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UBC Theses and Dissertations

Consequences of bacillus subtilis in iron deficiency 1968

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CONSEQUENCES OF BACILLUS .SUBTILIS IN IRON DEFICIENCY by WALTER JOSEPH PETERS .B.S.A. (Microbiology), U n i v e r s i t y of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL . FULFILMENT OF . THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY .We accept t h i s t h e s i s as.conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1968 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 o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Depar tment o r by hi.is r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada ABSTRACT Cultures of Bacillus sub t i l l s growing in an iron-deficient medium produced coproporphyria III (coproporphyrin) and phenolic acids. (2,3-dihydroxybenzoylglycine (DHBG), 2,3-dihydroxybenzoie acid (DHB) , or "both). (DHB(G) refers to DHB or DHBG, or both compounds). Phenolic acid production was proportional to the amount of iron present, and occurred logarithmically, parallelling growth. In the presence of DHB, lower levels of iron inhibited phenolic acid production, so that the actual inhibition of synthesis may involve the FQ J ; ( D H B ( G ) ) 2 complex. Acoimulatlbn of DHB(G) was influenced by the levels of aromatic amino acids, anthranilic acid, and histidine in the medium. In vitro experiments demonstrated that DHB was formed from chorismic acid. In vivo and in vitro experiments with strain B - 1 V 7 1 showed that DHB was coupled to added glycine to form DHBG. Disappearance of DHB(G) was observed in a l l strains studied, but oxidation did not occur. Phenolic acid production always preceded coproporphyrin production. Phenolic acids have very strong affinities for ferric iron. Their production may therefore allow the scavenging of the last traces of iron from the medium for hemin synthesis. The relationship between phenolic acid and coproporphyrin production was borne out by the following observations: (i) a higher level of iron was required to prevent coproporphyrin production than phenolic acid production (ii) the Fe"3 (DHB(G))^ complex was a more potent inhibitor of coproporphyrin production than iron alone ( i i i ) a mutant blocked at £-aminolevulinic acid synthetase did not produce phenolic acids during iron-deficient growth (iv) serine auxotrophs produced much lower levels of copro- porphyrin and phenolic acids than the wild-type strain (v) some mutants defective in phenolic acid production produced low levels of coproporphyrin, whereas one strain of this type produced elevated levels of coproporphyrin. Compounds known to inhibit normal functioning of the tricarboxylic acid cycle decreased coproporphyrin production in a l l strains studied. These inhibitors reduced DHBG excretion, but had no effect on DHB production. A number of analogs of DHB inhibited DHB(G) accumu-. lation to varying degrees, depending upon their structure. The most potent inhibitors were m-substituted derivatives of benzoic acid. Two sideramines, ferrichrome and ferrioxamine, inhibited DHBG,.production in strain B-1471. The inhibitory action of f e r r i - chrome was shown to be due to its ability to mediate cellular uptake of low levels of iron. The capacity of B. subtilis for iron uptake was increased about 20-fold by growing the cells in an iron-deficient medium. Under these conditions, the addition df low levels of phenolic acids increased both the rate and extent of iron uptake. Mutants unable to synthesize normal levels of phenolic.acid were shown to have a reduced capacity for iron uptake after growth in an iron-deficient medium. Mutants resistant to 8-hydroxyquinoline had an increased capacity for iron-uptake under these conditions. i i i TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS 5 ;I. B a c t e r i a l s t r a i n s 5 . 1. Wild type s t r a i n s . . . . . . . . . . . . . . . 5 2. Mutant s t r a i n s . . . . . . . . . . . . . . . . . 5 (a) Spontaneous mutants . . . . . . . . . . 5 (b) NTG-induced mutants . . . . . . . ' 5 I I . M e d i a . . . . . . . . . . . . 9 I I I . C u l t u r a l conditions. ; . . : . . . . . . . 10 IV. D e t e r m i n a t i o n s . . . . . . . . . . 10 .1.' Growth. . . . . . . . . . . . . . . . . . . . . . 10 2. Phenolic acids .. . . .. .. .. .. .. . 11 3. Coproporphyrin I I I . . . . . . . . 11 •;k. I d e n t i f i c a t i o n of g l y c i n e . . . . . . . 12 5 . Protein concentration . . . . . . . . 12 V. Preparation of extracts'. 12 : VI. Enzyme assays. . . . . . . . . . . . . . . . . . . . 13 . 1. Synthesis of DHB.. .. .; .. .. .. .. .. . 13 .2. Synthesis of DHBG . 13 . VII. Respiration. studies. . . . . . . . . . . . . . . • 13 VIII. Iron transport.studies. . . . . . . .: . 13 . 1. Preparation of c e l l s 13 2. Iron uptake . . . . . . . . . . . . . 14 i v Table of Contents (Continued) Page (a) I r o n - d e f i c i e n t c e l l s . . . lh (b.) Iron-suff i c i e n t . c e l l s . . . . . . . lh 3. Assay of i r o n uptake . . . . . . . . . . . lh IX. Chemicals . . . . . . . . . . . . . 15 RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . 17 Section I: General properties of phenolic a c i d excretion. . . . . . . . . . . . . 17 : .1. Excretion of phenolic acids by wi l d type s t r a i n s . . ; . . . . . . . . . . . . . 17 2. Influence of i r o n on DHBG production. . . . . . . 17 .3 . Fe :(DHBG)^ complex and the co n t r o l of DHBG production.. .. . .. .. .; .. .. .. . 20 h. E f f e c t of aromatic amino acids on DHBG production. . . . . . . . . . . . . . . . . 20. 5. Source of glycine i n DHBG. . . . . . . . . . . . . - 2k ,6. Variations among st r a i n s of B. ' s u h t i l i s . . . . . 27 . 7. E f f e c t of h i s t i d i n e on phenolic a c i d excretion. . . . . . . . . . . . . . . . . . . . . . . 31 .8. In v i t r o syntheses. 31 (a) Formation of DHB . . . . . . . . . . . . . . . . 31 (b) Formation of DHBG. . . . . . . . . . . . . . . . 31 ,9. Metabolism of DHB(G) b y . s t r a i n B-1U71.. . . . 35 1 0 . Metabolism of DHB(G) by s t r a i n s W-23 and WB-746 . 35 V Table of Contents (Continued) Page 11.. Properties of mutant s t r a i n s . 37 (a) 5 methyl tryptophan r e s i s t a n t s t r a i n s .; . .-37 (b) Aromatic auxotrophs ........ 37 (c) HQ r e s i s t a n t mutants . . . . . . . . . . . . . . 37" DISCUSSION . . . . . . . . . . . . . kO Section I I : The production of coproporphyrin and i t s r e l a t i o n s h i p to the production of phenolic acids. • k6 Introduction . . . . ; . . . . . ; . . . . . . . . ; . . . 46 1. Production of.coproporphyrin and phenolic . acids by wild-type.strains. . . . . . . . . . . . . . h6 • 2. I n h i b i t i o n of coproporphyrin production by i r o n . . . . . . . . . . . . . . . . . . . . . . . . . 50 3. I n h i b i t i o n of coproporphyrin production by hemin . . . . 50 k. I n h i b i t i o n of coproporphyrin production by aromatic amino a c i d s 5 2 5. Coproporphyrin and phenolic a c i d production by mutants of s t r a i n B-1471 . . . . . . . 52 (a) Glycine auxotrophs. . ; . . . ; . . . . 52 (b) Serine auxotrophs . . . . . . . . . . . . . . 52 (c) ALA . auxotrophs . . . . . . . . . . . . . . . . . . 5̂ (d) DHB auxotrophs . . . . . . . . . . . . . 58 DISCUSSION 62 v i Table .of Contents (Continued) Page Section I I I : I n h i b i t i o n of phenolic a c i d production i n B_. '. s u b t i l j s . 66 Introduction.; .; . . .. .. .; .. .. .. .. .. . . 66 1. Analogs of DHB. . . . . . . . . . . . . . . . . 66 .2. Compounds a f f e c t i n g the functioning of the TCA cycle . . . . . . . . . . . . . . . . . . 66 3. E f f e c t of sideramines on phenolic a c i d and porphyrin-production'by s t r a i n B-1U71 • . .: . 68 DISCUSSION. • 72 Section.IV: E f f e c t s of aeration and glucose concentration on growth and phenolic a c i d excretion . . . . . . . . . • 7^ Introduction. . . . . . . . . . . . . . . . . . . . jk .1. E f f e c t of aeration on phenolic a c i d production . . . . . . . . : . . ; . . . . : . lh 2. E f f e c t of glucose concentration i n phenolic a c i d excretion. . . . . . . . . . . . . . . 76 3. Oxidation studies. . ; . . . . . ; . . . . . . . 76 D I S C U S S I O N . . . . . . . . . . . . . . . . . . . . . . . 79 Section V: Iron transport and phenolic acids . . . 8 l Introduction. . . . . . . . . . . . . . . . . . . . 8 l .1. Preliminary experiments. . . . . . . . . . . . . 8 l 2. Experiments with i r o n - d e f i c i e n t cultures. Iron transport as a function of culture . . . 8 l .3. Iron transport as a.function of energy. . . . . . 82 . v i i Table .of Contents (Continued) Page h. The e f f e c t of temperature on i r o n transport. . ; . 8 2 5. Incorporation of i r o n i n t o TCA-insoluble material; . ; . . . . . ; . . . . ; . . . . 86 6. Iron transport as a function of i r o n . concentration.; . .; . . . . . . . . . . 8 6 7..The e f f e c t of c i t r a t e on i r o n uptake .; . .. . . 86 8. The e f f e c t of phenolic acids on i r o n U_P*fcc3,lCS • • • • , • • • • • • • • 90 9. Experiments with i r o n ^ s u f f i c i e n t cultures.. . ; . ; . 9h 10. Experiments with mutant s t r a i n s . ; .; . . . .; . 9h DISCUSSION .; . .; .; . . . . . . . . . . . 9h Section VI:.Control of i r o n transport . . . . . . . 99 Introduction . . . . . . . . . . . .. .. . . 99 .1.. E f f e c t of heme-iron requirement on transport co,|p3,cx"by • • • • . • • • • • 99' . 2. E f f e c t of ferrichromc 99 3. HQr mutants . . . . . . . . . . . . . 101 DISCUSSION . .; . . . . . . . . . . . .. . 105 GENERAL DISCUSSION . . . . . . . . .. . . . 107. LITERATURE CITED . . . . . . . . . . . . . . . I l l v i i i LIST OF TABLES Table T i t l e Page I B. s u b t i l i s w i l d type s t r a i n s 6 I I D e s c r i p t i o n o f B. s u b t i l i s mutant s t r a i n 7 - 8 I I I E x c r e t i o n of p h e n o l i c ac ids by w i l d type s t r a i n s " 18 IV D i s t r i b u t i o n o f g l y c i n e - 1 - ^ C 28 V C h a r a c t e r i s t i c s o f p h e n o l i c a c i d e x c r e t i o n by 29 s t r a i n s o f B. s u b t i l i s i n i r o n - d e f i c i e n c y V I E f f e c t o f h i s t i d i n e on p h e n o l i c a c i d p roduc t i on 32 i n w i l d type s t r a i n s V I I I n v i t r o s yn thes i s of DHB 33 V I I I I n v i t r o s yn thes i s of DHBG ' 3*+ IX I n h i b i t i o n of the p r o d u c t i o n o f coproporphyr in 53 and p h e n o l i c ac ids by hemin i n s t r a i n B-lk-71 X P roduc t i on of coproporphyr in and p h e n o l i c ac ids . 55 by se r i ne auxotrophs de r i v ed f rom s t r a i n B-1471 XI Products exc re ted by " r e v e r t a n t s " o f DHB auxo- 59 t rophs de r i v ed f rom s t r a i n B-1471 X I I Coproporphyr in p r o d u c t i o n i n Dhb-4 6 l X I I I E f f e c t s of analogs o f DHB on p h e n o l i c a c i d and 67 coproporphyr in p r o d u c t i o n XIV . E f f e c t o f i n h i b i t o r s a c t i n g a t the l e v e l o f the 69 TCA c y c l e on p h e n o l i c a c i d and coproporphyr in p r o d u c t i o n XV I n h i b i t i o n o f the p r o d u c t i o n o f coproporphyr in 71 by s ideramines i n s t r a i n B - l V f l XVI E f f e c t of a e r a t i o n on growth and p h e n o l i c a c i d 75 p r o d u c t i o n i n w i ld- t ype s t r a i n s LIST OF FIGURES Figure T i t l e 1 Growth and production of DHBG by strain B-1471 19 2 Effect of iron added at zero time on the production 21 of DHBG by strain B-1471 3 Effect on DHBG production of adding iron or DHB to 22 strain B-1471 -4 4 Effect of simultaneous addition of 3-5 x 10 M DHB 23 and 1 mg of iron per l i t r e on DHBG production by strain B-1471 5 Effect of adding end product amino acids on DHBG 25 production by strain B-1471 6 Effect of adding anthranilic acid on the production 26 of DHBG by strain B-1471 7 Effect of iron, added at zero time, on the production 30 of DHB(G) by strains WB-746, B-1471 and W-23 8 Metabolism of DHBG by strain B-1471 after addition 36 of iron 9 Production of DHBG by MTr strains of B-1471 38 10 Scheme of aromatic biosynthesis 42 11 Growth and production of phenolic acids and copro- 47 porphyrin by strain B-1471 12 Growth and production of phenolic acids and copro- 48 porphyrin by strain W-23 13 Growth and production of phenolic acids and copro- 49 porphyrin by strain WB-746 14 Effect on coproporphyrin production of adding iron 51 and DHB(G) 15 Effect of ALA supplementation on coproporphyrin and 57 phenolic acid production by hem-1 16 Growth and coproporphyrin production by dhb-4 60 17 Effect of sideramine supplementation on phenolic 70 acid excretion i n strain B-1471 X LIST OF FIGURES - continued Figure T i t l e Page 18 Growth of strain ¥B-jk6 i n iron-deficient medium 77 containing 0.3% glucose 19 Oxidation capacities of strain WB-746 grown in iron- 78 deficient medium containing 0.3% glucose 20 Iron uptake as a function of physiological age 83 21 Iron uptake as a function of energy 8>k 22 Effect of temperature on iron uptake 85 23 Incorporation of iron into TCA-insoluble material 87 2k Rate of iron uptake as a function of iron concen- 88 tration 25 Binding of iron to cells at 0 C 89 26 Effect of growth without citrate on iron uptake 91 27 Effect of phenolic acids on iron uptake 92 28 Effect of phenolic acids on uptake at lower levels 93 of iron 29 Iron uptake i n mutant strains 95 30 The F e 3 + (DHBG)3 complex 97 31 Strain differences i n iron uptake capacities 100 32 Effect of ferrichrome on iron transport 102 33 Effect of ferrichrome on iron uptake i n strain B-1471 103 3k Iron uptake by strain HQ,r-l 10k ACKNOWLEDGEMENTS To Professor R. A. J. Warren for his unique and enthusiastic approach to sc i e n t i f i c discovery, and for his demonstration of the framework on which science must be bu i l t . To Professor E. W. Rester for allowing me to draw upon his experience and knowledge of Bacillus subtilis. To Professor F. Gibson for providing many of his experimental observations prior to their publication. To W. W. Kay for valuable assistance with the planning and interpretation of transport experiments. To A. M. B. Kropinski whose general knowledge of microbial meta- bolism was continuingly available. To the other co-workers of Room 14 for constant discussion and criticism which, in many cases, led to experimental elucidation. To Mrs. Rosemary Morgan for generously typing this thesis. To Mrs. Rita Rosbergen for overcoming the administrative d i f f i c u l t i e s encountered during the past two weeks. INTRODUCTION An a l t e r a t i o n of metabol ism as a consequence of i r o n d e p r i v a t i o n has been r e p o r t e d f o r a number of m i c r o b i a l spec i e s : I n many i n s t a n c e s , t h i s a l t e r a t i o n i s man i fes ted by the p r o d u c t i o n of h i g h l e v e l s o f f e r r i c i r o n complexing agents ( 4 0 ) . Thus, U s t i l a g o sphaerogena (17) produced the complex t r ihydroxamates , f e r r i ch rome and f e r r i c h r o m a A, wh i l e B a c i l l u s megaterium produced a secondary monohydroxamic a c i d ( " s c h i z o k i n e n " ) (10) under i r o n d e f i c i e n c y f Other organisms have been shown to exc re te p h e n o l i c ac ids when grown i n a medium c o n t a i n i n g l i m i t i n g i r o n . Mycobacter ium smegmatis produced s a l i c y l i c a c i d ; Aerobacter aerogenes and a s t r a i n of E s c h e r i c h i a c o l i produced 2 ,3-d ihydroxybenzo ic a c i d (DHB) ( 5 0 ) ; another s t r a i n of E. c o l i produced 2 ,3-d ihydroxybenzoy l se r ine (DHBS) ( 1 7 ) . E x t r a c t s o f t h i s second s t r a i n of E. c o l i formed DHBS from DHB and s e r i n e , and the l e v e l o f t h i s enzyme (DHBS synthetase) was dependent upon the l e v e l o f i r o n i n the growth medium (17)» B rot and,Goodwin (6) have suggested t ha t i r o n may ac t as a corepressor i n the system c o n t r o l l i n g the s yn thes i s o f DHBS synthe tase . I t o i c a c i d , 2 ,3-d ihydroxybenzoy lg l y c ine (DHBG) was found to be exc re ted by B a c i l l u s s u b t i l i s ( 1 8 ) . Accumula t ion of t h i s compound depends upon the l e v e l o f i r o n i n the medium: c u l t u r e s devo id o f i r o n d i d not grow; h i g h l e v e l s o f i r o n prevented i t s p r o d u c t i o n ( 2 7 ) ; low l e v e l s of i r o n l e a d t o the e x c r e t i o n of more than 200 mg of DHBG per l i t r e o f c u l t u r e f l u i d (40) . Dur ing the pas t few yea r s , a r e l a t i o n s h i p has been demonstrated between pheno l e x c r e t i o n and aromat ic metabol ism. P i t t a r d et a l 2 (47, 48) showed tha t washed c e l l suspensions of a number of aromat ic amino a c i d auxotrophs of A. aerogenes exc re ted o-dihydroxy pheno ls , amongst them DHB, when incubated i n m in ima l medium. In one such s t r a i n , NC3, b l o cked at a n t h r a n i l a t e synthetase , f o rma t ion of o- d ihydroxy phenols was i n h i b i t e d comple te l y by exogenous t r yp tophan . Furthermore, i t has been shown t h a t w h i l e a w i l d type s t r a i n of A. aerogenes produced DHB o n l y under i r o n d e f i c i e n c y , a mutant o f t h i s s t r a i n b l o cked a f t e r a n t h r a n i l a t e synthetase produced DHB r ega rd l e s s of the l e v e l o f i r o n i n the medium (5l)« The p r e c i s e a l t e r a t i o n of aromat ic metabol ism brought about by i r o n d e f i c i e n c y and caus ing the f o rma t i on of p h e n o l i c ac ids i s u n c l e a r . Rat ledge and Winder (52) have suggested t ha t M. smegmatis may s yn thes i ze s a l i c y l i c a c i d f rom s h i k i m i c a c i d by steps analogous t o t h o s e . i n v o l v e d i n a n t h r a n i l i c a c i d f o rma t ion ( 6 0 ) , but w i t h the i n t r o d u c t i o n of a h y d r o x y l group i n s t e a d of an amino group. Under i r o n d e f i c i e n c y , the h y d r o x y l - i n s e r t i o n r e a c t i o n cou ld be accentuated, p o s s i b l y by a requirement f o r i r o n by a competing pathway. Th is sugges t ion was supported by the obse r va t i on t ha t e i t h e r magnesium or i r o n was r e q u i r e d f o r the convers ion of s h i k i m i c acid-5-phosphate t o a n t h r a n i l i c a c i d by an e x t r a c t o f E. c o l i (59)« Work w i t h +2 A. aerogenes, however, has i n d i c a t e d t h a t Fe i ons i n h i b i t an th r an - i l i c a c i d s yn thes i s ( 5 3 ) . I n a d d i t i o n , an i r o n requirement has .no t been shown f o r the a n t h r a n i l a t e synthetase prepared f rom E. c o l i (5, 5 8 ) . Cox and Gibson (12) showed t h a t DHB was a growth f a c t o r f o r c e r t a i n m u l t i p l e auxotrophs of E. c o l i , but o n l y i n i r o n - d e f i c i e n t medium. Young et a l (67) found tha t e x t r a c t s o f E. c o l i were 3 capable of fo rming DHB f rom chor ismate , and t ha t the a c t i v i t y o f these p r epa ra t i ons was dependent upon the l e v e l o f i r o n . I n many microorganisms, growth i n i r o n - d e f i c i e n t media leads to a d i s tu rbance of p o r p h y r i n metabol ism (3l). When U. sphaero- gena was grown i n i r o n - l i m i t i n g medium, no p o r p h y r i n compounds were exc r e t ed . E x t r a c t s prepared from these c e l l s con ta ined l e s s S-amino levu l inate dehydratase a c t i v i t y than e x t r a c t s f rom i r o n s u f f i c i e n t c e l l s (29). I n o ther organisms, there was e x c r e t i o n of h i g h l e v e l s o f a p o r p h y r i n . B. s u b t i l i s (18), Corynebacter ium d i p h t h e r i a e (23) and Mic rococcus l y s o d e i k t i c u s (62) produced coproporphyr in I I I . Furthermore, i t has been r epo r t ed (25) t ha t the p r o d u c t i o n of coproporphyr in I I I by B. s u b t i l i s was always accompanied by the e x c r e t i o n of l a r g e amounts of DHBG. As DHBG has a v e r y s t rong a f f i n i t y f o r f e r r i c i r o n (26) i t has been suggested t h a t t h i s p h e n o l i c a c i d may be exc re ted i n t o the medium to make i r o n a v a i l a b l e t o the c e l l (40 ) . I n t h i s r ega rd , i t i s i n t e r e s t i n g t h a t o-dihydroxy phenols- are r e q u i r e d f o r the growth of M. l y s o d e i k - t i c u s (56). S i m i l a r l y , low l e v e l s o f these compounds have been shown to r ep l a ce the t y r o s i n e requirement of a spec ies of S a r c i n a (22). The work desc r i bed i n t h i s t h e s i s was undertaken i n an attempt t o de f ine an o v e r a l l approach to the study of i r o n - d e f i c i e n c y i n B. s u b t i l i s . I t was necessary , f i r s t o f a l l , t o determine whether DHB(G) was produced du r i ng a c t i v e growth of the organism or i f i t accumulated o n l y du r ing the s t a t i o n a r y phase. I f i t were produced o n l y dur ing the s t a t i o n a r y phase i t would be much harder t o des ign and i n t e r p r e t 1+ experiments on the control of i t s production. Aromatic compounds which, like DHB, are synthesized by the c e l l from chorismic acid, were examined for their effects on DHB production. Such studies would indicate whether or not DHB production was regulated by the control mechanisms known to operate i n the aromatic pathway in B. s u b t i l i s . The relationship of DHB(G) production to coproporphyrin production was then examined. Iron is required for the formation of hemin and of non-heme iron proteins. It was possible, there- fore, that the production of coproporphyrin and DHB(G) might be related i n some other way besides a lack of iron. Analogs of DHB were tested for their effects on DHB production. Those analogs which were markedly inhibitory might then be useful i n studying the enzymology of DHB(G) synthesis and i t s control. DHB and DHBG are known to bind iron strongly (26) so that a possible function of these compounds might be to serve as iron- transport factors. A study was made, therefore, of iron uptake by B. subtilis and of the effects of DHB(G) on this process. In addition, the effects of sideramines on DHB(G) production and on iron uptake were studied. Sideramines are thought to function as iron transport factors i n other microorganisms (39? 41). 5- MATERIALS AND METHODS I. Bacterial strains 1 . Wild type strains The source of each wild-type strain employed is shown in Table I. Strain WB-746 was selected (4-5) as a spontaneous prototrophic revertant of a tryptophan auxotroph strain (68). 2. Mutant strains Mutant strains were selected as spontaneous derivatives or as NV-methyl-N^-nitro-N-nitrosoquanidine (NTG) -induced mutants (Table I i ) . (a) Spontaneous mutants Strains resistant to 8-hydroxyquinoline (HQ1"), 5-methyltryptophan (MTr), and to various antibiotics were selected by spreading log phase cells on minimal medium supplemented with a level of inhibitor which prevented the growth of the wild type strain. The supplementation level required for each class of mutants is presented i n the text. (b) NTG-induced mutants Washed, log phase cells were suspended i n tris-^maleate buffer (l) and treated with NTG according to the procedure of Lorence and Nester (33 ) . Auxotrophs were selected as minute colonies on minimal medium containing a limiting level of the appropriate supplement: serine- glycine auxotrophs, 0.3 M-g serine per .ml; i-aminolevulinate (ALA) 6 Table 1 : B. s u b t i l i s w i l d type s t r a i n s S t r a i n Source B-1471 J . B. Ne i l ands , U n i v e r s i t y o f C a l i f o r n i a , Be rke ley . WB-746 E. ¥. Nes te r , WB-443 U n i v e r s i t y o f Washington, S e a t t l e . W-23 J . S p i z i z e n , Sc r ipps C l i n i c and Research Foundat ion, La J o l l a , C a l i f o r n i a . 6051 American Type Cu l tu r e C o l l e c t i o n (ATCC) 6633 ATCC 6455 ATCC I2696 ATCC 11+807 ATCC 7 Table II: Description of B. subtilis mutant strains Strain Genotype Enzyme defect Source trp-1 trp anthranilate synthetase B - l 4 7 i a trp-l-MT r-l trp~MTr anthranilate synthetase trp-1 trp-l-MT r-2 trp~MTr anthranilate synthetase trp-1 trp-2 trp tryptophan synthetase B - l 4 7 l a trp-3 trp InGP synthetase B - l 4 7 i a trp-4 trp anthranilate synthetase B-l471 a trp-5 trp tryptophan synthetase B - l 4 7 l a trp-6 trp tryptophan synthetase B - i 4 7 l a trp-7 trp PRA isomerase B - l 4 7 l a hem 1 ALA." ALA. synthetase B - l 4 7 l a phe-1 phe B - i 4 7 i a phe-2 phe B - l 4 7 l a MT r-l MTr B-i47l b MTr-2 MTr B-i47i b HQ1" 1-6 prototroph B-i47i b ser 1-4 ser B-i47l a d h b - 1 - 5 prototrophic revert- ants'3 of DHB auxo- troph a of B-1471 aro-1 shk B - i 4 7 i a SB-168 trp" InGP synthetase E.W. Hester, WB-746 SB-194 trp anthranilate synthetase E.W. Hester, WB-746 SB-194-MTt-l trp anthranilate synthetase SB-194b SB-194-MTr-2 trp anthranilate synthetase SB-194b SB-30 tyr trp E.W. Hester, WB-746 WB 2102 shk" DAHP synthetase E.W. Hester, WB-746 SB 167 shk" DHQ synthetase E.W. Hester, WB-746 MT r-l MTr WB-746b MTr-2 r MT WB-746b 8 Table II (continued) Strain Genotype Enzyme defect Source MT' prototrophs r MT a: spontaneous derivative; b: NTG - induced derivative. Abbreviations used: InGP, indoleglycerol phosphate; PRA, phosphoribo- sylanthranilic acid; Shk, shikimic acid; DAHP, 3-deoxy-D-arabino-heptu- losonic acid 7-phosphate; DHQ,, dehydroquinic acid; ALA, -aminolevulinic acid. 9 auxotrophs, 0.1 ug ALA per ml, with 10 g e i t r i c acid per l i t r e replacing glucose as the carbon source; DHB auxotrophs, 0.05 M-g DHB per ml, with citrate being omitted from the medium i n this case because i t has been shown that citrate can replace DHB as a growth requirement for a multiple aromatic auxotroph of Escherichia c o l i (67). Before plating for mutant selection, the mutagenized cells were diluted and plated on the appropriate selection medium to score for survivors. To avoid lysis of the treated cells during the incubation of plates for scoring survivors, i t was necessary to store the cells at h C in minimal medium containing 5$ glycerol (33). Under these conditions about 90$> of the cells remained viable three days. A l l strains were stored on Difco TAM sporulation agar slants i n screw-cap v i a l s . II. Media Difco trypticase soy broth supplemented with 1% Difco yeast extract (TSY medium) was used for the growth of inocula. The minimal medium used for the production of porphyrin and phenolic acids was a modification of the medium of Neilands and Garibaldi (42). It contained, i n g per l i t r e : KgHPO^, 1.0; ammonium acetate, 3.0; MgSOĵ . TH^Oj 0.08; glucose, 10.0. It was supplemented with 5 .ml per l i t r e of a 10% solution of Difco yeast extract i n d i s t i l l e d water, autoclaved with alumina prior to use (10), and with 0.1 .ml per l i t r e of the Neurospora trace elements solution of Vogel (65), from which the iron was omitted. The medium was made up with glass-distilled water, and was adjusted to pH 7.4 before autoclaving. 10 The glucose was ster i l i z e d separately as a hO'fo solution in glass- d i s t i l l e d water. Extraction of the medium with 8-hydroxyquinoline (HQ,) (65) yielded erratic results, presumably due to microquantities of HQ, remaining i n the medium after chloroform extraction. This procedure was therefore not employed. III. Cultural conditions A l l glassware was autoclaved twice with glass-distilled water prior to use. Erlenmeyer flasks (250 ml), f i t t e d with side arms and containing 25-ml quantities of the medium, were incubated at 37 C i n a New Brunswick Metabolyte water bath (Hew Brunswick Scientific Co., New Brunswick, N."J.), rotating at 250 rev/min. A l l experiments were performed by use of a 0. 5$> inoculum from a ik-hr culture, which was one transfer away from the stock slant. The time of onset of DHB(G)* production was constant under these conditions. Additions to flasks were made at the times mentioned in the text. IV. Determinations 1. Growth Growth was followed turbidimetrically, by use of a Klett-Summerson colorimeter with a540 f i l t e r . Readings were converted to c e l l numbers by use of a standard curve prepared with the organism. * DHB(G) refers to DHB, or DHBG, or both compounds. 11 For higher densities i t was necessary to dilute the culture with Q complete medium. A c e l l density of 10 per .ml gave a reading of 42 Klett units. Growth curves were followed during a l l experiments. 2. Phenolic acids DHB(G) was assayed by adding 0.5 ml of a f e r r i c iron solution (0.5 mg per ml) to 2.0 ml of cell-free medium adjusted to pH 7.6, centrifuging, and measuring the optical density (0.D) of the super- natant at 510 .mu, 1.0 cm light path, with a Beckman Model B spectro- photometer. Blank corrections were always made, using 2.0 .ml of c e l l free medium and 0.5 ml d i s t i l l e d water. Under these conditions, 1.0 O.D. unit represented a concentration of 3^4 ng DHB(G) per ml of culture supernatant. Production of DHB and DHBG was confirmed spectrophotometrically and chromatographically (27) by comparison with authentic specimens. Descending chromatographic analyses were performed using two solvent systems; (l) n-butanol, acetic acid, water 4/1/5; (2) t-butyl alcohol, methyl ethyl ketone, water, diethyl amine 1 0 / l 0 / 5 / l . The RF values are given i n Table V. 3. Coproporphyrin II I * . A 2.0 ml sample of culture supernatant was adjusted to pH 5.0 with acetic acid. The coproporphyrin was extracted with a known volume of ether. The absorption of the ether extract was measured at 408 mu. using a Beckman DB spectrophotometer ( 1 5 ) . Care was taken to ensure that, at the time of measurement, the volume of * Coproporphyrin III is referred to as coproporphyrin. 12 the ether was equal to that originally added to the acidified culture supernatant. 4. Identification of glycine Electrophoresis was performed with a model D high-voltage electrophorator (Gilson Medical Electronics, Middleton, Wise), using 2.5$ formic acid and ,7.8$ acetic acid buffer (pH 1.9), at 2000 v for 45 min. Glycine was identified by co-electrophoresis with an authentic specimen. 5. Protein concentration Protein was assayed by the method of Lowry et a l ( 3 5 ) . V. Preparation of extracts After 12-14 hr growth, cultures were harvested by centri- fugation at 20 C, washed once with complete medium, and the cells resuspended at a concentration of 1 g wet weight per 5 ml i n 0.05 M potassium phosphate - 0.01 M mercaptoethanol, pH 7.5. Lysozyme (100 ug per ml), deoxyribonuclease (10 u.g per ml) and MgCl^ (5 x 10 M) were added to the suspension and the mixture incubated for 30 min at 37 C. The resulting extract was sonicated for 2 min at a probe intensity of 70'.' using a Biosonik probe oscillator, (Bromwill Scientific, New York, N.Y.), and then centrifuged at 25,000 x g for 15 min at 4 C. The supernatant f l u i d was used directly for enzyme assays. VI. Enzyme assays 13 1. Synthesis of DHB The formation of DHB from chorismic acid was measured by the method of Young et a l ( 6 7 ) . DHB production was estimated at a stan- dard curve prepared using commercial DHB ( 6 7 ) . Under these conditions, an O.D. at 3 l 8 mu. of 0.1 represented 0.12 umoles DHB. 2. Synthesis of DHBG The synthesis of DHBG from DHB and glycine was determined by the method of Brot et a l ( 7 ) , except that glycine was substituted for serine i n the reaction mixture. VII. Respiration studies Cells were harvested at 20 C at the times indicated i n the text, +2 washed twice with 0.005 M Mg - 0.1 M tris(hydroxymethyl)amino.methane (Tris)-chloride (pH 7.k), and resuspended in the same buffer at a concentration of approximately 5 ™g dry weight of cells per ml. Respiration studies were performed at 37 C in Warburg vessels which +2 contained a f i n a l concentration of 0.005 M Mg - 0.05 M Tris pH 7.4, one .ml of c e l l suspension, and 5 umoles of acetate or citrate. VIII. Iron transport studies 1, Preparation of cells Cultures were grown to a density of 5.5 x 10 cells/ml (unless stated otherwise), then used immediately for transport studies. For iron-sufficient growth, the medium was supplemented with 1 ug of iron/ml. 2. Iron uptake (a) Iron-deficient cells Ten .ml of culture were transferred to a 250 ml Erlenmeyer flask. The flask was incubated at 37 C (unless stated otherwise) in a New Brunswick Model G-77 Metabolyte water bath (New Brunswick Scientific Co., New Brunswick, N.J.), rotating at 100 rpm. Additions were made after 10 min. (b) Iron-sufficient cells Ten ml of culture were f i l t e r e d through a 0.45 n Millipore membrane. The cells were washed with 10 volumes of prewarmed, iron-deficient medium, and the membrane transferred to 10 .ml prewarmed iron-deficient medium i n a 250 ml Erlenmeyer flask. The cells were resuspended by gently blowing medium over the membrane with a pipette. The flask was incubated as for iron- deficient cells, with additions again being made after 10 min. In both cases, additions were made in a total volume of 0.5 ml glass-distilled water. When uptake was measured at 0 C, glycerol was added to a concentration of 5$ to prevent ly s i s of the cell s . Unless stated otherwise, iron was used at a concentration of 5.0 mug/ml. 3. Assay of iron uptake Samples of 1.0 ml were withdrawn with hypodermic syringes, f i l t e r e d through 0.45 LI Millipore membranes and the cells washed with 2.0 ml prewarmed, iron-deficient medium. The membranes were dried, placed i n vials containing 1 0 ml s c i n t i l l a t i o n f l u i d (Liquifluor, New England Nuclear Corp.) and assayed for radio- activity i n a liq u i d s c i n t i l l a t i o n counter (Nuclear Chicago Model 2 2 5 ) . Unless stated otherwise, the rate of uptake was measured over the interval 3 min to 8 min after iron addition. Radioactivity i n the cold TCA-insoluble fraction of cells was measured by the method of Roberts et a l ( 5 3 ) . A 1 . 0 ml sample was added to 1 . 0 ml of 10% TCA at 0 C. After 3 0 min at 0 C, the suspension was f i l t e r e d through a 0,1*5 u Millipore membrane, and the membrane washed with two volumes of iron-deficient medium. The membrane was dried and assayed for radioactivity. IX. Chemicals Chorismic acid was purified from the cultural supernatant of A. aerogenes 6 2 - 1 according to the method of Gibson ( 1 9 ) except that the acid was precipitated from the Dowex effluent as the barium salt ( l 8 ) . Free chorismic acid was obtained by sedimenting most of the barium as the phosphate salt, and by subsequent addition of Dowex-50 to the supernatant. DHBG was synthesized by a modification of the dicyclohexylcarbodiimide method of Sheehan and Hess ( 2 6 ) . Phosphoribosylanthranilic acid (l-(O-carboxyphenylamino)-l-d-D-ribulose-5-phosphate) was synthesized according to the method of Doy ( 1 3 ) . Ferrichrome was obtained from J. B. Neilands of the University of California, Berkeley. The iron was removed from this compound by the method of Emery and Neilands (ik). Ferrioxamine was obtained from W. Keller-Schierlein, of Eldg. Technische Hochschule, Zurich, Switzerland. The following chemicals were obtained from commercial sources: o- and m-tyrosine, ^-aminolevulinic acid (Sigma Chemical Co.); 2 - and 3-fluoro, and 2,3-dimethoxybenzoic acid, DHB, NTG (Aldrich Chemical Co.); m-hydroxybenzoic acid, a-picolinic acid, and hippuric acid ( j . T. Baker Chemical Co.); MT, 3-hydroxyanthranilic acid, hemin, serine-l-^C (specific activity 1 0 . 3 nc per umole) (Calbiochem Co.); s a l i c y l i c acid (The British Drug Houses, Ltd.); ^Fe as FeCl^, specific activity 2 5 - 5 M-c/ng (international Chemical and Huclear Co.); g l y c i n e - 1 - ^ C (specific activity 2 u.c per umole) (Merck & Co.). RESULTS Section I: - General properties of phenolic acid excretion 1. Excretion of phenolic acids by wild type strains A number of wild type strains of B. subtilis were grown i n iron- deficient medium to determine the compounds produced under these conditions. The strains f e l l into three groups according to their patterns of phenolic acid production: strains of group I produced only DHBG, those of group II only DHB, and those of group III both of these compounds (Table III). A strain representing each group was selected for further study, i.e. B-1471 (group I), WB-746 (group II) and W-23 (group III). 2. Influence of iron on DHBG production Production of DHBG by strain B-1471, i n the absence of added iron, started after about 8 hrs of growth and continued logarithmically, parallelling growth, u n t i l the early stationary phase was reached. Significant production of DHBG did not occur i n the stationary phase. If the medium was supplemented with 1 mg of iron per l i t r e , production of DHBG was inhibited completely (Fig. l ) . At concentrations below 150 ug of iron per l i t r e , DHBG production was inversely proportional to the level of iron i n the medium. As the level of iron added was increased from 0 to 150 ug per l i t r e of medium, the production of DHBG began proportionately later.in the growth period (Fig. 2 ) . DHBG production was inhibited completely by the addition of 150 ug of iron per l i t r e . Table I I I . Excretion of phenolic acids by wild type strains Group Strain DHB DHBG level produced (mg/l) I B - 1 4 7 1 _ 3 0 0 6 6 3 3 - + 5 0 6455 - + 6 0 1 2 6 9 6 - 1 5 0 I I WB - 7 4 6 + - 1 0 0 0 S B - 4 4 3 + - 1 0 0 I I I w - 2 3 + + 2 5 0 6 0 5 1 + + 2 0 0 1 4 8 0 7 + + 1 0 0 HOURS •Figure 1. . Growth and production of DHBG by s t r a i n B-1471. C e l l s were grown i n the absence (©) and presence (0) of 1 mg of added i r o n per l i t r e . DHBG was produced under conditions of i r o n d e f i c i e n c y (X) . • ..- but was not produced i n the presence of added i r o n . 3 . Fe : (DHBG)^ complex and the control of DHBG production When added to an iron-deficient culture at any time "between 5 and 9 hrs after inoculation, 1 mg of iron per l i t r e did not completely inhibit DHBG production. The addition of DHB or DHBG to an iron-' deficient medium at zero time had no effect on subsequent DHBG production. (Fig. 3 ) . - 4 The. simultaneous addition of 3 . 5 x 1 0 M DHB and 1 mg of iron per l i t r e , 5 hrs after inoculation, resulted i n the complete inhibition of DHBG production (Fig. 4 ) . In fact, 8 0 ug of iron per l i t r e was - 4 equally effective i n the presence of 3 . 5 x 1 0 M DHB. When the iron-DHB mixture was added 7 or 9 hrs after inoculation, i t s inhibitory effect on DHBG production was not f u l l y manifested for about 3 hrs (Fig. 4 ) . It appeared therefore, that between 5 and 7 hrs i n the growth cycle, an iron-deficient culture became committed to the production of some DHBG, regardless of subsequent addition of an iron-DHB mixture. 4 . Effect of aromatic amino acids on DHBG production The excretion of DHB by certain aromatic amino acid auxotrophs of A. aerogenes ( 4 3 ) prompted investigation of the effects of aromatic intermediates and end products on the production of DHBG by B. subtilis. None of the supplements, at the levels used, affected the growth rate of cultures. Supplementation of media with p-hydroxybenzoic acid or p-amino- benzoic acid, at concentrations up to 1 0~^M, had no effect on subsequent DHBG production. Addition of tryptophan at a concentration - 4 of 1 0 M caused earlier and significantly higher production of DHBG 21 8 9 10 II 12 13 14 HOURS Figure 2. Effect of iron added at zero time on the production of DHBG by strain B-1V71. The levels of iron added •were, i n ng per l i t r e : 20 (0), 50 (©), 80 (A), and 150 ( A ) . No iron was added to the control flask (X). 0.8 H O U R S Figure 3. Effect on-DHBG production of adding iron or DHB to strain B-1471. DHB (3-5 x 10 M) was added at zero time (X). Iron ( l mg per l i t r e ) was added at-5 hr (©), 7 hr (A), and ' 9' hr ( A ) . . NO iron or DHB was added to the control flask (0). 2 3 i - 4 Figure 4 . Effect of- simultaneous addition of 3 . 5 x 1 0 M DHB or DHBG and 1 mg of iron per l i t r e on DHBG production "by strain B - 1 4 7 1 . The additions were made at 5 hr (X), 7 hr (©), and 9 hr (A). Wo phenolic acids or iron was added to the control flask (.0). 2k than i n the control flask. As the level of tryptophan was increased beyond 10 ̂ M, DHBG accumulation was inhibited (Fig. 5 ) . Phenyl- -k alanine and tyrosine reduced DHBG accumulation at 10 M, and to a greater extent at higher concentrations. Tyrosine was a more effective inhibitor than phenylalanine (Fig. 5 ) . These inhibitory effects were not additive. The percentages of inhibition observed for each amino acid at 10 "̂ M,.were tryptophan, 25; phenylalanine, 27; and tyrosine, 52. When a l l three amino acids were present together at 10 the inhibition was 50$. Inhibition of DHBG production, comparable to that seen with tyrosine, was produced by anthranilic acid. The production of DHBG was inversely proportional to the level of anthranilic acid i n the medium (Fig. 6). This inhibition occurred i n the presence of tryptophan, so that the effect of anthranilic acid was not produced as a consequence of i t s depriving the c e l l of tryptophan (Fig. 6). 5« Source of glycine i n DHBG A volume of 200 ml of medium was inoculated and incubated as described i n Materials and Methods. Immediately after the in i t i a t i o n of DHBG synthesis, 50 umoles of glycine-l-^C (specific activity, 2 uc per umole) were added. Four hours later, the cells were removed by centrifugation and the DHBG was purified according to the method of Ito and Neilands ( 2 9 ) . A 30-mg sample (wet weight) of cells was fractionated according to the procedure of Roberts et a l (14). Radioactivity of the various fractions was measured by use of a Nuclear Chicago model l 8 l A planchet counter (Table IV). The purified DHBG gave a single spot when chromato- 0 . 6 Figure .5. Effect of adding end product amino acids on DHBG production by strain B-1471. Flasks were supplemented at zero time with: IQ-h M tryptophan (0); 10-^ M, 10-3 M phenylalanine,., or 1Q-3 M tryptophan ( a ) ; 10-3 M tyrosine (•); 2 x 10-3 M tyrosine, or 2 x 10"3._M of each of tyrosine, tryptophan and phenylalanine ( A ) . ... No"'additions were made to the control flask. (X). 2 6 0.5-1 ^ 0 . 4 - o 0 . 3 - < 0 . 2 d 0 . 1 - 0 8 9 10 II 12 13 H O U R S Figure 6 . Effect of adding antrhanilic acid on the production of DHBG by.strain B - 1 4 7 1 . Anthranilic acid was added at zero time at levels'of 1 0 M ( 0 ) , 1 0 . - 3 M (A), and 2 x 1 0 " 3 M (©). Identical curves were obtained when 1 0 - ^ - M tryptophan was added to the anthranilic acid supplemented flasks. .No aromatic addition was made to the control flask (.X).' 2 7 graphed on paper with three different solvent systems ( 2 9 ) . A sample of the purified DHBG was hydrolysed i n 6 N HC1 for 1 6 hrs in a sealed, evacuated tube at 1 2 0 C. The hydrolysate was chromato- graphed in the above systems, and also was subjected to high voltage paper electrophoresis. Labelled DHB was not detected. The label was found only in the glycine moiety. 6 . Variations among strains of B. subtilis By use of chromatographic and spectrophotometric methods (see Materials and Methods), two other wild-type strains of B. subtilis were examined for compounds produced during iron-deficient growth. Strain WB-746 produced DHB and smaller quantities of catechol. Strain W - 2 3 produced 'JHBG i n i t i a l l y , but after about 1 0 . 5 hrs of growth, appeared unable to maintain the DHBG conjugation system and began producing DHB (Table V). Strain W - 2 3 exhibited the same responses to the concentration of iron, anthranilic acid, and aromatic amino acids i n the medium as strain B-1V71. Strain WB-746, however, exhibited significantly different responses. In this strain, DHB synthesis was independent of the level of added iron up to a concentration of 1 2 0 ug per l i t r e . At 1 5 0 |ig of added iron per l i t r e , the quantity of DHB produced was almost as high as that obtained with the two other strains i n the absence of added iron (Fig. 7 ) . At higher levels of iron, DHB production by strain WB-746 was inhibited completely. Unlike strains B - 1 4 7 1 and W - 2 3 , production of DHB by strain WB-746 was not stimulated by low levels of tryptophan. In addition, the inhibition of pro- duction by high levels of tryptophan and by anthranilic acid was Table IV. Distribution of g l y c i n e - 1 - C Fraction 1 0 count s/min Total culture 5 . 0 2 C e l l supernatant f l u i d 2 . 0 3 Coproporphyrin III 0 . 5 9 0 Purified DHBG 0 . 6 0 0 Cells 2 . 9 6 Cold trichloroacetic acid-soluble 0 . 0 4 5 Alcoho1-soluble 0 . 0 0 5 Ether-alcohol 0 . 0 0 0 Hot trichloroacetic acid-soluble 0 . 8 9 7 Residual 2 . 0 6 Table V. Characteristics of phenolic acid excretion by strains of B . subtilis i n iron deficiency Characteristics of phenolic •Strain acid excretion •B-1471 WB-746 w - 2 3 Phenolic acid produced DHBG DHB DHBG and DHB Rp in solvent 1 0 . 8 3 0 . 8 8 0 . 8 3 0 . 8 8 i n solvent 2 0 . 4 7 0 . 6 9 0 . 4 7 O . 6 9 Effect of aromatic supplement- ation on excretion: Control 100$ 1 0 0 $ 1 0 0 $ - 4 1 0 M tryptophan 1 2 4 $ 1 0 0 $ 1 2 0 $ _ 3 1 0 M tryptophan 7 5 $ 8 9 $ ' 7 5 $ - 3 1 0 M tyrosine 48$ 7 4 $ 5 0 $ - 3 1 0 M phenylalanine 7 3 $ 9 0 $ 7 5 $ _ 3 1 0 M anthranilate 5 8 $ 84$ 5 5 $ - 3 2 x 1 0 M anthranilate 2 1 $ 7 8 $ 2 5 $ 30 Figure "7. only about 50$ of that observed i n the two other strains (Table V). 7« Effect of histidine on phenolic acid excretion When 20 u,g histidine per .ml was added at zero time to each of the wild-type strains, phenolic acid excretion was decreased about 25$ i n B-11+71 and W-23, and about 50$ in WB-746 (Table VI). Wester has demonstrated a regulative involvement of histidine in aromatic bio- synthesis (1+3) J i n a l l cases, the effect of histidine supplementation was overcome by tyrosine supplementation at 20 ug per ml (1+3). Histidine inhibition of phenolic acid excretion was not relieved by tyrosine; instead, the inhibitory effect was additive (Table VI, Fig. 5 ) . These results may reflect an additional unknown involvement of histidine i n aromatic metabolism. 8. In vitro syntheses (a) Formation of DHB Cell-free extracts prepared from strains B-ll+71 or WB-746 converted chorismate to DHB when incubated under the conditions described by Young et a l (67). (Table VII). WB-746 extracts were twice as active as B-1471 extracts. The addition of 10 % anthran- ila t e completely inhibited the formation of DHB (Table VII). (b) Formation of DHBG Extracts of B-ll+71 formed DHBG from DHB or from chorismate in the presence of glycine when incubated under the conditions described by Brot et a l (7 ) . (Table VIII). There was no formation of DHBS when serine was substituted for glycine. The addition of F e + 3 had Table VI. Effect of histidine on phenolic acid production in wild type strains Strain Supplement (20 ug/ml) % Phenolic acids excreted B-1471 100 B-1471 histidine 75 B-1471 histidine & tyrosine 55 W-23 - 100 W-23 histidine 70 W-23 histidine & tyrosine 50 WB-746 - 100 WB-746 histidine 50 WB-746 histidine & tyrosine 4o 33 Table VII. In vitro synthesis of DHB Extract Source Reaction Mixture DHB Formed (umoles) 1. WB-746 Complete 0.32 2. - chorismate 0.02 3. Complete, at 0 C 0.03 4. _3 Complete + 1 0 M anthran- 0.01 ilate 5- , " -3 Complete + 10 M of each 0.13 of tryptophan, tyrosine and phenylalanine 6. B-1471 Complete 0.13 Synthesis of DHB from chorismate. The complete reaction mixture contained:crude extract(3.0 mg protein), 50 umoles Tris-Cl buffer (pH 8.0), 1.0 umole chorismate, 1.0 umole NAD, and 5.0 umoles MgCl^ in a total volume of 1.0 ml. Table VIII. In vitro synthesis of DHBG Extract Source Reaction Mixture 4 / 10 counts/min in DHBG 1. B-1471 Unsupplemented 0.08 2. + chorismate 3.1 3. +3 + chorismate + Fe 3.0 4. + chorismate, at 0 C 0.10 5. + DHB 8.2 6. WB-746 + chorismate 0.05 7. + DHB o.o4 Synthesis of DHBG. Each reaction mixture contained: crude extract (3.0 mg protein), 10 umoles Tris-Cl buffer (pH 7.4), 0.1 umoles -^C-l-gly (0.2 u.c), and 1.0 umole ATP. Additions were made as indicated: 0.1 umole DHB, 0.5 umole chorismate, Fe +3 0.05 umole. The total volume of each reaction mixture was 1.0 ml. The incubation time was 1 hr at 37 C no effect on DHBG formation from chorismate. . WB - 7 4 6 extracts were incapable of DHBG formation (Table VIII). 9 . Metabolism of DHB(G) by strain B - 1 4 7 1 When DHB was added to cultures of strain B - 1 4 7 1 at zero time, some of i t disappeared from the medium. In the absence of added iron, this disappearance continued u n t i l the culture started to produce DHBG (Fig. 3 ) . When iron, to a concentration of 1 mg per l i t r e , was added with the DHB, a similar rate of disappearance was observed for 1 2 hrs (the OD at 5 1 0 mu dropped from 0 . 5 5 to 0 . 3 5 ) . Ho further change occurred during the next 8 hrs. If iron and DHB were added between 5 and 9 hrs there was no disappearance of DHB before l 6 hrs (Fig. 4 ) . The most marked disappearance of DHB(G) occurred i n the early stationary phase. During this period, the rate of disappearance was proportional to the concentration of iron i n the medium (Fig. 8 ) . Attempts were made to detect oxidation of DHB(G) in Warburg experiments. Washed cells were tested after growth with and without added iron and i n the presence or absence of DHB(G). In no case was oxidation observed. 1 0 . Metabolism of DHB(G) by strains W - 2 3 and WB-746 Strain W - 2 3 metabolized DHB(G) and the pattern of disappearance was similar to that seen in strain B - 1 4 7 1 . Strain WB-746, however, did not metabolize DHB(G), not even when supplemented with 1 .mg of iron per l i t r e . Oxidation of DHB(G) was not observed with washed cells of either of these strains. 0 . 5 n F igu re 8. Metabo l i sm o f DHBG by s t r a i n B-1471 a f t e r a d d i t i o n o f i r o n . An 8 0 - L i g amount of i r o n per l i t r e (&) and. 1 mg o f i r o n per l i t r e (0) were added to f l a s k s a t 10 n r . No i r o n was added t o the c o n t r o l f l a s k (X ) . 3 7 1 1 . Properties of .mutant strains (a) 5-methyltryptophan resistant (MTr) strains Resistance to MT ( 3 7 ) i n B. subtilis ( 4 5 ) causes derepression of the tryptophan biosynthetic enzymes ( 4 3 ) . When MT mutants of B - 1 4 7 1 were grown in iron-deficient medium, a l l produced lower levels of phenolic acids than the corresponding parent strains (Fig. 9 ) » MT mutants selected from anthranilate synthetase-less (ant ) strains of B - 1 4 7 1 (Table II), produced the same level of phenolic acids as MT-sensitive ant strains (Fig. 9 ) - Similar DHB(G) excretion patterns were observed i n MTr and ant mutants of WB - 7 4 6 and W - 2 3 . (b) Aromatic auxotrophs Phenolic acid production by supplemented aromatic auxotrophs blocked at one or more steps after the synthesis of chorismate (Table II) was comparable to that of the corresponding parent strain. Multiple aromatic auxotrophs (Table II), however, produced no phenolic acids i n iron-deficient medium supplemented with the required aromatic end-products. ( 2 0 ug per ml tryptophan, tyrosine and phenylalanine; 2 ug per ml p-aminobenzoic acid and p-hydroxybenzoic acid; and 2 0 ug per ml shikrmic acid). (c) HQ, resistant mutants ( H Q R ) HQ is a powerful iron-binding compound ( 6 6 ) . Growth of the three wild-type strains was inhibited by HQ at a concentration of 0 . 0 1 ug per .ml. Spontaneous mutants of B - 1 4 7 1 were obtained, however, which were resistant to 1 0 ug HQ per ml (Table II). Five days were 9 10 II 12 13 HOURS Figure 9» Production of DHBG by MTr strains of B-147J,. DHBG excretion was measured by: B-1471 (0); MT -1 and MTr-2 (X); Trp-1 (•).; and Trp-l-MT r-l (®). required for colony formation under these conditions, although the growth rate of these mutants i n the absence of HQ, was the same as the parent strain. Mutants of W - 2 3 or WB - 7 4 6 resistant to 1 0 ug HQ per ml were not obtained, but mutants resistant to 0 . 1 ug HQ, per ml were obtained (Table II). In a l l strains, one mutant 7 8 colony was obtained for every 1 0 to 1 0 cells plated. When HQr mutants of W - 2 3 or WB - 7 4 6 were grown in iron-deficient medium in the absence of HQ, normal levels of phenolic acids were produced. Under these conditions HQ mutants of B - 1 4 7 1 produced only very low levels of DHBG. Supplementation of media with 0 . 1 ug HQ/ml restored DHBG excretion in these mutants to the level of the parent strain. Three series of spontaneous derivatives of B - 1 4 7 1 were selected which were resistant to: (a) albomycin ( 4 9 ) , (b) actinomycin ( 4 9 ) and (c) catechol. These strains produced DHBG at the same level as the parent strain. 4 0 D I S C U S S I O N Under conditions of iron-deficiency, DHB(G) production by B. subtilis i s associated with active growth of the organism. It is not a metabolic by-product accumulating during the stationary phase. The control of DHB(G) production by iron appears to 3+ involve the Fe : (DRT^G)).^ complex, and a c r i t i c a l level of this effector complex is required to stop production. The complex may function directly, as a corepressor, a feedback inhibitor, or as both, or i t may function indirectly, by f a c i l i t a t i n g the transport of iron into the c e l l . The results of adding iron to iron-deficient cultures showed that after a certain time the cells are committed to the production of D H B ( G ) , and the addition of iron does not reverse this commitment. 3+ These results also support the suggestion that the Fe : ( D H E ^ G ) ) ^ complex is involved i n the control of DHB(G) production. If iron is not added u n t i l the culture has started to produce D H B ( G ) , production is stopped more effectively than by the addition of iron prior to the onset of production (Fig. 3). DHB and DHBG +3 were equally effective as the Fe :(phenolic acid)^ complex. B. subtilis u t i l i z e d glycine as a nitrogen source, but was unable to cleave the glycine from DHBG for i t s nitrogen requirement. Therefore DHBG was not being converted to DHB under the conditions described. From the levels of DHB(G) accumulated in the medium, i t is obvious that an iron deficiency either (i) causes a severe distortion of aromatic biosynthesis in B. subtilis, or ( i i ) allows the c e l l 1+1 to u t i l i z e a metabolic pathway which is not functional i n the presence of sufficient iron. The length of time required for the shutoff of D H B ( G ) production, i n the presence of the Fe 3 +: (DKE^G))^ complex, suggests that repression is involved (see Fig. 3 and 1 + ) , thereby favouring the second alternative. This alternative agrees with the results obtained with E. c o l i , where the activities of extracts for the synthesis of DHB from chorismate were dependent on the level of iron i n the growth medium ( 6 7 ) , The production of DHB(G) was influenced markedly by the exogenous levels of aromatic amino acids. These effects could be direct, the amino acids acting on the enzymes specific to DHB synthesis, or they could be indirect, decreasing the amount of chorismate available for DHB synthesis. A direct effect i s unlikely because these amino acids do not inhibit DHB synthesis by extracts. (Table VII). This suggests that the production of DHB i n vitro is regulated by the mechanisms which form part of the general system of control of aromatic biosynthesis in B. subtilis ( 2 8 , kh, 1 + 5 ) . That portion of aromatic biosynthesis relevant to the present study is presented in Fig. 1 0 . At low levels of exogenous tryptophan (Fig. 5 ) DHBG synthesis was initiated earlier and occurred to a higher extent than in the control flask. This phenomenon is expected, since low levels of tryptophan would inhibit anthranilate synthetase and prephenate (PPA) dehydratase ( 1 + 4 ) . The increased levels of chorismate would then be available for DHB synthesis; and, i f the enzyme(s) responsible for DHB synthesis ( 6 7 ) was derepressed, efficient u t i l i z a t i o n of chorismate might prevent PPA accumulation. High exogenous levels of tryptophan and the other aromatic amino k2 SCHEME OF AROMATIC BIOSYNTHESIS E-4-P PEP DAHP SYNTHETASE DAHP DHB •CHORISMATE \ SHK ANT ANT SYN- THETASE PPA P£A PPA DEHYDRA/XDEHYDRO- TASE GENASE PHENYb ALANINE TYROSINE TRYPTOPHAN * F i gu re 10. A b b r e v i a t i o n s : E-4-P, erythrose-4-phosphate; PEP, phosphenol • . py ruva te ; DAHP, d-arab inoheptu loson ic ac id-7-phosphate; SHK, sh i k ima te ; PPA, prephenate;. P P , ' pheny lpy ruva te ; HPP, 4-hy- droxypheny lpyruvate ; ANT, a n t h r a n i l a t e ; PRT, phosphor i bo ' s y l t r a n s f e r a s e ; PRA, p h o s p h o r i b o x y l a n t h r a n i l a t e ; PRL, phosphor- i b o s y l i somerase; CAR, l- (o-carboxyphenylamino)-1-d-D-r ibu- lose-5-phosphate; INGP S; i n d o l e g l y c e r o l phosphate synthetase ; . . T. S., t r yp tophan synthetase . ^3 acids may have caused direct feedback inhibition and repression at the level of DAHP synthetase ( 2 8 ) . The greater inhibition of DHBG- accumulation by phenylalanine and tyrosine, when compared to tryptophan may reflect the higher sensitivity of DAHP synthetase to inhibition by PPA than by chorismate ( 2 8 ) . The inhibition of DHB(G) production by anthranilic acid was not caused by an indirect effect on tryptophan biosynthesis, since the inhibition was s t i l l observed i n the presence of try- ptophan. In addition, the inhibition of DHB(G) production was not caused by the conversion of anthranilate to tryptophan, because the inhibition has been observed i n mutants blocked at any step between anthranilate and tryptophan. (Table II). Anthranilate, however, inhibited the synthesis of DHB from chorismate by extracts (Table V i i ) . Cell-free extracts of strains B-1471 and WB-746 converted chorismate to DHB. Extracts of B-1471 formed DHBG from chorismate or DHB i n the presence of ATP. The addition of coenzyme A had no effect on DHBG formation. Extensive studies were not done on these i n vitro syntheses because they were being conducted in other laboratories ( 6 , 7 , 2 1 ) . The formation of DHB but not DHBG, by strain WB-746 may result from the r i g i d control of glycine synthesis i n this strain. The observation that WB-746 did not form appreciable amounts of copro- porphyrin III under iron deficiency provides further evidence for this s t r i c t control. Strains B-1471 and W-23 did excrete copro- porphyrin III under these conditions. It is also significant that strain WB-746 consistently produced greater quantities of DHB(G) than strains B - 1 4 7 1 or W - 2 3 (Fig. 8 ) . Evidence is presented in Sections II and IV that glycine production may be related to an oxidatively functional tricarboxylic acid (TCA) cycle. The disappearance of DHB(G) from cultures remains to be explained. An enzyme which decarboxylates DHB to catechol was purified from Aspergillus niger. Oxygen was not required for this reaction ( 6 l ) . A pseudomonad, capable of using DHB as the sole carbon source, cleaved the aromatic ring of DHB with the uptake of 1 mole of oxygen. Decarboxylation did not occur prior to oxidation ( 5 5 ) . Although DHB(G) was metabolized by strains W - 2 3 and B - 1 4 7 1 , i t was not possible to demonstrate either decarboxylation or oxidation of DHB by whole cells of these strains after growth under a variety of conditions. Cultures appear to be able to metabolize DHB(G) during the early log phase and the early stationary phase, but not during the late log phase, the time during which DHB(G) is produced i n iron-deficient cultures (Fig. 3 , 4 and 8 ) . DHB was present i n a strain of A. aerogenes (blocked after anthranilate synthetase) after 1 1 hrs of growth i n a quinic acid medium but not after 2 4 hrs. Catechol, on the other hand, was present after 2 4 hrs but not after 1 1 hrs ( 5 l ) . A series of B - 1 4 7 1 mutants (Table II), which were blocked at each of the steps of tryptophan biosynthesis, were examined for the compounds produced under iron-deficiency. Although each of these auxotrophs produced normal levels of DHBG and showed the usual responses to iron, none of them produced catechol during the 20-hr growth and incubation period. r The i n a b i l i t y of MT strains to produce normal levels of phenolic acids may reflect intracellular chorismate depletion by.the derepressed tryptophan biosynthetic enzymes. In addition, excessive tryptophan production would tend to inhibit DAHP synthetase (28) further decreasing the chorismate available for DHB synthesis ( 6 7 ) . This interpretation was supported by the observation (Fig. 9) that a l l ant strains of B-1471 produced the same level of phenolic acids whether they were resistant to or sensitive to MT. The excretion properties of HQ, strains of B-1471 i n the presence and absence of HQ indicated that these mutants may have a lower requirement for iron. This theory was supported by subsequent iron-uptake studies (Section IV). 46 Section II: - The production of coproporphyrin and i t s relationship to the production of phenolic acids. Introduction Strains B-1471 and ¥-23 produced DHBG and coproporphyrin during logarithmic growth (Table III, Fig. 1, p.43). Both products accumulated only i n iron deficient medium. Since glycine is required for the synthesis of both compounds, strains auxotrophic for glycine were selected and were grown in iron deficient medium to determine alterations in their excretion capacities. Since an iron requirement has been demonstrated in porphyrin bio- synthesis ( 3 0 , 3 2 ) , a strain blocked at the f i r s t step of porphyrin biosynthesis (£-aminolevulinate synthetase) was selected. By growing this mutant in the presence and absence of o*-aminolevulinate (ALA), the effect of heme iron requirement on phenolic acid excretion was studied. Strains lacking the a b i l i t y to synthesize normal levels of phenolic acids were selected to further elucidate the relationship between phenolic acid and coprophorphyrin production. 1. Production of coproporphyrin and phenolic acids by wild-type strains Strains B-1471 and W-23 showed similar responses to iron-deficiency, Phenolic acid production started when the cultures reached a density Q of about 7.0 x 10 viable cells per ml, and continued logarithmically, parallelling growth (Fig. 11 and 1 2 ) . Coproporphyrin production started about 1 hr after the f i r s t appearance of phenolic acids and hi Figure 11. Growth and production of phenolic acids and Coproporphyrin by s t r a i n B-1471. C e l l number (0); phenolic acids ( A ) ; and coproporphyrin (X). kQ 0 2 4 6 8 10 12 14 16 18 H O U R S Figure 12. Growth and production of phenolic acids and coproporphyrin by s t r a i n W-23. C e l l number (0); phenolic acids ( A ) ; and coproporphyrin (X). ^ 9 Figure 13. Growth and production of phenolic acids and coproporphyrin by s t r a i n WB - 7 4 6 . C e l l number (X); phenolic acids (A); and coproporphyrin (X). also continued.logarithmically (Fig..11 and 1 2 ) . Phenolic a c i d production s t a r t e d e a r l i e r i n s t r a i n .WB-746, and continued at a f a s t e r rate than i n the two other s t r a i n s ( Fig. 1 3 ) . Conversely, coproporphyrin production started l a t e r , occurred at a slower r a t e , and d i d not p a r a l l e l growth (Fig. 1 3 ) . 2. I n h i b i t i o n of coproporphyrin production by i r o n The addition of various l e v e l s of i r o n at zero time t o . s t r a i n B-1471 showed that while 150 ug per l i t r e completely i n h i b i t e d phenolic a c i d production (Fig. 2 ) , a l e v e l i n excess of 200 ug per l i t r e was required to i n h i b i t coproporphyrin production. The rate of growth was not s i g n i f i c a n t l y a l t e r e d by the addition of i r o n . As the l e v e l of added i r o n was increased above 200' yg per l i t r e the time of appearance of coproporphyrin was delayed, and at the higher concentrations occurred i n the stationary phase. The addition of 1 mg i r o n p e r . l i t r e a f t e r 7 hrs incubation r e s u l t e d i n an 85% decrease i n t o t a l coproporphyrin production by s t r a i n B - l 4 7 1 ( F i g . 1 4 ) . I f t h i s amount of i r o n was added a f t e r 8 hrs incubation, production was decreased by 43%, and i f added ;after.9 hrs, by 30% (Fig. 14). The addition of 3.5 x 10~^M DHB or DHBG alone d i d not cause a s i g n i f i c a n t decrease i n coproporphyrin production. I f 1 mg i r o n per l i t r e was added with the phenolic a c i d , however, there was a r a p i d cessation of coproporphyrin production (Fig. 1 4 ) . .3. I n h i b i t i o n of.coproporphyrin production by.hemin The a d d i t i o n of low. l e v e l s of hemin.to iron-rdeficient cultures 51 H O U R S Figure lh. • E f f e c t on coproporphyrin production of adding i r o n and , DHB(G"> • One mg i r o n per l i t r e was added at 7 hr (®); . 8hr.(*0> or 9 hr (0). DHB(G) at 50 mg per l i t r e and 1 mg i r o n per l i t r e were added at 8 hr (A). • No • ' additions were made to the c o n t r o l f l a s k ( X ) . The additi o n of 50 mg of DHB(G) alone had no e f f e c t ( X ) . 52 of strain B-1471 at zero time resulted i n later production of copro- porphyrin and phenolic acid (Table IX). Under these conditions, coproporphyrin was eventually produced at the normal level but the level of phenolic acid accumulated was markedly reduced (Table IX). There was no inhibition of growth at 0.5 - 1.0 ug hemin per ml. Growth, however, was inhibited above 1.0 ug of hemin per ml. 4. Inhibition of coproporphyrin production by aromatic amino acids Whereas accumulation of phenolic acids was inhibited by adding aromatic amino acids and anthranilic acid to the medium at zero time (Table V), coproporphyrin production was not influenced by supple- mentation of the medium with these acids at concentrations up to 2 x 10 Similarly, histidine supplementation decreased the excretion of phenolic acids (Table VI) but had no effect on copro- porphyrin production. 5. Coproporphyrin and phenolic acid production by mutants of strain B-1471 (a) Glycine auxotrophs Repeated attempts to isolate glycine auxotrophs were unsuccessful. (b) Serine auxotrophs Glycine is known to be synthesized via serine i n E. c o l i (64). Therefore, attempts were made to isolate serine auxotrophs so that their capacity for coproporphyrin and phenolic acid production could be examined. Three absolute serine auxotrophs, ser-1, ser-2 and Table IX. Inhibition of the production of phenolic•acids and coproporphyrin by hemin i n strain B-1471. O.D. 510 O.D. 4o8 14 hr 18 hr 24 hr 16 hr 20 hr 24 hr 1. Control 0.50 0.51 0.48 1.4 1.5 1.6 2. + 0.5 ug hemin per ml 0 0.3 0.35 0 0.9 1.5 3. + 1.0 ug hemin per ml 0 0 0.20 0 0.4 1.4 4. + 2.0 ug hemin per ml growth inhibited Hemin was added to cultures at zero time. The growth rate was unaffected by hemin supplementation at 1.0 ug per ml. 5 ^ s e r - 3 , and one leaky serine auxotroph, s e r - 4 , were isolated. Glycine would not replace serine as a growth.requirement for any of the strains. None of the strains produced phenolic acids, as determined by colorimetric assay, when grown under conditions of iron deficiency in minimal medium supplemented with 3 0 ug serine per ml. Copro- porphyrin was produced by s e r - 1 , s e r - 2 and s e r - 3 at about 1 0 $ and by ser-k at about 1 5 $ of the level produced by the wild-type (Table X). When the culture supernatants from these experiments were concentrated and the phenolic acids extracted into ethyl acetate, chromatographic examination of the extracts revealed low levels of DHB but no DHBG (Table X). These experiments were repeated using 5 0 - 1 0 0 ug glycine per ml in addition to the serine supplement. Under these conditions, the results were qualitatively and quantitatively similar to those observed with serine alone. Spontaneous prototrophic revertants were selected from each of the serine auxotrophs. A l l revertants tested ( 2 for each auxotrophic strain) produced normal levels of coproporphyrin and phenolic acids under iron-deficiency. However, chromatographic examination of the culture supernatants showed that the revertants produced both DHB and DHBG (Table X). (c) ALA auxotrophs Only one ALA auxotroph, designated hem-1, was isolated i n several attempts. In unsupplemented glucose medium, hem-1 had a doubling time of 1 9 0 min. When the medium was supplemented with Table X. Production of coproporphyrin and phenolic acids by serine auxotrophs derived from strain B - 1 4 7 1 . Strain Per Cent Production Cop Phenolic acids * DHB DHBG* B - 1 4 7 1 1 0 0 1 0 0 0 1 0 0 Ser 1 - 3 1 0 5 1 0 0 0 Ser 4 1 5 5 1 0 0 0 Revertants of Ser 1 - 4 1 0 0 1 0 0 7 0 - 8 0 2 0 - 3 0 *• Expressed as per cent of tot a l phenolic•acids produced 5.0 ug ALA. per ml, the doubling time was 130 min. This strain would not grow i n unsupplemented medium containing citrate as sole carbon source. When grown under conditions of iron-deficiency i n the test (i.e. glucose) medium, hem-1 showed unusual behavior. In unsupple- mented medium, neither phenolic acids nor coproporphyrin was produced. Supplementation of the medium with ALA led to the production of both coproporphyrin and phenolic acids. However, the responses to different levels of ALA showed that low levels led to the production of phenolic acids but not coproporphyrin, and that higher levels led to the production of coproporphyrin and a decreased quantity of phenolic acids (Fig. 1 5 ) . Furthermore, when glycine or serine (50 - 100 ug per ml) was added to unsupplemented medium, phenolic acid excretion by hem-1 was restored to 10$ of the level i n the parent strain, while coproporphyrin accumulation remained unaffected. Chromatographic examination of culture supernatants showed that hem-1 produced only DHBG. Protoporphyrin IX would not support the growth of hem-1 with citrate as sole carbon source, even i n the presence of 1 ug of iron per l i t r e . Similarly, supplementation of the glucose medium with 0.5 - 1.0 ug protoporphyrin IX per ml did not increase the growth rate, as seen with ALA, nor did i t allow the production of phenolic acids and coproporphyrin during iron-deficient growth. It should be pointed out that the growth of strain B-1471 was inhibited by 0.5 ug per ml of protoporphyrin IX. Ten spontaneous revertants of hem-1 were selected on citrate medium. A l l showed normal production of phenolic acids and copro- porphyrin under iron-deficiency. 57 r 2 . 4 5 10 15 jig AL per ml. CO o < d d Figure 15. E f f e c t of ALA supplementation on porphyrin and phenolic a c i d production by hem-1. The. l e v e l s of coproporphyrin (0) and phenolic acids (X)' . were measured a f t e r 2k hrs growth. 5 8 (d) DHB auxotrophs Only five strains, dhb-1, dhb-2, dhb-3, dhb-4 and dhb-5, which required DHB for growth were isolated i n many attempts. When f i r s t isolated, supplementation of the minimal medium with 0 . 2 ug DHB per ml resulted i n the appearance of normal colonies. However, a l l five strains rapidly lost their dependence on DHB for growth. The designations dhb-1, etc. were retained for these revertant strains because they produced low levels of phenolic acids. A "revertant" from each strain was screened for coproporphyrin and phenolic acid production under iron deficiency. In each case, the production of DHBG was considerably diminished compared to the production by the wild-type (Table XI). "Revertants" from four of the strains showed a decreased production of coproporphyrin (Table Xi). A "revertant" from strain dhb-4, however, 'showed very interesting properties. Under conditions of iron-deficiency i t produced a several-fold higher level of coproporphyrin than the wild-type .(Fig. l6). Subsequently, i t was found to produce coproporphyrin in the presence of iron, iron plus D H B ( G ) , or ferrichrome (Table XTl). The wild- type strain did not produce coproporphyrin under any of these conditions. Strain dhb-4 also had an extended lag period when inoculated into iron-deficient medium (Fig. 14). Supplementation of the medium with 2 mg iron per l i t r e , 2 mg iron with 5 0 mg DHB(G) per l i t r e , or 4 mg ferrichrome per l i t r e did not shorten this lag. The lag was shortened by the addition of 0 . 5 $ yeast extract. It should be pointed out that a l l the dhb-less strains grew more slowly than the wild-type strain in iron-deficient medium. Table XI. Products excreted by "revertants" of DHB auxotrophs derived from strain B - 1 4 7 1 . Excretion level (per cent of parent strain) Strain DHBG Coproporphyrin Dhb-1 5 2 0 Dhb-2 2 0 8 . Dhb-3 1 0 1 5 Dhb-4 5 3 0 0 Dhb-5 5 5 6o 3 . 0 - w 2.0-1 0_ bJ O 0 > _ 1.0- Q - f i — Q — 4 . 0 e - 3 . 0 CO o f- - 2 . 0 < • d -1 .0 Figure l6.. Growth and coproporphyrin production by dhb-U. C e l l number ( 0 ) ; coproporphyrin (X). Note that : the scale f o r O.D. at k08 mu does not correspond to that given i n Figure 11-13. Table XII. Coproporphyrin production i n Dhb-4. Per Cent Control 100 3+ + 2 mg Fe per l i t r e 100 + 50 mg per l i t r e DHB(G) + 2 mg Fe per l i t r e 100 + k mg per l i t r e ferrichrome 100 Discussion The results indicate that when cultures of B. subtilis strains B - 1 4 7 1 and ¥ - 2 3 became iron-deficient, the cells f i r s t started to produce phenolic acids. About an hour later the production of coproporphyrin was started. Phenolic acids have very strong a f f i n i t i e s for f e r r i c iron ( 2 6 ) , and i t has been suggested that they serve as iron solubilization factors ( 4 0 ) . The production of DHB(G) may thus f a c i l i t a t e scavenging of the last traces of iron from the medium for the synthesis of heme and non-heme iron proteins. Once the available iron was taken up by the c e l l , hemin would not have been formed anymore, resulting i n a loss of control over porphyrin biosynthesis, which was manifested by the production of high levels of coproporphyrin. In R. spheroides, hemin was shown to inhibit ALA synthetase ( 9 ) . In addition, i n the same organism ( 3 0 , 3 2 ) and in T. vorax ( 3 0 ) , iron appeared to be required for the conversion of coproporphyrinogen III to proto- porphyrin IX ( 9 ) . Loss of control over porphyrin synthesis i n B. subtilis would be expected to lead to the accumulation of coproporphyrinogen III, which would oxidize spontaneously to coproporphyrin III, either before or after excretion. This interpretation is supported by the observation that phenolic acid production was init i a t e d before coproporphyrin production, by the effect of hemin, and by the effect of iron at 1 5 0 ug per l i t r e on phenolic acid and coproporphyrin production. It is supported also by the properties of hem-1; only phenolic acids were produced with low levels of added ALA, whereas both phenolic acids and coproporphyrin were produced with higher levels. Only the higher levels of ALA would have allowed porphyrin synthesis i n excess of iron. Phenolic acid production appeared also to be related to the demand for iron. In the absence of added ALA, hem-1 did not produce phenolic acids, suggesting that the iron available i n the medium was sufficient to satisfy the non-heme iron requirements of the culture. In the presence of ALA, additional iron was required for heme synthesis, and phenolic acid production was initiated. Compared with strains B-1471 and W-23, strain WB-746 showed an earlier onset of phenolic acid production, and a later onset of coproporphyrin production. It has been shown (Section IV) that the rate of uptake of iron by cells of strain WB-746 i s much slower than the rates i n strains B-1471 and W-23. This suggests that under the experimental conditions, iron was not made avail- able to cells of strain WB-746 at a rate fast enough to satisfy non-heme iron requirements. In addition, the tricarboxylic acid (TCA) cycle i n strain WB-746 appears to be oxidatively inoperative when high levels of glucose are present (Section III). This could lead to a reduced demand for heme iron, and a late i n i t i a t i o n of porphyrin production. The patterns of inhibition of coproporphyrin production by 1.0 .mg iron per l i t r e were in good agreement with those obtained for inhibition of phenolic acid production (Fig. 3 ) . The results obtained by adding iron during growth showed that once a culture became iron-deficient, i t was committed to the synthesis of some phenolic acid and coproporphyrin, even after the addition of iron. 64 The much more rapid inhibition obtained by the addition of iron and Biffin) suggested that DHB(G) might help in carrying iron into the c e l l . Neilands ( 4 2 ) suggested some time ago that the addition of DHBG to the medium could make iron more available to cultures of B. subt i l i s. This has now been demonstrated (Section V). The serine auxotrophs made no DHBG when grown in medium supplemented with serine, suggesting that the conversion of serine to glycine may have been the limiting step in the synthesis of DHBG from DHB (Table VIII). However, when these auxotrophs were grown in the presence of both serine and glycine, again no DHBG was formed. It has been shown that labelled glycine added to the medium was incorporated into the glycine moiety of DHBG (Table IV), implying that B. subtilis can take up glycine from the medium. The results obtained with the serine auxotrophs are d i f f i c u l t to interpret in light of this observation. However, i t is possible that B. subtilis has a common active transport system for glycine and serine. If this system is inefficient, and transports serine i n preference to glycine, the results could be explained by the c e l l being unable to transport glycine at a rate fast enough to support growth, and serine only at a rate fast enough to support growth but not DHBG production. The fact that the i n a b i l i t y to synthesize serine prevents the production of the normal level of phenolic acids and reduces the production of coproporphyrin suggests that there may be a linked system of control involving glycine-serine, phenolic acids and coproporphyrin. Since coproporphyrin is produced by the serine auxotrophs, although at low levels, i t appears that glycine is used preferentially for porphyrin biosynthesis. The fact that supple- mentation of the medium with glycine or serine led to the production of some phenolic acid by hem-1 is of significance i n this regard. The low level of coproporphyrin production by some strains defective i n phenolic acid production again suggests a relationship between these compounds. The properties of strain dhb-4 cannot be explained at present, but they also support the existence of such a relationship. The fact that the extended lag period observed with this mutant in iron-deficient medium could not be reduced by supplementation of the medium with iron or iron-binding compounds suggested that solubilization of iron was not the factor limiting growth. Genetic analyses were attempted, to further explain the relation- ship between the excretion capacities of mutant strains. DNA extracted from strains B-1471 and WB-746 was used to transform (35) the auxotrophs derived from B-1471, but none of the strains was competent, even after addition of the "competence inducing factor" isolated from B. subtilis l68 I ( 2 ) . 66 Section III: - Inhibition of phenolic acid production i n B. subtilis Introduction In bacteria, certain compounds inhibit growth by acting as false regulatory metabolites of biosynthetic pathways (37). Thus, i n E. c o l i (37) and B. subtilis (46), 5-methyltryptophan has been shown to mimic tryptophan as a feedback inhibitor of anthranilate synthetase (37) and as a repressor of synthesis of the enzymes of the tryptophan pathway. Mutants resistant to this compound are either (i) insensitive to feedback inhibition or ( i i ) derepressed for the tryptophan bio- synthetic enzymes (37> 46). Various compounds structurally related to DHB(G) were therefore tested for their capacity to inhibit phenolic acid production. Such inhibitors might be useful in the isolation of mutants defective i n the control of phenolic acid synthesis. DHB(G) production was related not only to the level of iron i n the medium, but also to the level of iron required by the c e l l for heme biosynthesis. Because heme biosynthesis requires an operative TCA cycle, compounds known to inhibit the operation of this cycle i n B. subtilis were tested for their effects on phenolic acid production. 1 . Analogs of DHB The compounds tested as analogs of DHB(G) varied widely in their effects on phenolic acid and coproporphyrin production. Table XIII l i s t s the analogs i n order of their effectiveness as inhibitors. 2. Compounds affecting the functioning of the TCA cycle Some of the compounds tested are known to inhibit sporulation in Table XIII. Effect of analogs of DHB on phenolic acid and coproporphyrin production. Per cent accumulation Supplement Strain B - 1 4 7 1 Strain W - 2 3 Strain WB-746 ( 2 0 0 ug/ml) DHBG Cop* DHB(G) Cop DHB Cop None 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 Benzoic acid 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 3-hydroxy- 5 0 1 0 0 5 0 1 0 0 55 1 0 0 benzoate 2,3-dimethoxy- 6 5 1 0 0 5 0 1 0 0 75 1 0 0 benzoate 2-fluoroben- 6 5 1 0 0 4 0 1 0 0 6 5 1 0 0 zoate 3-fluoroben- 0 40 0 8 0 1 0 1 0 0 zoate Dipicolinic 0 55 0 5̂ 2 0 4o acid 3 -hydr oxyanth - 0 1 0 0 0 1 0 0 0 1 0 0 ranilate * Cop refers to coproporphyrin B a c i l l i , possibly because they inhibit the functioning of the TCA cycle. Glutamic acid ( 6 2 ) and a-ketoglutaric acid (24) inhibited sporulation i n B. subtilis by repressing the synthesis of aconitase. a-picolinic acid i s an iron chelating agent which has been shown to inhibit sporulation i n B. cereus ( 1 6 ) by preventing derepression of aconitase. m-Tyrosine inhibits sporulation i n B. subtilis ( 4 ) , but i t s mechanism of action is unknown. The compounds tested, and their effects on phenolic acid and coproporphyrin production are shown'in Table XIV. 3. Effect of sideramines on phenolic acid and porphyrin production by strain B - 1 4 7 1 . Sideramines are f e r r i c trihydroxyamates which can act as growth factors for certain bacteria ( 3 9 ) 5 possibly by transporting iron into the c e l l ( 4 0 ) . Micrococcus lysodeikticus w i l l grow in minimal medium i f a dihydroxyphenol, such as catechol ( 5 6 ) or protocatechuic acid ( 5 6 ) , or a sideramine, such as ferrichrome ( 4 l ) , is present. Therefore, the effects of sideramines on phenolic acid and copro- porphyrin production were examined in strain B - 1 4 7 1 . There are two types of sideramine ( 4 9 ) , and a representative of each group was chosen for study. When added at zero time, 0 . 4 ug ferrichrome per ml or 0 . 2 ug ferrioxamine per ml inhibited phenolic acid production completely, and delayed and reduced coproporphyrin production (Fig. 1 7 , Table XV). The level of ferrichrome used was equivalent to the addition of 2 0 ug iron per l i t r e . The addition of this much iron alone did not inhibit phenolic acid or coproporphyrin production by strain B - 1 4 7 1 (Fig. 2 and Section II). When the same levels of the 6 9 Table XIV. Effect of inhibitors acting at the level of the TCA cycle on phenolic acid and coproporphyrin production. Per cent accumulation Strain B - 1 4 7 1 Strain W - 2 3 Strain WB-746 Inhibitors DHBG Cop DHB(G) Cop DHB Cop None 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 0 . 0 5 $ a-ketoglutaric acid 7 5 7 0 8 0 7 0 1 0 0 8 0 or glutamic acid 0 . 1 0 $ cn-ketoglutaric acid 5 5 6 0 5 0 1 0 0 5 0 or glutamic acid a-picolinic acid 0 0 1 0 3 0 1 0 0 8 0 m - tyrosine 0 0 0 0 0 0 0 - tyrosine 6 0 0 2 0 8 0 6 0 8 5 A l l TCA cycle inhibitors were added at zero time. None of the supple- ments significantly affected growth except m - tyrosine, which retarded growth slightly i n W B - 7 4 6 . Corrections were made for this growth impairment (see text). 0.6^ 0.5- 9 10 II 12 13 14 15 16 HOURS Figure 17. E f f e c t of sideramine supplementation on phenolic a c i d excretion i n s t r a i n B-1471. Ferrichrome (0.4 ug per ml) or ferrioxamine (0.2 :' ' Ug per ml) was added at zero time (A) or a f t e r 8 hr growth (X). No.additions were made to the c o n t r o l f l a s k (0). Table XV. Inhibition of the production of coproporphyrin by sideramines i n strain B-1471 O.D. at 408 mu 12 hr 14 hr 18 hr 24 hr 1. Control 0.45 1.1 1.5 1.6 2. 0.4 ug ferrichrome per ml added at zero time 0 0 0.45 0.60 3. 0.4 ug ferrichrome per ml added at 8 hours 0 0 0.4o 0.50 4. 0.2 ug ferrioxamine per ml added at zero time 0 0 0.65 O.90 5. 0.2 ug ferrioxamine per ml added at 8 hours 0 0 0.80 1.2 The growth rate was not affected by supplementation with ferrichrome or ferrioxamine. 7 2 sideramines were.added to cultures after 8 hr growth under iron- deficient conditions, phenolic acid production was stopped rapidly (Fig. 1 7 ) and coproporphyrin production was delayed and reduced (Table XV). Discussion It appears that there are two classes of compounds affecting phenolic acid production i n iron-deficient cells of B. subtilis. Compounds of Class 1 inhibit both coproporphyrin and phenolic acid production. Those of Class 1 1 inhibit only phenolic acid production. The production of phenolic acids by B. subtilis is dependent upon the level of iron i n the medium. It is dependent also on the iron requirements of the c e l l . If &-aminolevulinate synthesis is blocked by mutation, phenolic acids are not produced under iron- deficiency, presumably because the non-heme iron requirements are met by the residual iron i n the medium (Fig. 1 5 ) . Therefore, i t is not surprising that compounds affecting the functioning of the TCA cycle inhibit both coproporphyrin and phenolic acid production. None of the compounds of Class 1 1 inhibited the growth of B. subtili s. This suggests that phenolic acids are not essential for growth under the experimental conditions used. Mutants isolated from B. subtilis strain B - 1 4 7 1 as DHB auxotrophs lost their dependence on DHB within one or two transfers. When examined, however, such "revertants" were found to produce very low levels of phenolic acid under iron-deficiency. Some of these strains also produced low levels of coproporphyrin, whereas one of them produced increased levels (Table Xl). Phenolic acids, therefore, are not essential for growth or coproporphyrin production, under the experimental conditions employed. The most effective inhibitors i n Class II are meta-substituted benzoic acids. Dipicolinic acid i s , i n effect, such a compound. It i s interesting that anthranilate (Table V) and m-hydroxybenzoic acid inhibited phenolic acid production about 50 per cent, whereas 3-hydroxyanthranilic acid inhibited i t 100 per cent (Table XIV). The effects of these inhibitors on DHB synthesis by cell-free extracts are being investigated. The inhibitory effect of sideramines is probably due to their f a c i l i t a t i n g the uptake of iron by the c e l l , a conclusion (Section VI) which is supported by the failure of desferri-ferrichrome to 3+ produce the inhibition seen with ferrichrome. Since Fe : ( D H B ( G ) ) . is a more effective inhibitor of phenolic acid production than iron alone, i t would seem that any compound able to form a f e r r i c iron complex to which the c e l l i s permeable w i l l be able to inhibit phenolic acid production. This affords an experimental approach to the study of the specificity of the iron uptake mechanism(s) of B. subtilis (Section V). Section IV: - Effects of aeration and glucose concentration on growth and phenolic acid excretion Introduction A l l three wild type strains which were studied extensively produced phenolic acids during logarithmic growth i n iron-deficient medium containing high levels ( l per cent) of glucose (Table V), One of these strains (WB-746), however, excreted only DHB and no DHBG under these conditions (Table V). Unlike strains B - 1 4 7 1 and W - 2 3 , the accumulation of phenolic acid i n WB-7̂ -6 was not affected by inhibition of the TCA cycle (Table XIV). It was found sub- sequently that WB-746 excreted coproporphyrin at lower glucose levels, and that excretion was not initia t e d u n t i l the late station- ary period (Fig. 1 3 ) . These observations suggested that the TCA cycle i n WB-746 was only p a r t i a l l y functional under the conditions employed. Because high levels of glucose have been shown to repress the operation of this cycle i n B. subtilis ( 1 1 , l6, 1 7 ) , phenolic acid and coproporphyrin production i n WB-746 were studied in medium containing lower levels ( 0 . 3 per cent) of glucose. In addition, the effect of aeration on phenolic acid production was studied i n the three strains. 1 . Effect of aeration on phenolic acid production When strains B - 1 4 7 1 and W - 2 3 were grown in s t i l l cultures, containing 1 per cent glucose, the generation time was 1 9 0 min and the maximum c e l l density resulting over a 2 4 hr period was only Q 7.5 x 1 0 cells per ml (Table XVl). Under these conditions, no 7 5 Table XVI. Effect of aeration on growth and phenolic acid production i n wild-type strains. Strains Aeration Generation time (min) Max. c e l l density (cells per ml) O.D. at 5 1 0 mu O'.D. at 4 0 8 mu B - 1 4 7 1 maximum 1 3 0 2 . 8 x 1 0 9 0 . 5 5 1 . 6 B - 1 4 7 1 s t i l l culture 1 9 0 Q 7 . 5 . x 1 0 0 0 . 2 W - 2 3 maximum 1 3 0 2 . 8 x 1 0 9 0 . 4 5 1 . 6 W - 2 3 s t i l l culture 1 9 0 8 . 0 x 1 0 8 0 0 . 3 WB-746 maximum 1 9 0 2 . 8 x 1 0 9 1 . 2 1 . 3 WB-746 s t i l l culture 1 9 0 2 . 7 x 1 0 9 1 . 1 0 . 0 5 Determinations were made over a 2 4 hr growth period, and the maximum values are reported. 7 6 DHB(G-) and only 1 0 per cent of the normal level of coproporphyrin accumulated (Table XVT). Under aerobic conditions, the generation time of these strains was 1 3 0 min, the maximum c e l l density achieved was 2 . 8 x 1 0 ^ per ml, and phenolic acids and coproporphyrin were produced (Table XVT). When WB - 7 4 6 was grown i n s t i l l culture, no coproporphyrin was produced; but the generation time, maximum c e l l density and production of DHB were identical to those obtained under aerobic growth conditions (Table XVI). 2 . Effect of glucose concentration on phenolic acid excretion When WB - 7 4 6 was grown i n medium containing 0 . 3 per cent glucose, a diauxic growth curve was observed (Fig. 1 7 ) . The f i r s t 1 0 hr growth period was followed by a 5 hr plateau, during which there was no increase i n c e l l number; this was followed by a second period of logarithmic growth. . DHB production was initia t e d after 8 hr incubation, and continued logarithmically for 3 hr. The DHB con- centration then decreased linearly during the plateau period. Coproporphyrin was produced only during the second logarithmic growth period (Fig. 1 8 ) . 3 . Oxidation studies Because the growth medium contained 0 . 1 $ citrate and 0 . 3 $ acetate, the oxidation of these substrates by WB-746 was studied. Cells harvested and washed (see Materials and Methods) after 1 0 hr growth- (Fig. 1 8 ) showed a lag of 1 . 5 hr before acetate and citrate were oxidized (Fig. 1 9 ) . Even after this period, oxidation of these substrates proceeded very slowly. Extracts prepared from these LU Q_ 00 LU O 0 5 10 15 2 0 HOURS Figure 18. Growth of s t r a i n WB-746 i n i r o n - d e f i c i e n t medium containing 0.3$ glucose. C e l l number (0), DHB (X) and coproporphyrin (^) were measured over a 2k hr incubation per iod . 78 Figure 19. Oxidation c a p a c i t i e s of s t r a i n WB-746 grown i n i r o n - d e f i c i e n t medium containing .0.3% glucose. • C e l l s harvested a f t e r 10 hr oxidized acetate (©) • and c i t r a t e (&) a f t e r a $0 min lag. C e l l s harvested after.17 hr growth oxidized acetate (0) and c i t r a t e without a l a g (A). 1 0 hr cells were also unable to oxidize acetate or citrate. In contrast, cells harvested after 1 7 hr growth (Fig. 1 8 ) oxidized acetate and citrate without a lag. At no time during growth were cells able to oxidize DHB(G) in the presence or absence of 1 mg Fe per l i t r e . The extended plateau (Fig. 1 8 ) suggested that iron deficiency may have prevented the formation or functioning of TCA cycle enzymes. The addition of 1 mg iron per l i t r e at 9 hr, however, did not reduce this plateau period. Unlike strain WB - 7 4 6 , strains B - 1 4 7 1 and W - 2 3 did not display diauxic growth when grown in medium containing 0 . 3 per cent glucose, and were able to oxidize acetate and citrate without a lag when harvested at any time after 7 hr incubation. Discussion The i n a b i l i t y of strain WB - 7 4 6 to produce coproporphyrin during logarithmic growth in iron-deficient medium (Fig. 1 3 ) appeared to be related to i t s i n a b i l i t y to oxidize acetate and citrate i n the presence of glucose. Prior to and during subsequent acetate and citrate oxidation, the level of DHB i n the culture supernatant decreased; this decrease was independent of the level of iron i n the medium. Ho oxidation of DHB was observed by cells harvested at any time during growth. It is possible that during the plateau preceding acetate and citrate oxidation (Fig. 1 7 ) DHB served, v i a the quinone form, as an electron transport by-pass, circumventing the normally operative cytochrome system. Gray et a l ( 2 3 ) have indicated that when an organism with a high aerobic and anaerobic rate of glycolysis was grown with adequate ' glucose, enough ATP would become available from the Embden-Meyerhof pathway to minimize the role of the TCA cycle i n energy production. These workers further suggested that the TCA cycle enzymes are induced or repressed i n three groups, each under independent control: enzymes involved in the synthesis of (a) tricarboxylic acids, (b) 5-carbon dicarboxylic acids, and (c) 4-carbon dicarboxylic acids. In a medium containing adequate glucose, the synthetic portion (i.e. steps leading to the synthesis of a-ketoglutarate) of the cycle would predominate. This appears to be the case in W B - 7 4 6 . Glucose was much less inhibi- tory to the oxidative functioning of the TCA cycle i n strains B - 1 4 7 1 and W - 2 3 . The production of high levels of DHB but no DHBG in s t i l l cultures (Table XIV) again reflects the relationship of DHBG production to TCA cycle activity. 81 Section V: - Iron transport and phenolic acids Introduction Phenolic acids have very strong a f f i n i t i e s for f e r r i c iron ( 2 6 ) , and i t has been suggested that they may be involved in iron transport in B. subtilis (40). The production of phenolic acids was related not only to the level of iron in the medium (Fig. 2 ) , but also to the iron requirements of the c e l l (see Section I i ) . Therefore, an examination was made of the uptake of iron by B. subtilis, and the effects of phenolic acids on this process. 1 Preliminary experiments Labelled iron was added to sterile medium and the mixture incubated as described in Materials and Methods. Samples were removed at intervals over a 50 min period and passed-through 0.45 U Millipore membranes. No radioactivity was retained on the membranes. Therefore, insoluble iron compounds, which might be retained on the membranes, were not being formed during the experiments described below. For uptake experiments, i t was found that washing of c e l l samples with 10.0 ml medium removed no more iron from the membranes than washing with 2.0 ml. The smaller volume was used in a l l experiments. 2. Experiments with iron-deficient cultures. Iron transport as a function of culture age The rate of iron uptake was proportional to c e l l concentration, g i.e. culture age, up to a density of 2.0 x 10 /ml. At higher densities, the curve became sigmoid (Fig. 2 0 ) . It should be emphasized that these experiments employed growing cells, and that the culture medium contained traces of iron. I t was possible that the increased rate of transport observed above a c e l l density of 2.0 x 10 /ml reflected the development of an increased capacity to take up iron i n response to the approach to an iron-deficient state. A l l subsequent experiments were performed using a c e l l density of 5«5 x 10 /ml. In the absence of added iron, cultures started to produce DHBG at a c e l l density of about 7-0 x 10 /ml (see (Fig. l l ) . 3. Iron transport as a function of energy The addition of iron led to an i n i t i a l rapid binding of label to the cells (Fig. 2 l ) . This was followed by a slower, linear rate of uptake u n t i l about 30 min, after which time the rate decreased (Fig. 2 1 ) . A mixture of sodium azide and iodoacetamide inhibited the slower phase of uptake, but was without effect on the i n i t i a l rapid binding of iron (Fig. 2 l ) . The gradual decrease in bound iron i n the presence of the inhibitors was the result of c e l l l y s i s . k. The effect of temperature on iron transport The binding of iron to the cells was temperature independent (Fig. 2 1 ) . The slower rate of uptake was temperature dependent, with an optimum at 37 C (Fig. 2 2 ) . Measurements at temperatures above 1+5 C were not made because of c e l l l y s i s . 83 F i g u r e 20. I r o n uptake as a f u n c t i o n o f p h y s i o l o g i c a l age. I r o n was added t o c u l t u r e samples a f t e r growth t o the dens i t y , i n d i c a t e d . Each p o i n t represents the r a t e o f uptake measured over the i n t e r v a l 3-8 min a f t e r i r o n a d d i t i o n . Qk cn LU Q. CL 3 CD lO IX. 2 .0 0> 10 2 0 30 4 0 MINUTES Figure 21. Iron uptake as a f u n c t i o n of energy. The rate of i r o n uptake was measured at 37 C (0), o- C (A), at 37 C a f t e r pre-incubation f o r 30 min with 30 mM sodium azide and 1 mM iodoacetamide (X), and .at 37 C a f t e r the ad d i t i o n of 0.1$ c i t r a t e (A). 0 . 3 i 20 2'5 3'0 35* 4 0 ~45 TEMPERATURE • F i g u r e 2 2 . • E f f e c t of temperature on i r o n uptake. Culture. samples were pre-incubated at the appropriate temperature f o r 10 min before the a d d i t i o n of i r o n . 5 . Incorporation of iron into TCA-insoluble material A significant fraction of the iron bound to the cells, and of that subsequently taken up, was insoluble i n cold 5 $ TCA (Fig. 2 3 ) . This was true also of the iron bound at 0 C. 6 . Iron transport as a function of iron concentration The rate of iron uptake at 3 7 C was proportional to the iron concentration, up to a level of kO mug/ml (Fig. 24). As the concentration was increased beyond 4 0 mug/ml, the rate of uptake appeared to decrease (Fig. 2k). Further examination of uptake at high iron concentrations showed that a period of very rapid uptake, lasting about 6 - 7 min, was followed by a period during which iron was lost very rapidly from the cells. This loss of iron was not observed at lower iron concentrations (i.e. at 5 mug/ml), not even when cells were allowed to take up iron for 3 0 min, were fil t e r e d , washed with iron-free medium, and then incubated i n iron- free medium for a further 3 0 min. The binding sites on the cells were saturated (i.e. Fe uptake at 0 C) at an iron concentration of 2 0 0 mug/ml (Fig. 2 5 ) , while the maximum rate of uptake was obtained at kO mug iron/ml. 7 . The effect of citrate on iron uptake The medium used contained sodium citrate. It has been reported that citrate could replace the DHB requirement of a multiple aro- matic auxotroph of Escherichia c o l i ( 6 7 ) . When citrate was omitted from the medium, the rate of iron uptake by cells was increased about 4-fold, and the amount of iron taken up was increased about cc u C L LJ 0. ID 3 . 0 i 2 . 0 1.0 / / 0 / . X , .0. T T .0 10 2 0 3 0 4 0 50 MINUTES F i g u r e . 2 3 . I n c o r p o r a t i o n o f i r o n i n t o TCA- inso lub le m a t e r i a l . Whole c e l l s at 37 C (A), TCA- . i n s o l u b l e f r a c t i o n at 37 C ( 0 ) , whole. ' c e l l s a t 0 C (X) , and TCA- inso lub le f r a c t i o n at O.C («).. 88 ug Fe PER L I T R E Figure 2h. Rate of i r o n uptake as a funct ion of i r o n concentration. I ron -de f i c ien t c e l l s ( 0 ) , i r o n - s u f f i c i e n t c e l l s (X). Uptake rates were measured over the i n t e r v a l 2-6 min a f te r i r o n add i t ion . 89 0 itiO 2 b 0 3 0 0 . 4 0 0 5 0 0 ^ig Fe P E R L I T R E Figure 25. Binding of i r o n to c e l l s at 0 C as a fu n c t i o n of i r o n concentration. Iron d e f i c i e n t c e l l s (0), i r o n s u f f i c i e n t c e l l s (X). Uptake, was measured 3 min a f t e r a d d i t i o n of i r o n . 4 0 $ (Fig. 2 6 ) . There was also a 2 - 3 fold increase in the amount of iron bound by the cells at 0 C (Fig. 2 6 ) . The addition of 5 - 3 0 0 ug DHB(G)/ml to cultures grown in the absence of citrate caused a 2 0 $ increase in the level of iron uptake (Fig. 2.6). The addition of 0 . 1 $ citrate at zero time or after 3 0 min incubation, however, did not affect iron uptake by cells growing i n iron- deficient, citrate-containing medium (Fig. 2 1 ) . 8 . The effect of phenolic acids on iron uptake The addition of 5 ug/ml of DHB or DHBG at the same time as 5 mug of iron/ml resulted i n a 4 - f o l d increase i n the rate of iron uptake (Fig. 2 7 ) . Increased concentrations of phenolic acid up to 3 0 0 ug/ml did not give any further stimulation of the rate of uptake. It should be pointed out that the phenolic acids were mixed with the iron and l e f t at room temperature to equilibriate for 1 5 min ( 2 6 ) prior to their addition to the cell s . In the absence of a phenolic acid about 5 5 $ of the added iron was taken up by the ce l l s . In the presence of a phenolic acid about 9 ° $ was taken up (Fig. 2 7 ) . Phenolic acid in the range 5 - 3 0 0 ug/ml decreased the binding of iron at 0 C by about 5 0 $ . If less than 5 ug/ml phenolic acid was added, the rate of uptake was s t i l l elevated, but there was some decrease i n the amount of iron taken up by the cells (Fig. 2 7 ) . The addition of 5 ug/ml of phenolic acid with 0 . 5 mug iron/ml did not affect the rate of uptake, but i t did increase the amount of iron taken up from 3 0 $ to 7 0 $ (Fig. 2 8 ) . The binding of iron at 0 C again was reduced i n the presence of phenolic acid (Fig. 2 8 ) . on LU Q. LU in ° 6 " 10 20 30 40- 50 MINUTES Figure 26. E f f e c t of growth without c i t r a t e on i r o n uptake. Uptake was measured at 37 C with 5 Ug DHB(G)/ml (X), and with no addition (A). Binding was at 0 C with 5 ug DHB(G)/ml (O), and with no addi t i o n ( A ) . MINUTES .Figure 2 7 . Effect of phenolic acids on iron uptake. Iron was added to a l l flasks at 5 mug/ml. Uptake was measured at 3 7 C with 5 - 3 0 0 ug • • . ' DHB(G)/ml ( A ) , 1 u g DHB(G)/ml ( 0 ) , 0 . 5 ug DHB(G)/ml.(A), and with no addition (X). . Binding was measured at 0 C with 5 - 3 0 0 ug DHB(G)/ml (A),, and with no addition (t). A A A A A A 0 10 20 30 40 50 60 MINUTES Figure 28. E f f e c t of phenolic acids on uptake at lower l e v e l s of i r o n . Iron was added to a l l f l a s k s at 0.5 mug/ml. Uptake was measured at 37 C with 5 ug DHB(G)/ml (0), and with no ad d i t i o n (X). Binding was measured at 0 C'with 5 g: DHB(G)/ml (A), and with no addi t i o n (•). 9 . Experiments with iron-sufficient cultures Cells grown in iron-sufficient medium bound less than 5 $ of the level of iron bound by iron-deficient cells at 0 C (Fig. 2 5 ) . In addition, the rate of uptake was greatly reduced as a consequence of growth i n iron-sufficient medium (Fig. 2 4 ) . The addition of phenolic acids to iron-sufficient cells did not affect either the binding or the rate of uptake of iron. 1 0 . Experiments with mutant strains After growth i n iron-deficient medium, the rates of iron uptake in strains dhb-1, dhb-4 and dhb-5 were less than 2 0 $ of the rate observed i n strain B - 1 4 7 1 (Fig. 2 9 ) . The binding of iron at 0 S was reduced by 8 0 $ i n these strains (Fig. 2 9 ) . In the presence of 5 0 'ug DHB(G)/ml, the rate of uptake was increased 1 0 - f o l d i n strain dhb-4, but the rates i n strains dhb-1 and dhb-5 were unchanged (Fig. 2 9 ) . Discussion The results indicate that B. subtilis possesses a system for the transport of iron, which has the properties of an active trans- port system ( 3 ) , being temperature and energy-dependent. Growth under conditions of iron-deficiency results in a considerable increase in the capacity of the transport system. In fact, the iron-deficient c e l l develops this capacity to such an extent that eventually i t can no longer retain the high levels of iron taken up when increased quantities are added to the medium. In addition to the transport system, cells of B. subtilis can bind iron at 0 C, 95 rr LU LU 3 C7> 3.0- 2.0- 0 10 20 M INUTES Figure 29. Iron uptake i n mutant s t r a i n s . S t r a i n dhb-1, dhb-U,. and dhb-5: without addition at 37 C (©), and at 0 C (X). Strains dhb-1 and dhb-5 with 50 ug DHB(G)/ml at 37 C (0). S t r a i n dhb-k with 50 ug DHB(G)/ml at 37 C (•). and this binding also i s increased considerably by growth under iron-deficiency. The addition of a phenolic acid to iron-deficient cells stimulated the rate and increased the level of iron transported. At lower iron concentrations, only the level of iron transported was altered by phenolic acid addition. Mutants unable to produce normal levels of phenolic acid showed a decreased rate of transport and a decreased binding capacity. This suggests that phenolic acids are involved directly in iron transport. At 3+ neutral pH, the 3:1 complex of DHBG:Fe is favoured (Fig. 30) (ho), so that the transport system would be able to recognize the complex i n the presence of a great excess of free DHBG. The properties of the mutant strains suggest that phenolic acids may also be involved i n the binding of iron to the c e l l . The transport system may be inducible because the capacity for transport increases during iron-deficiency. If i t i s inducible, the question of the nature of the inducer becomes important. It 3+ is unlikely to be the Fe (DKLXG)).^ complex because the increased capacity i s the consequence of a lack of iron. An attractive alternative i s that the system is repressible, with the corepressor 3+ being an iron complex, possibly Fe (DHE^G)).^ Citrate can bind iron, and could serve to make limiting iron more available to the c e l l , thereby postponing the onset of iron- deficiency. In the present case, the omission of citrate from the medium led to an increased capacity for iron transport. Almost half of the iron taken up by the cells was insoluble in cold TCA. This was true also of the iron bound to the cell s . 97 The nature of this "insoluble" iron is not clear at present, but i t i s being investigated. Attempts are being made to isolate mutants t o t a l l y unable to synthesize phenolic acids so that their capacities for iron transport and binding may be studied. 99 Section VI: - Control of iron transport Introduction Phenolic acid production started earlier and continued at a faster rate i n WB - 7 4 6 than i n B - 1 4 7 1 (Fig. 1 1 , . 1 3 ) . Conversely, coproporphyrin production started later and occurred at a slower rate in WB-746 than i n B - 1 4 7 1 (Fig. 1 1 , 1 3 ) . Oxidative functioning of the TCA cycle in WB-746 was inhibited by 0 . 3 $ glucose (Fig. 1 8 ) , while that of B - 1 4 7 1 was not. Hem-1 had no oxidative TCA cycle in unsupplemented media, but the normal functioning of this cycle was restored following supplementation with ALA (Fig. l 4 , p. ). These properties prompted an investigation of the relationship between iron transport capacity and heme-iron requirement. 1 . Effect of heme-iron requirement on transport capacity When uptake studies were conducted, the rate and extent of iron uptake in B - 1 4 7 1 greatly exceeded that in WB-746 or of hem-1 i n unsupplemented medium (Fig. 3 1 ) . When hem-1 was grown in medium supplemented with 5 Hg ALA per ml, Its iron uptake capacity approached that of the parent strain (Fig. 3 1 ) . 2 . Effect of ferrichrome + 3 Addition of the phenolic acid Fe complex inhibited phenolic acid (Fig. 4 ) and coproporphyrin production (Fig. l 4 ) by strain B - 1 4 7 1 . This was probably the result of phenolic acids increasing iron uptake (Fig. 2 7 ) . The addition of low levels ( 0 . 4 ug per ml) of ferrichrome to iron-deficient cultures of B - 1 4 7 1 at zero time or after 1 0 0 0 10 2 0 3 0 4 0 MINUTES Figure 3 1 * - • .Strain differences i n iron uptake capacities. Iron uptake was measured i n strains B - 1 4 7 1 ( 0 ) and WB - 7 4 6 (^). Hem-1 growing i n the absence of ALA had a low iron uptake capacity (©). When hem-1 was grown i n . the presence of 5 Ug ALA per ml, i t s uptake capacity (X) was comparable to that of the parent strain ( 0 ) . 8 hr growth, inhibited DHBG (Fig. 1 7 ) and coproporphyrin production (Table XIV). The level of iron provided by this supplementation is about 2 0 ug per l i t r e , a level which alone caused no inhibition of excretion (Fig. 2 ) . Ferrichrome has been implicated as an iron solubilization factor for several microorganisms (39? 41). F e r r i - chrome and desferri-ferrichrome were tested therefore for their effects on iron transport. When cells were incubated with 5 mug ^%e per ml, 5 5 $ of this iron was available to the c e l l over a 4 0 min incubation period (Fig. 3 2 ) . When 0 . 4 ug ferrichrome per ml was added simultaneously with the radioactive iron, there was a 3 0 $ decrease in the amount of label incorporated into the cell s . Pre- 59 incubation of the ferrichrome with Fe for one hr, which would allow about 2 2 $ of the ferrichrome-bound unlabelled iron to exchange with Fe ( 3 4 ) resulted i n only an 1 8 $ decrease in the amount- of radioactive label incorporated compared to cells i n the absence of ferrichrome (Fig. 3 2 ) . The addition of desferri-ferrichrome simultaneously with ^ % e , made 99$ °f the labelled iron available to the cells within 1 5 min incubation (Fig. 3 3 ) . r 3 . HQ, mutants The properties of strains HQr-l-6 (Section I) suggested that these strains required less available iron than the parent strain. To check this theory, iron transport studies were conducted. When these strains were grown in the absence of HQ, the rate and extent of iron uptake were significantly increased (Fig. 3 4 ) . Supple- mentation of media with 0 . 1 ug HQ per ml (which did not affect the ;; growth rate) further increased the rate and extent of transport (Fig. 3.0 cr LU CL LU CL 2.0 CD LO £ - 1 . 0 - 10 2 0 3 0 M I N U T E S T 4 0 Figure 32. - E f f e c t of ferrichrome on i r o n transport.- •Ferrichrome .'• (0.4 ug/ml) was pre-incubated f o r 1 hr with' i r o n ( A ) , added simultaneously with i r o n (0), or ferrichrome , (0.8 ug/ml) was added simultaneously with i r o n (X). Wo ferrichrome was added to the c o n t r o l f l a s k (8) . 103 if) LU O LU Q_ LU Q_ 3 CD ID 0) Li- en 4.0 10 20 30 MINUTES 40 Figure 33• E f f e c t of ferrichrome on i r o n uptake i n s t r a i n B-1471. C e l l s were grown to a v i a b l e density of 5. 5 x 10" per ml p r i o r to study. 59Fe (5 mug per ml) -and d e s f e r r i - . ferrichrome (0.4 ug per ml) were then added (at zero . time (©). Ferrichrome was- omitted from the c o n t r o l f l a s k (0). CO o 5 , 0 LLI UJ < Q_ 3 L L . 4 . 0 - 3 . 0 2. .0" -—© 8' 1.01 1 - o 10 2 0 3 0 4 0 MINUTES F igu re 3 4 . I r o n uptake by s t r a i n H Q r - l . Uptake o f ^^Fe'oy s t r a i n B-1471 (0) was compared to uptake by s t r a i n H Q r - l grown i n . the absence (X) and presence •'(©) o f 0.1 u-g/ml HQ. 'Discussion A requirement for heme-iron appeared to cause an increase in the transport capacity of ce l l s . The inhibitory effect of low levels of ferrichrome on DHBG and coproporphyrin excretion i n B - 1 4 7 1 appeared to be related to i t s a b i l i t y to drastically increase both the rate and extent of iron uptake. The observations that uptake 5 9 of 5 mug Fe per ml was decreased by ferrichrome addition, and was increased by supplementation with desferri-ferrichrome may indicate that: (i) ferrichrome was competing at the active site for free iron transport, or ( i i ) upon entering the c e l l , ferrichrome caused inhibition of free iron uptake. It has been suggested ( 4 l ) that ferrichrome is active i n the insertion of iron into porphyrin. Thus, ferrichrome may have provided iron directly for heme synthesis i n B - 1 4 7 1 , allowing normal iron sufficient porphyrin biosynthesis, thereby eliminating the requirement for DHBG synthesis. Results with B - 1 4 7 1 (Section II) indicated that i f iron were available for heme synthesis, neither coproporphyrin nor DHBG would accumulate. The extent of iron transport under the experimental conditions employed allows speculation concerning the level of extracellular iron remaining prior to the excretion of DHBG and coproporphyrin (Fig. l l ) by this strain. Transport studies were conducted under conditions identical to those used for studying excretion products, +3 except that 5 Mg Fe per l i t r e were added to cells approximately one hour prior to the onset of phenolic acid excretion (Fig. l l ) . Of this level of added iron, 4 5 $ was unavailable to cells i n the absence of phenolic acids (Fig. 2 6 ) , so that the extracellular iron concentration prior to DHBG excretion could be estimated to be 5 ug per l i t r e . Excretion of DHBG by cells would then f a c i l i t a t e solubilization of about 90$ of this iron (Fig. 2.6) preceding the onset of coproporphyrin production (Fig. 1 1 ) . Only about 0.35 Hvg per l i t r e of the remaining iron would subsequently be available to the c e l l (Fig. 27), which accounts for the excretion of very high levels of coproporphyrin (Fig. l l ) . r HQ mutants had an increased iron uptake capacity compared to the parent strain. When these mutants were grown in the presence of 0.1 ug HQ per ml, their iron transport a b i l i t y was increased further. HQ -1-6 were unable to excrete normal levels of DHBG or coproporphyrin i n the absence of HQ (Section I) . In the presence of 0.1 ug HQ per ml, however, these products did accumulate i n the medium (Section I ) . It remains to be determined i f HQ forms a complex with iron which then (a) cannot be taken up by the c e l l , or (b) can be taken up by the c e l l but from which the iron cannot be released. GENERAL DISCUSSION Although the factor exerting the greatest effect on phenolic acid production by B. subtilis was the level of iron i n the medium, production was influenced also by other aromatic compounds syn- thesized from chorismic acid and by the requirements of the c e l l for iron. The effects of the aromatic amino acids i n reducing phenolic acid production were to be expected because i t is known that the aromatic biosynthetic pathway in B. subtilis i s subjected to feed- back inhibition by these amino acids ( 2 8 , 3 3 ) - Repression of the enzymes of the pathway has been demonstrated recently ( 4 3 ) and i t is significant that i t appears to be mediated most strongly by tyrosine, the amino acid exerting the greatest degree of inhibition of phenolic acid production. The aromatic amino acids would reduce the level of chorismic acid available for DHB synthesis. The marked inhibitory effect of anthranilic acid appeared to be directly on DHB synthesis from chorismic acid. That the production of phenolic acids was related to the actual iron requirements of the c e l l was shown by the properties of strain B - 1 4 7 1 hem-1. This strain, blocked at the f i r s t step of porphyrin biosynthesis, did not produce phenolic acids under iron-deficiency. In addition, s t i l l cultures of strains B - 1 4 7 1 and W - 2 3 did not produce phenolic acids under iron-deficiency. Such cultures would have had a low capacity for porphyrin biosynthesis. The production of DHB by strain WB-746 under these conditions may have reflected the decreased iron uptake capacity of this strain. It 108 was possible that, i n spite of these observations, the relationship between phenolic acid production and a requirement for iron for hemin biosynthesis was more apparent than real, with phenolic acid and coproporphyrin production occurring independently i n response to an iron deficiency. However, hemin inhibited phenolic acid production. It has been shown that hemin i s probably unable to satisfy the non-heme iron requirements of certain bacteria (39)> suggesting that i t does not readily release i t s iron inside the cell s . It was significant, also, that phenolic acid production started before coproporphyrin production. . This aspect of the problem is to be extended by examining mutants blocked after ALA for their capacities for phenolic acid production. It is intended also, to use strain B-1471 hem-1 to determine the heme-iron, the -non-heme iron and the t o t a l iron requirements of strain B-1471 under various conditions. The relative importance of iron for hemin biosynthesis can then be assessed and the relationship of phenolic acid production to porphyrin biosynthesis worked out in detail) DHB and DHBG have strong a f f i n i t i e s for f e r r i c iron (2.6), so that they could chelate low levels of iron in the medium. The development of an increased capacity for iron uptake by cells grown in an iron-deficient medium, and the enhancement of this capacity by phenolic acids, supported the idea that the acids were serving to scavenge the last traces of iron from the medium. The production of phenolic acids could have been initi a t e d when the level of iron inside the c e l l f e l l below a c r i t i c a l value. It remains to be determined what this value might be and whether i t represents total, free or bound iron. Perhaps the most interesting aspect of this problem is the question of how the level of iron in the medium controls the production of phenolic acids. There are two major pos s i b i l i t i e s : the enzymes for DHB synthesis are either repressible or inducible. The nature of the corepressor or inducer is not clear at this point. It might be that iron i t s e l f i s a corepressor, with a c r i t i c a l level being required to activate the repressor. The Fe :(DHB(G ) ) 2 complex might be the corepressor; i n this case i t would be necessary to postulate that sideramines inhibit phenolic acid production by carrying iron into the c e l l to allow formation of the Fe :(DHB(G ) ) 2 ̂ ^P-1-625-' Alternatively, the system might be induced by free D H B ( G ) . In the presence of sufficient iron, a l l the D H B ( G ) could be i n the Fe^ +: (DHB(G) ) complex; as the level of iron f a l l s below the c r i t i c a l level, free DHB(G) could appear to induce the system. It is unlikely that the m-substi- tuted benzoic acids inhibited phenolic acid production by acting as corepressors since none of them could complex iron, and the medium was iron-deficient. They could, however, have competed with free DHB(G) to prevent induction. The increased capacity for iron uptake seen in iron-deficient cells could have resulted from the appearance of a specific permease in response to the i n i t i a t i o n of phenolic acid production. This point must be investigated further, and an attempt made to determine i f such a permease is under the same system(s) of control as the enzymes involved i n DHB synthesis. The system can be subjected to genetic analysis, using transformation and 110 or transduction. Attempts w i l l be made to isolate mutants i n which the production of phenolic acids i s no longer controlled by iron. Ferrichrome and ferrioxamine served as effective "inhibitors" of phenolic acid production, and ferrichrome served very effectively to carry iron into the c e l l . If a specific permease was involved i n carrying f e r r i c iron complexes into the c e l l , i t might be character- ized by being relatively non-specific with regard to the ligands binding the iron. The specificity of the iron uptake mechanism seen i n iron- deficient cells could be examined using phenolic compounds closely and distantly related to DHB and DHBG. I l l LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. 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