, 1968; Glover, 1978). Davies (1970) suggested that i t i s the presence of c h e l a t e d i r o n which s t i m u l a t e d c h l o r o p h y l l p r o d u c t i o n . The strong c o r r e l a t i o n between pr o d u c t i o n of Csaky p o s i t i v e compounds and s t i m u l a t e d f l u o r e s c e n c e may be of use as a s c r e e n i n g procedure for siderophore producing phytoplankton. T h i s procedure c o u l d 126 perhaps dete c t siderophore p r o d u c t i o n by c e l l s which produced siderophore d u r i n g the s t a t i o n a r y phase of growth, but i t would not be s u i t a b l e f o r d e t e c t i n g siderophore p r o d u c t i o n d u r i n g e x p o n e n t i a l growth. Fur t h e r work i s r e q u i r e d to c o n f i r m the r e l a t i o n s h i p between siderophore p r o d u c t i o n and c e l l u l a r f l u o r e s c e n c e . Our data c h a r a c t e r i z e the production of p r o r o c e n t r i n by the marine d i n o f l a g e l l a t e , Prorocentrum minimum. Two other s p e c i e s P. mar i a e - l e b o u r iae and grac i l e , although l e s s thoroughly s t u d i e d , showed s i m i l a r p a t t e r n s of p r o d u c t i o n . Our i n a b i l i t y to s t i m u l a t e p r o r o c e n t r i n p r o d u c t i o n i n the two other Prorocentrum s p e c i e s may be due to our i n a b i l i t y to c r e a t e s u i t a b l e c o n d i t i o n s for i r o n - l i m i t e d growth. The v e r i f i c a t i o n of an a c t i v e , high a f f i n i t y , i r o n t r a n s p o r t system in marine d i n o f l a g e l l a t e s i s an important step in our understanding of t r a c e metal c o n t r o l of phytoplankton growth. If these compounds are s p e c i e s s p e c i f i c , then the e c o l o g i c a l advantage of p r o r o c e n t r i n p r o d u c t i o n under i r o n -s t r e s s i s immediately e v i d e n t . However, the r o l e of a siderophore as a f a c t o r c o n t r o l l i n g i n t e r s p e c i e s competition i s more complicated. C e r t a i n a l g a l e x c r e t i o n products, of which p r o r o c e n t r i n i s one example, form complexes with other metal ions at low c o n c e n t r a t i o n s . T h i s w i l l serve e i t h e r to d e t o x i f y or i n c r e a s e the a v a i l a b i l i t y the metals (Degens, 1970; Barber, 1973; Smith, 1976). T h i s most c e r t a i n l y i s the case for hydroxamate-type s i d e r o p h o r e s . Compounds l i k e p r o r o c e n t r i n , while h i g h l y s e l e c t i v e f o r i r o n , a l s o form s t a b l e complexes with 127 C u ( l l ) (Anderegg et a l . , 1963). The a b i l i t y to d e t o x i f y copper would most c e r t a i n l y be. important in areas where exchangeable i r o n i s very low. 1 28 VI I I . EXTRACELLULAR HYDROXAMATE-SIDEROPHORE PRODUCTION BY  NERITIC EUKARYOTIC MARINE PHYTOPLANKTON. A. ABSTRACT The production of hydroxamate-type siderophores by eu k a r y o t i c marine n e r i t i c phytoplankton i s presented. Four s p e c i e s were i n v e s t i g a t e d for siderophore p r o d u c t i o n under i r o n -s u f f i c i e n t and i r o n - l i m i t i n g c u l t u r e c o n d i t i o n s . T h a l a s s i o s i r a  pseudonana and D u n a l i e l l a t e r t i o l e c t a produced e x t r a c e l l u l a r siderophores under i r o n - l i m i t i n g c u l t u r e c o n d i t i o n s . There was no siderophore production in two s p e c i e s , O l i s t h o d i s c u s l u t e u s and Skeletonema costatum. • An inc r e a s e i n i n t r a c e l l u l a r siderophore c o n c e n t r a t i o n was not observed. In sp e c i e s which produced siderophores, there was an inc r e a s e in the i_n v i v o f l u o r e s c e n c e a s s o c i a t e d with the disappearance of the e x t r a c e l l u l a r siderophore from the c u l t u r e medium. While the extent of siderophore p r o d u c t i o n v a r i e d between these two s p e c i e s , the p a t t e r n of pr o d u c t i o n and the a s s o c i a t e d r a p i d i n c r e a s e i n in_ v i v o f l u o r e s c e n c e i n d i c a t e that the i r o n -a c q u i s i t i o n system i s s i m i l a r to the system in Prorocentrum minimum. 129 B. INTRODUCTION Iron i s an e s s e n t i a l m i c r o n u t r i e n t f o r a l l phytoplankton. In marine waters, i r o n may l i m i t the growth of phytoplankton, s i n c e most of the i r o n i s in a b i o l o g i c a l l y u n a v a i l a b l e form, e i t h e r a s s o c i a t e d i n l a r g e organic complexes (Sugimura et a l . , 1978) or as almost t o t a l l y i n s o l u b l e f e r r i c hydroxide aggregrates. Under i r o n l i m i t i n g growth c o n d i t i o n s most microorganisms u t i l i z e a high a f f i n i t y i r o n a c q u i s i t i o n system. The mechanism of i r o n a c q u i s i t i o n i n v o l v e s the pro d u c t i o n of i r o n ( 1 1 1 ) - s p e c i f i c e x t r a c e l l u l a r o r g a n i c s (siderophores) to scavenge i r o n at low c o n c e n t r a t i o n s . The i r o n - s i d e r o p h o r e complex i s a c t i v e l y taken up by the c e l l . The i r o n i s r e l e a s e d and u t i l i z e d i n c e l l u l a r metabolism (Neilands, 1974). I t has been suggested that a l l a e r o b i c microorganisms can produce siderophores (Neilands, 1974), yet co n t r o v e r s y surrounds the e x i s t e n c e of siderophore p r o d u c t i o n by marine e u k a r y o t i c phytoplankton. Marine b a c t e r i a (Goyne and Carpenter,1974) and marine cyanobacter i a (Estep e_t a_l. , 1975; Armstrong and Van Baalen, 1979) can produce s i d e r o p h o r e s . Murphy et §_1.,(1976) suggested that hydroxamate siderophores produced by f r e s h water c y a n o b a c t e r i a enabled them to out compete a s s o c i a t e d e u k a r y o t i c phytoplankton, which were unable to produce a siderophore. However, there e x i s t s some evidence f o r the pro d u c t i o n of siderophores by e u k a r y o t i c phytoplankton. Spencer et. a l . (1973) i s o l a t e d a hydroxamate-1ike compound from a non-axenic c u l t u r e of the marine diatom, Chaetoceros s o c i a l i s . C r i t i c s 130 have suggested that the siderophore may have o r i g i n a t e d from the b a c t e r i a , r a t h e r than the diatom. S i m i l a r l y , Armstrong and Van Baalen (1979) found hydroxamate-type compounds produced by the marine diatom, C y l i n d r o t h e c a sp. T h i s m a t e r i a l was not c h a r a c t e r i z e d f u r t h e r . Other r e s e a r c h e r s have claimed that axenic e u k a r y o t i c phytoplankton, u n l i k e p r o k a r y o t i c phytoplankton, are unable to produce siderophores (Swallow et a_l. , 1978; McKnight and Morel, 1979). Since, the production of low molecular weight i r o n ( I I I ) c h e l a t o r s was c o n s i d e r e d u n l i k e l y by eu k a r y o t i c phytoplankton, Anderson and Morel (1980) suggested the a c q u i s i t i o n of i r o n (II) as a a l t e r n a t i v e mechanism. A siderophore from the marine d i n o f l a g e l l a t e , Prorocentrum  minimum has r e c e n t l y been i s o l a t e d (Chapter V). T h i s compound, given the t r i v i a l name p r o r o c e n t r i n , had chemical and s p e c t r o s c o p i c c h a r a c t e r i s t i c s t y p i c a l of low molecular weight hydroxamate-containing siderophores and i t was found both i n t r a c e l l u l a r l y and e x t r a c e l l u l a r l y . I r o n - l i m i t e d growth c o n d i t i o n s s t i m u l a t e d the production of e x t r a c e l l u l a r p r o r o c e n t r i n . I t was argued that the i n a b i l i t y of past r e s e a r c h e r s to detect hydroxamate siderophore i n c u l t u r e s of e u k a r y o t i c marine phytoplankton was due t o : 1) The i n a b i l i t y to achieve i r o n -l i m i t e d growth c o n d i t i o n s and 2) the f a i l u r e to concentrate siderophores p r i o r to a n a l y s i s f o r hydroxamate. In t h i s chapter, siderophore production in a few species of e u k a r y o t i c phytoplankton from three major a l g a l c l a s s e s i s 131 examined. I t has been suggested that n e r i t i c phytoplankton have a l a r g e r requirement f o r i r o n than oceanic s p e c i e s (Menzel and Ryther, 1961; Ryther and Kramer, 1961; Johnston, 1964; Lewin and Chen, 1971). In order to f a c i l i t a t e a c h i e v i n g i r o n - l i m i t e d growth c o n d i t i o n s only n e r i t i c s p e c i e s were chosen. C. MATERIALS AND METHODS A l l i s o l a t e s were obtained from the North East P a c i f i c C u l t u r e (NEPCC), U n i v e r s i t y of B r i t i s h Columbia. Species examined i n c l u d e d Skeletonema costatum (NEPCC # l 8 ( a ) ) , T h a l a s s i o s i r a pseudonana (clone 3H) (NEPCC #58), 0 1 i s t h o d i s c u s  l u t e u s (NEPCC #278) and D u n a l i e l l a t e r t i o l e c t a (NEPCC #1). c u l t u r e s , with the exception of 0. l u t e u s , were axenic (Chapter I I I ) . C e l l s were grown in i r o n - d e f i c i e n t e n r i c h e d , n a t u r a l seawater medium (ESNW-Fe) d e s c r i b e d i n Chapter V. The seawater was t r e a t e d with c h a r c o a l (24h, constant s t i r r i n g ) to remove d i s s o l v e d o r g a n i c s . To reduce the b i o l o g i c a l l y a v a i l a b l e i r o n , the c h a r c o a l t r e a t e d seawater was r e p e a t e d l y heated and f i l t e r e d by the procedure of Lewin and Chen (1971) p r i o r to enrichment. C e l l s were c u l t u r e d in 6 L or 10 L f l a s k s with constant s t i r r i n g (60 rpm) and were incubated at 18 *C under constant d a y l i g h t f l u o r e s c e n t lamps p r o v i d i n g 120 yE-m .s . C e l l growth was monitored by in v i v o f l u o r e s c e n c e using a Turner Designs Model 10 fluorometer. C e l l s were harvested by f i l t e r i n g 1 L of c u l t u r e through g l a s s f i b e r f i l t e r s (GF/C). The remaining c e l l s were removed by passing the f i l t r a t e through 0.45 ym membrane f i l t e r s . F i l t r a t i o n pressure was kept below 250 mm Hg vacuum. 1 32 D i s s o l v e d o r g a n i c s were c o l l e c t e d from the c e l l - f r e e f i l t r a t e by the procedure o u t l i n e d i n Chapter 7 and b r i e f l y o u t l i n e d as f o l l o w s . The f i l t r a t e was a c i d i f i e d to pH 2.0 with c o n c e n t r a t e d HC1 and passed over a 25 X 2 cm column of XAD-2 r e s i n . The column was d e s a l t e d with 150 ml of d e i o n i z e d , d i s t i l l e d water and the absorbed o r g a n i c s were removed from the column with 300 ml of MeOH. The MeOH f r a c t i o n was reduced to dryness by r o t a r y e v a p o r a t i o n . Organics were p a r t i t i o n e d between CHC1 3 a n d H 20. To t e s t f o r siderophores, a mo d i f i e d Csaky t e s t ( G i l l a m , et a l . , 1981) was employed on the water s o l u b l e organic f r a c t i o n . Thorough t e s t i n g has shown that the Csaky t e s t i s d i a g n o s t i c f o r hydroxamate-type sid e r o p h o r e s , as opposed to c a t e c h o l - t y p e s i d e r o p h o r e s . D. RESULTS Growth r e s u l t s are presented i n F i g s . 24-27. For two of the four s p e c i e s , there was l i t t l e d i f f e r e n c e between the f l u o r e s c e n c e of the c e l l s growing i n i r o n - s u f f i c i e n t medium and i r o n - d e f i c i e n t medium. Skeletonema costatum ( F i g . 24) and 0. l u t e u s ( F i g . 25) showed ex p o n e n t i a l i n c r e a s e s in f l u o r e s c e n c e and a p l a t e a u of f l u o r e s c e n c e a f t e r i r o n l i m i t a t i o n at day 6 and day 5, r e s p e c t i v e l y . The two other s p e c i e s , T. pseudonana ( F i g . 26) and D. t e r t i o l e c t a ( F i g . 27), showed f l u o r e s c e n c e p a t t e r n s of i r o n -d e f i c i e n t c u l t u r e s which d i f f e r e d s i g n i f i c a n t l y from the inc r e a s e in f l u o r e s c e n c e i n i r o n - s u f f i c i e n t medium. When i r o n 1 33 F i g u r e 24 - Inf l u e n c e of i r o n on the growth of S. costatum. Comparison of growth and the production of e x t r a c e l l u l a r hydroxamate siderophores of S. costatum grown i n batch c u l t u r e . C e l l s were grown i n : a) i r o n - s u f f i c i e n t , or b) i r o n - d e f i c i e n t media. Days 1 34 F i g u r e 25 - Inf l u e n c e of i r o n on the growth of 0. l u t e u s . 'Comparison of growth and the p r o d u c t i o n of e x t r a c e l l u l a r hydroxamate siderophores of 0. l u t e u s grown i n batch c u l t u r e . C e l l s were grown i n : a) i r o n - s u f f i c i e n t , or b) i r o n - d e f i c i e n t media. 1 35 F i g u r e 26 - Inf l u e n c e of i r o n on the growth o f T. pseudonana. Comparison of growth and the pr o d u c t i o n of e x t r a c e l l u l a r hydroxamate siderophores of T. pseudonana grown in batch c u l t u r e . C e l l s were grown i n : a) i r o n - s u f f i c i e n t , or b) i r o n -d e f i c i e n t media. 30-r Days 136 F i g u r e 27 - Inf l u e n c e of i r o n on the growth of D. t e r t i o l e c t a . Comparison of growth and the production, of e x t r a c e l l u l a r hydroxamate siderophores of D. t e r t i o l e c t a grown in batch c u l t u r e . C e l l s were grown in:- a) i r o n - s u f f i c i e n t , or b) i r o n -d e f i c i e n t media. 30-r Days 1 37 was s u f f i c i e n t both s p e c i e s e x h i b i t e d the t r a d i t i o n a l p a t t e r n of an e x p o n e n t i a l i n c r e a s e i n f l u o r e s c e n c e with time, f o l l o w e d bya p l a t e a u when i r o n l i m i t a t i o n o c c u r r e d . F l u o r e s c e n c e f o r i r o n -l i m i t e d growth was s i m i l a r but e x h i b i t e d a second i n c r e a s e i n f l u o r e s c e n c e about three days l a t e r , probably due to the onset of i r o n l i m i t a t i o n . In the case of T. pseudonana, the second i n c r e a s e i n f l u o r e s c e n c e was s h o r t - l i v e d and f l u o r e s c e n c e decreased w i t h i n the next two days. A p a t t e r n of f l u o r e s c e n c e s i m i l a r to T. pseudonana and D. t e r t i o l e c t a was seen i n i r o n - l i m i t e d c u l t u r e s of P. minimum (Chapter 7). I t was suggested that the r a p i d i n c r e a s e in fl u o r e s c e n c e d u r i n g the s t a t i o n a r y growth p e r i o d may be used as a d i a g n o s t i c f e a t u r e to estimate the temporal p a t t e r n of siderophore p r o d u c t i o n i n batch c u l t u r e , p r o v i d i n g i r o n - l i m i t e d growth was achieved. To i n v e s t i g a t e the l i n k between e x t r a c e l l u l a r hydroxamate-type siderophore p r o d u c t i o n and the r e s u l t i n g i n c r e a s e i n f l u o r e s c e n c e , we analyzed f o r the pr o d u c t i o n of e x t r a c e l l u l a r siderophores by growing the s p e c i e s under i r o n - d e f i c i e n t c o n d i t i o n s . The p a t t e r n s of e x t r a c e l l u l a r hydroxamate-type siderophore f o r the two s p e c i e s which d i d not show the s t i m u l a t i o n of f l u o r e s c e n c e are presented in F i g s . 24 and 25. Neither S. costatum nor 0. l u t e u s produced s i g n i f i c a n t amounts of Csaky p o s i t i v e siderophore. T h a l a s s i o s i r a ( F i g . 26), however, produced a l a r g e amount of e x t r a c e l l u l a r , Csaky p o s i t i v e m a t e r i a l immediately before the in c r e a s e in f l u o r e s c e n c e . D. t e r t i o l e c t a ( F i g . 27) produced l e s s 1 38 e x t r a c e l l u l a r C s a k y - p o s i t i v e m a t e r i a l , than T. pseudonana, but the r e l a t i o n s h i p to the s h i f t i n ini v i v o f l u o r e s c e n c e was the same. There was no i n t r a c e l l u l a r Csaky p o s i t i v e m a t e r i a l produced by any of the s p e c i e s . None of the c u l t u r e s produced s i g n i f i c a n t amounts of Csaky p o s i t i v e siderophore when i r o n was suf f i c i e n t . E. DISCUSSION In Chapter VII i t was suggested that the p a t t e r n of growth measured by rn v i v o f l u o r e s c e n c e , c o u l d be used as a d i a g n o s t i c f e a t u r e to p r e d i c t hydroxamate-type siderophore p r o d u c t i o n . C r i t i c a l examination of T. pseudonana, S. costatum, 0. l u t e u s , and D. t e r t i o l e c t a , combined with previous a n a l y s i s of P. minimum (Chapter V I I ) , strengthens the r e l i a b i l i t y of the p r e d i c t i v e value of t h i s method. Since f l u o r e s c e n c e values r e q u i r e no c o n c e n t r a t i o n or i s o l a t i o n steps, t h i s f e a t u r e should a i d i n l a r g e surveys of siderophore production by e u k a r y o t i c phytoplankton. The i n a b i l i t y of S. costatum and 0. l u t e u s to produce Csaky p o s i t i v e siderophore may be due to our i n a b i l i t y to decrease the a v a i l a b l e i r o n to low enough c o n c e n t r a t i o n s s i g n i f i c a n t l y low to s t i m u l a t e siderophore p r o d u c t i o n . Since a l l of the s p e c i e s were grown in i d e n t i c a l medium, t h i s may suggest that s p e c i e s which d i d not produce siderophores have a low requirement f o r i r o n , r a t her than the s p e c i e s ' i n a b i l i t y to produce siderophore. F u r t h e r examination of these two species i s r e q u i r e d to d i f f e r e n t i a t e between these two processes. 139 The p r o d u c t i o n of a hydroxamate-type siderophore by D. t e r t i o l e c t a and T. pseudonana i s of great i n t e r e s t . McKnight and Morel (1978) were unable to induce the same T h a l a s s i o s i r a c lone to produce s i g n i f i c a n t . q uantities of high a f f i n i t y c h e l a t o r s . They noted that micromolar amounts of a weak c h e l a t i n g l i g a n d was produced. Our r e s u l t s c o n f i r m the n e c e s s i t y to concentrate the o r g a n i c s p r i o r to a n a l y s i s and to induce p r o d u c t i o n by c a r e f u l c o n t r o l of a v a i l a b l e i r o n . The amount of Csaky p o s i t i v e compound produced by T. pseudonana i s in the same range as the maximum values recorded f o r Prorocentrum s p e c i e s (Chapter V I I ) . As d i s c u s s e d in the p r e v i o u s chapter, the a b s o l u t e amount of m a t e r i a l w i l l be a f u n c t i o n of the number of hydroxamate u n i t s per siderophore, the e f f i c i e n c y of o x i d a t i o n d u r i n g the Csaky t e s t and the e f f i c i e n c y of recovery from the medium. Thus, l i t t l e s i g n i f i c a n c e can be attached to the absolute amount recorded f o r T. pseudonana. The nature of the u n c e r t a n t i e s in our estimate i s such that siderophore c o n c e n t r a t i o n s would be underestimates. The p a t t e r n of production of Csaky p o s i t i v e compounds by T. pseudonana and D. t e r t i o l e c t a are i d e n t i c a l in nature to the p a t t e r n of p r o r o c e n t r i n (the hydroxamate-type siderophore) p r o d u c t i o n by P. minimum. The mechanism for i r o n a c q u i s i t i o n i s s i m i l a r to that d i s c u s s e d f o r P. min imum (Chapter V I I ) . T h i s mechanism i n v o l v e s de novo s y n t h e s i s of e x t r a c e l l u l a r siderophore a f t e r the onset of i r o n l i m i t a t i o n . The residence time of t h i s e x t r a c e l l u l a r m a t e r i a l i s short because the Csaky r e a c t i v e m a t e r i a l i s r a p i d l y removed from the medium. 1 40 S e v e r a l c o n c l u s i o n s are c l e a r . The p r o d u c t i o n of the e x t r a c e l l u l a r hydroxamate-type compounds by T. pseudonana and D. t e r t i o l e c t a v e r i f i e s that siderophore p r o d u c t i o n i s not unique to d i n o f l a g e l l a t e s (or to Prorocentrum s p e c i e s ) . Phytoplankton from three major a l g a l c l a s s e s , B a c i l l a r i o p h y c e a e , Chlorophyceae, and Dinophyceae, can be induced to produce s i m i l a r , but not n e c e s s a r i l y c h e m i c a l l y i d e n t i c a l , i r o n ( I I I ) c h e l a t o r s . Newly a c q u i r e d i r o n may s t i m u l a t e in v i v o f l u o r e s c e n c e by removing the i r o n - i n h i b i t i o n of c h l o r o p h y l l b i o s y n t h e s i s . Iron d e f i c i e n c y i n h i b i t s the a c t i v i t y of a - a m i n o - l e v u l i n i c a c i d synthetase, the f i r s t enzyme of the heme b i o s y n t h e t i c pathway (Li g h t and Clegg, 1974). Iron from the t r a n s p o r t e d i r o n - s i d e r o p h o r e complex c o u l d remove the i n h i b i t i o n , i n i t i a t i n g de novo c h l o r o p h y l l b i o s y n t h e s i s . Since i r o n a c t s at the enzyme l e v e l of b i o s y n t h e s i s , only a small i n c r e a s e i n a v a i l a b l e i r o n would be necessary to s i g n i f i c a n t l y i n c r e a s e i_n v i v o f l u o r e s c e n c e . The c a t a l y t i c s t i m u l a t i o n of f l u o r e s c e n c e appears to be a r e l i a b l e i n d i c a t o r of temporal p a t t e r n s of siderophore p r o d u c t i o n under the i r o n - d e f i c i e n t batch c u l t u r e c o n d i t i o n s . I n i t i a l s c r e e n i n g by f l u o r e s c e n c e reduces the time-consuming i s o l a t i o n of the siderophore c o n t a i n i n g water f r a c t i o n and enables the time of production to be p r e d i c t e d under d e f i n e d growth c o n d i t i o n s . Fluorescence p a t t e r n s corresponded to siderophore p r o d u c t i o n i n P. minimum , T. pseudonana and D. t e r t i o l e c t a . The use of other growth parameters, such as 141 c e l l c o n c e n t r a t i o n s , does not provide the s e n s i t i v i t y of the f l u o r e s c e n c e technique. Two s p e c i e s f a i l e d to produce siderophore i n our experiments. The lack of s i g n i f i c a n t amounts of e x t r a c e l l u l a r Csaky p o s i t i v e compounds was v e r i f i e d f o r both 0. l u t e u s and S. costatum. 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Aquat. S c i . 38:864-867. 190. T y l e r , M.A. and H.H. S e l i g e r . 1978. Annual subsurface t r a n s p o r t of a red t i d e d i n o f l a g e l l a t e to i t s bloom area: water c i r c u l a t i o n p a t t e r n s and organism d i s t r i b u t i o n i n Chesapeake Bay. Limnol. Oceanogr. 23:227-246. 191. Uchida, T. 1977. E x c r e t i o n of a d i a t o m - i n h i b i t o r y substance by Prorocentrum micans Ehrenberg. Jpn. J . E c o l . 27:1-4. 192. V a l i e l a , I., L. Koumjian, T. Swain, J.M. T e a l and J.E. Hobbie. 1979. Cinnamic a c i d i n h i b i t i o n of d e t r i t u s f e e d i n g . Nature 280:55-57. 193. Warren, R.A.J, and J.B. N e i l a n d s . 1964. M i c r o b i a l degradation of ferrichrome compounds. J . Gen. M i c r o b i o l . 35:459-470. 194. Weinberg, E.D. 1970. B i o s y n t h e s i s of secondary m e t a b o l i t e s : r o l e s of t r a c e metals. Adv. M i c r o b i a l . P h y s i o l . 4:1-44. 195. Wilson, D.P. 1951. 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M i c r o b i o l . 7:119-124. 158 APPENDIX A - ANTIBACTERIAL PROPERTIES OF THE g-DIKETONE USING MARINE BIOASSAY SPECIES A. I n t r o d u c t i o n To e s t a b l i s h the g-diketone as a b i o l o g i c a l l y important metabolite,, the a n t i b a c t e r i a l nature must be examined. P r e l i m i n a r y o b s e r v a t i o n s i n d i c a t e d that the g-diketone was i n h i b i t o r y to Staphylococcus aureus (Andersen e_t aJ.. , 1980). Since one of the c r i t i c i s m s a g a i n s t previous r e s e a r c h (see Chapter 1) was the use of bioassay s p e c i e s from h a b i t a t s that were widely d i f f e r e n t than the m e t a b o l i t e - p r o d u c i n g s p e c i e s , the a n t i b a c t e r i a l nature of the g -diketone a g a i n s t marine b a c t e r i a was i n v e s t i g a t e d . b. M a t e r i a l s and Methods Four marine species were chosen as bioassay organisms. Two marine s p e c i e s , Pseudomonas ac i d i v a r i s and V i b r i o sp. were obtained from the American Type C u l t u r e C o l l e c t i o n (ATCC). The other two s p e c i e s , Flavobacterium sp. and a Chromobacterium sp., were i s o l a t e d from S c r i p p s i e l l a sweeneya c u l t u r e s . I n i t i a l taxonomic i d e n t i f i c a t i o n i s t e n t a t i v e , based e n t i r e l y on colony shape and c o l o r . B a c t e r i a l c u l t u r e s were maintained at 18°C i n l i q u i d broth c u l t u r e s (ISOL medium; B e l l and M i t c h e l l , 1972). Growth was monitored by r e c o r d i n g changes in absorbance of the c u l t u r e at 750 nm. I n i t i a l experiments were s i m i l a r to those of Chan et a l . (1980). ISOL medium with 1.5% agar was made i n t o p l a t e s and i n o c u l a t e d with a lawn of the bioassay s p e c i e s . A s i n g l e 1 cm g l a s s f i b e r f i l t e r d i s c , which had been i n o c u l a t e d with 100 yg of the g-diketone and a u t o c l a v e d , was placed in the center of the p l a t e . P l a t e s were incubated at 18 C and examined a f t e r 24 and 48 hours. A f t e r 48 hours the s i z e of the zone of i n h i b i t i o n , which i s seen as a c l e a r zone around the d i s c , was recorded. To examine the i n f l u e n c e of known c o n c e n t r a t i o n s of the g-diketone on growth of Chromobacteriurn, l i q u i d ISOL medium c u l t u r e s were employed. The h e t e r o t r o p h i c a c t i v i t y of n a t u r a l , b a c t e r i a l p o p u l a t i o n s was determined as f o l l o w s . A subsurface sample was c o l l e c t e d from two s t a t i o n s from the F r a s e r River plume in the S t r a i t of Georgia, o f f Vancouver, B.C. (March 18, 1982). The sample was separated i n t o 50 ml a l i q u o t s and d i f f e r e n t amounts of g-diketone were added. Two ml of l h C-glucose (sp. act.= 1 59 4.43) was added and a l l samples were p l a c e d i n running s u r f a c e water (7.5 C). A f t e r one hour of i n c u b a t i o n i n the dark, samples were f i l t e r e d through 0.22 ym Nucleopore f i l t e r s . The f i l t e r s were washed with 10 ml f i l t e r e d (0.22 urn) seawater, and p l a c e d i n s c i n t i l l a t i o n f l u o r . Counts were taken a f t e r 40 hours using a Unilux III s c i n t i l l a t i o n counter. Since b a c t e r i a l counts were not taken, r e s u l t s are given as the percent i n h i b i t i o n compared to the c o n t r o l (no added 3 - d i k e t o n e ) . C. R e s u l t s A l l b a c t e r i a formed smooth, uniform lawns w i t h i n 48 hours of i n o c u l a t i o n . The i n f l u e n c e of d i s c s impregnated with 100 p g g-diketone on b a c t e r i a l growth i s presented i n Table 12. One marine i s o l a t e ( Pseudomonas '* a c i d i v a r i s ) was i n s e n s i t i v e to g-diketone. Chromobacterium sp. was the most s e n s i t i v e based on the s i z e of the zone of i n h i b i t i o n . Since the d i s c bioassay i s not a r e l i a b l e p r e d i c t o r of the e f f e c t i v e c o n c e n t r a t i o n of i n h i b i t i o n , Chromatobactium sp. was grown in l i q u i d c u l t u r e with g-diketone c o n c e n t r a t i o n s ranging from 0 to 100 yg L 1 . At g-diketone c o n c e n t r a t i o n s g r e a t e r than 25 yg L l, c e l l y i e l d was i n h i b i t e d but the growth r a t e was u n a f f e c t e d ( F i g . 28). Complete s u p r e s s i o n of growth was not recorded i n t h i s range of g-diketone c o n c e n t r a t i o n s . Growth was monitored fo r 14 days to ensure that the b a c t e r i a c o u l d not adapt to the g-diketone c o n c e n t r a t i o n . The i n f l u e n c e of i n c r e a s i n g g-diketone c o n c e n t r a t i o n s on the h e t e r o t r o p h i c uptake of glucose i s presented i n F i g . 29. For a 50% i n h i b i t i o n of the short-term h e t e r o t r o p h i c uptake, 100 yg L 1 g-diketone was r e q u i r e d . However, c o n c e n t r a t i o n s as low as 20 yg L 1 i n h i b i t e d the h e t e r o t r o p h i c uptake (10% r e d u c t i o n ) and at g-diketone c o n c e n t r a t i o n s (50 y g L 1 ) t y p i c a l l y produced by l a b o r a t o r y c u l t u r e s of P. minimum, h e t e r o t r o p h i c uptake was i n h i b i t e d by about 15%. The g-diketone c o n c e n t r a t i o n s from l a b o r a t o r y c u l t u r e s would overestimate the a c t u a l c o n c e n t r a t i o n of e x t r a c e l l u l a r g-diketone produced because of the l a r g e d i s c r e p a n c i e s in c e l l c o n c e n t r a t i o n s . These p r e l i m i n a r y experiments have e s t a b l i s h e d the g -diketone as an a n t i b i o t i c m e t a b o l i t e a c t i v e a g a i n s t marine b a c t e r i a . The i n h i b i t i o n of growth of three of four marine i s o l a t e s and the i n h i b i t i o n of the h e t e r o t r o p h i c uptake of glucose i s s u f f i c i e n t evidence to v e r i f y the a n t i b a c t e r i a l p r o p e r t i e s of the 3-diketone. 160 Table XII - D e s c r i p t i o n and o r i g i n of b a c t e r i a l s p e c i e s examined. Q u a l i t a t i v e r e s u l t s of the d i s c bioassay i n terms of the s i z e the zone of i n h i b i t i o n (0-no i n h i b i t i o n ; +-<0.5 cm; ++->0.5 cm ATCC-American Type C u l t u r e C o l l e c t i o n . Size of zone of Species Origin Inhibition Pseudomonas acidivaris ATCC # 9355 0. Vibrio sp. ATCC # 14400 + Flavobacter sp. Scrippsiella sweeneya culture ++ Chromobacterium sp. Scrippsiella sweeneya culture ++ 161 F i g u r e 28 - Growth c h a r a c t e r i s t i c s of Chromobacterium sp. Growth was measured by changes i n absorbance at 750 nm, i n ISOL medium with v a r y i n g c o n c e n t r a t i o n s of the g-diketone. ( C o n t r o l ; 10 u g - L ' ^ o ; 25 u g - L ^ x ; 40 u g - L " 1 ^ ; 75 ug-L" 1 =A) . T 1 I -J— 1 1 10 20 30 40 50 6 0 Hours 162 F i g u r e 29 - The e f f e c t of the g-diketone on the h e t e r o t r o p h i c uptake of 1 I + C - g l u c o s e by a n a t u r a l p o p u l a t i o n . B a c t e r i a were c o l l e c t e d from the F r a s e r River plume, S t r a i t of Georgia, Vancouver, Canada. Arrow i n d i c a t e s the r e l a t i v e amount of the g-diketone produced by l a b o r a t o r y c u l t u r e s . 

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