UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Consequences of bacillus subtilis in iron deficiency Peters, Walter Joseph 1968-12-31

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
831-UBC_1968_A1 P48.pdf [ 4.75MB ]
Metadata
JSON: 831-1.0104612.json
JSON-LD: 831-1.0104612-ld.json
RDF/XML (Pretty): 831-1.0104612-rdf.xml
RDF/JSON: 831-1.0104612-rdf.json
Turtle: 831-1.0104612-turtle.txt
N-Triples: 831-1.0104612-rdf-ntriples.txt
Original Record: 831-1.0104612-source.json
Full Text
831-1.0104612-fulltext.txt
Citation
831-1.0104612.ris

Full Text

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 o f 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 a c c e p t t h i s t h e s i s as.conforming t o t h e required  standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1968  In p r e s e n t i n g  for  thesis  an a d v a n c e d d e g r e e  that  the  Study.  thesis  Library  for  agree  scholarly  or  publication  without  shall  I further  Department  or  this  at  in p a r t i a l  the U n i v e r s i t y of  make i t  that  freely  purposes  my w r i t t e n  this  thesis  may be g r a n t e d  for  permission.  Department The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  for  Columbia  It  is  financial  of  British  available  permission  b y hi.is r e p r e s e n t a t i v e s .  of  fulfilment  for  the  Columbia,  I  reference  and  extensive  by  the  requirements  copying  this  Head o f my  understood  gain  of  agree  shall  that  not  be  copying  allowed  ABSTRACT  Cultures of Bacillus sub t i l l s growing i n an iron-deficient medium produced coproporphyria III (coproporphyrin) and phenolic acids. (2,3-dihydroxybenzoylglycine or "both).  (DHBG),  2,3-dihydroxybenzoie acid  (DHB),  (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 ; ( D H B ( G ) ) 2 J  complex.  Acoimulatlbn of DHB(G) was influenced  by the levels of aromatic amino acids, anthranilic acid, and histidine i n the medium.  In vitro experiments demonstrated that DHB was formed  from chorismic acid. B-1V71  showed that  DHB  In vivo and i n vitro experiments with strain was coupled to added glycine to form  DHBG.  Disappearance of DHB(G) was observed i n a l l strains studied, but oxidation did not occur. Phenolic acid production always preceded coproporphyrin production. Phenolic acids have very strong affinities for f e r r i c 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 ( i i ) the Fe" (DHB(G))^ complex was a more potent inhibitor of coproporphyrin 3  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 coproporphyrin and phenolic acids than the wild-type strain (v) some mutants defective i n 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 i n 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 i n strain B-1471.  The inhibitory action of f e r r i -  chrome was shown to be due to i t s a b i l i t y 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 i n 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 i n an iron-deficient medium.  Mutants resistant to 8-hydroxyquinoline had an increased  capacity for iron-uptake under these conditions.  iii  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. W i l d t y p e s t r a i n s . . . . . . . . . . . . . . .  5  2. Mutant s t r a i n s . . . . . . . . . . . . . . . . .  5  II. M  e  (a) Spontaneous mutants  . . . . . . . . . .  (b) NTG-induced mutants  .  .  .  .  .  .  .  .  d  i  a  .  .  .  .  .  .  III. Cultural conditions. ; .  .  .: .  .. .  .  5  .  .  . .  .  2. P h e n o l i c  acids  .. .  3. C o p r o p o r p h y r i n I I I .  . .  .  V. P r e p a r a t i o n  .  10 10  .. .. .. .. .. .  11  .  .  .  .  .  .  .  . . . . . .  11 .  12  . . . . .  12  o f extracts'.  12  : V I . Enzyme a s s a y s . . . . . . . . . . . . . . . . . . . . . 1. S y n t h e s i s .2. S y n t h e s i s  o f DHB.. .. .; .. .. .. .. .. . o f DHBG  . 1. P r e p a r a t i o n 2. I r o n uptake  13 13 . 13  . VII.Respiration. studies. . . . . . . . . . . VIII. Iron transport.studies.  10  .  •;k. I d e n t i f i c a t i o n o f g l y c i n e . 5 . Protein concentration  5 9  IV. D e t e r m i n a t i o n s . . . . . . . . . . .1.' Growth. . . . . . . . . . . . . . . . . . . . .  '  . . . . . .  .. .. •  13  .: .  13  of cells . . . . . . . . . . . . .  13 14  iv  T a b l e o f Contents (Continued) Page (a)  Iron-deficient cells  .. .  (b.) I r o n - s u f f i c i e n t . c e l l s .  lh  . . . . . .  lh  3. Assay o f i r o n uptake . . . . . . . . . . . IX.  Chemicals .  RESULTS.  .....  .  .  .  .  .  .  .  ...........  .  .  .  lh  .  .......  . 15 .  17  Section I : General properties o f phenolic acid excretion. . . . . . . . . . . . .  17  :  .1. E x c r e t i o n o f p h e n o l i c a c i d s by w i l d type s t r a i n s .  .; .  .  .  .  .  . . .. .  2. I n f l u e n c e o f i r o n on DHBG p r o d u c t i o n . .3.  Fe  .. .  17  .....  . 17  .. .. .; .. .. .. .  20  :(DHBG)^ complex and t h e c o n t r o l  o f DHBG p r o d u c t i o n . .  .. .  h. E f f e c t o f aromatic amino a c i d s on DHBG p r o d u c t i o n . . . . . . . . . .  .......  20.  5. Source o f g l y c i n e i n DHBG. . . . . . . . . . . . .  - 2k  ,6. V a r i a t i o n s among s t r a i n s o f B. ' s u h t i l i s . . . . .  27  . 7.  Effect  o f h i s t i d i n e on p h e n o l i c  excretion. . . . . . . . . . .  acid 31  . . . . . . . . . . . .  31  .8. I n v i t r o s y n t h e s e s .  ,9. 10.  .........  31  ...............  31  (a)  F o r m a t i o n o f DHB . . . . . . .  (b)  F o r m a t i o n o f DHBG.  M e t a b o l i s m o f DHB(G) b y . s t r a i n  B-1U71.. . . .  35  M e t a b o l i s m o f DHB(G) by s t r a i n s W-23  and WB-746  .  35  V  T a b l e o f Contents  (Continued) Page  11.. P r o p e r t i e s o f mutant s t r a i n s .  37  (a) 5 m e t h y l t r y p t o p h a n r e s i s t a n t s t r a i n s (b) Aromatic  auxotrophs  .; .  .-37  ........  37  (c) HQ r e s i s t a n t mutants . . . . . . . . . . . . . . DISCUSSION .  .  .  .  .  .  .  S e c t i o n I I : The p r o d u c t i o n o f  .  .  .  .  .  37"  kO  .  coproporphyrin  and i t s r e l a t i o n s h i p t o t h e p r o d u c t i o n • k6  of phenolic acids. Introduction  .  .. .; .. .. .; .  .. .. .. .; .. .  46  1. P r o d u c t i o n o f . c o p r o p o r p h y r i n and p h e n o l i c . a c i d s by w i l d - t y p e . s t r a i n s .  h6  .............  • 2. I n h i b i t i o n o f c o p r o p o r p h y r i n  production  by i r o n . . . . . . . . . . . . . . . . . . . . . . . . . 3. I n h i b i t i o n o f c o p r o p o r p h y r i n  production  by hemin  k.  50  . . . .  I n h i b i t i o n of coproporphyrin by aromatic  amino  5. C o p r o p o r p h y r i n  a  50  production  c  i  d  s  5  and p h e n o l i c a c i d p r o d u c t i o n  by mutants o f s t r a i n  B-1471  (a) G l y c i n e auxotrophs.  . . . . . . .  .; .  .  .; .  (b) S e r i n e auxotrophs . . . . . . . . . .  .  .  52 .  . . . .  (c) ALA . auxotrophs . . . . . . . . . . . . . . . . . . (d) DHB DISCUSSION  2  auxotrophs . . . . . . . . . . . . .  52 52 5^ 58 62  vi  T a b l e .of Contents (Continued) Page Section I I I : I n h i b i t i o n of phenolic  acid  production  i n B_. '. s u b t i l j s . Introduction.;  .; .  1. Analogs o f DHB.  .  66  .. .. .; .. .. .. .. .. .  . . . . . . . .  .  . . . . . . . .  66 66  .2. Compounds a f f e c t i n g t h e f u n c t i o n i n g o f t h e TCA c y c l e .  .  .. .. .  .  .. .. .. .  3. E f f e c t o f s i d e r a m i n e s on p h e n o l i c  .. .  66  .: .  68  a c i d and  p o r p h y r i n - p r o d u c t i o n ' b y s t r a i n B-1U71 •  .  DISCUSSION. Section.IV:  • 72 E f f e c t s o f a e r a t i o n and g l u c o s e concentration acid excretion  Introduction.  on growth and p h e n o l i c .  .  .  . . . . . . . . .  .1. E f f e c t o f a e r a t i o n on p h e n o l i c production  .  .  .  .. .. .: .  2. E f f e c t o f g l u c o s e c o n c e n t r a t i o n acid excretion. . . . . . . 3. O x i d a t i o n  studies.  .; .  .  DISCUSSION..............  .1. P r e l i m i n a r y experiments.  .  .  .  • 7^ jk  acid .; .  .  .  .: .  lh  i n phenolic .  .  .  .  76  .; .  .  .. .. .  76  . . . . . . . . .  S e c t i o n V: I r o n t r a n s p o r t and p h e n o l i c Introduction. . . . . . . . . .  .  . . . . . . . . . .  . . . . .. .  .  acids  .  .  79 .  8 l  . . . . . . . . . .  8 l  . . . . . . . . . . . .  8 l  2. Experiments w i t h i r o n - d e f i c i e n t c u l t u r e s . I r o n t r a n s p o r t as a f u n c t i o n o f c u l t u r e .3. I r o n t r a n s p o r t as a . f u n c t i o n o f energy.  .  .  .....  .8l 82  . vii  T a b l e .of Contents  (Continued) Page  h.  The e f f e c t o f temperature on i r o n t r a n s p o r t .  5. I n c o r p o r a t i o n o f i r o n i n t o material;  .; .  .  .  .  .;  . 8 2  TCA-insoluble  .; .  .  .  .; .  .  .  .  .  .  .  86  6. I r o n t r a n s p o r t as a f u n c t i o n o f i r o n . concentration.;  .  .; .  .  .  .  .  7..The e f f e c t  o f c i t r a t e on i r o n uptake  8. The  o f p h e n o l i c a c i d s on  effect  U_P*fcc3,lCS •  •  •  •, •  •  Experiments w i t h mutant s t r a i n s . ;  DISCUSSION .; .  .; .; .  .  .  .  . 8 6  .; .  .. .  .  86  •  •  •  90  cultures.. . ; . ; .  9h  iron  9. Experiments w i t h i r o n ^ s u f f i c i e n t 10.  .  .  •  •  •  .; .  .  .  .; .  9h  .  .  .  .  9h  .  .  Section VI:.Control of i r o n t r a n s p o r t . . . . . . . Introduction .1.. E f f e c t  3. HQ  .  .  o f heme-iron  co,|p3,cx"by . 2. E f f e c t  .  •  •  •  .  .  .  .  .  .  .  .  .. .. . . 99  requirement on t r a n s p o r t •. •  •  •  •  •  of ferrichromc  99' 99  .  .  .  .  .  .  .  .  .  .  .  .  .; .  .  .  .  .  .  .  .  .  .  .  .. . 1 0 5  GENERAL DISCUSSION  .  .  .  .  .  .  .  .  .. .  LITERATURE CITED .  .. .  .  .  .  .  .  .  .  r  mutants  99  DISCUSSION .  .  .. .  . 101  . 107. . I l l  viii  LIST OF TABLES Table  Title  I  B. s u b t i l i s w i l d t y p e s t r a i n s  II  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  III  Page 6 7 - 8  E x c r e t i o n of phenolic acids by w i l d type strains"  18  Distribution of glycine-1-^C  28  Characteristics of phenolic acid excretion by 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  29  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 r o d u c t i o n i n w i l d type s t r a i n s  32  I n v i t r o s y n t h e s i s o f DHB  33  I n v i t r o s y n t h e s i s o f DHBG '  3*+  I n h i b i t i o n of the production of coproporphyrin and p h e n o l i c a c i d s b y hemin i n s t r a i n B-lk-71  53  P r o d u c t i o n o f c o p r o p o r p h y r i n and p h e n o l i c a c i d s . by s e r i n e auxotrophs d e r i v e d from s t r a i n B-1471  55  P r o d u c t s e x c r e t e d b y " r e v e r t a n t s " o f DHB auxot r o p h s d e r i v e d f r o m s t r a i n B-1471  59  C o p r o p o r p h y r i n p r o d u c t i o n i n Dhb-4  6l  E f f e c t s o f a n a l o g s o f DHB on p h e n o l i c a c i d and coproporphyrin production  67  E f f e c t of i n h i b i t o r s acting at the l e v e l of the TCA c y c l e on p h e n o l i c a c i d and c o p r o p o r p h y r i n production  69  XV  I n h i b i t i o n of the production of coproporphyrin by sideramines i n s t r a i n B - l V f l  71  XVI  E f f e c t o f a e r a t i o n o n growth and p h e n o l i c a c i d production i n wild-type strains  75  IV V VI VII VIII IX X XI XII XIII XIV  .  LIST OF FIGURES  Figure  Title  1  Growth and production of DHBG by s t r a i n  2  E f f e c t of iron added at zero time on the production of DHBG by s t r a i n  3  B-1471  21  B-1471  E f f e c t on DHBG production of adding iron or DHB to strain  19  B-1471  22  -4  4  E f f e c t of simultaneous addition of 3-5 x 10 M DHB and 1 mg of iron per l i t r e on DHBG production by s t r a i n B-1471  23  5  E f f e c t of adding end product amino acids on DHBG  25  production by s t r a i n 6  E f f e c t of adding a n t h r a n i l i c acid on the production of DHBG by s t r a i n  7  B-1471  E f f e c t of iron, added at zero time, on the production of DHB(G) by strains WB-746,  8  26  B-1471  Metabolism of DHBG by s t r a i n  30  B-1471 and W-23 B-1471  after addition  36  of i r o n  9  Production of DHBG by MT  r  strains of B-1471  38  10  Scheme of aromatic biosynthesis  42  11  Growth and production of phenolic acids and copro-  47  porphyrin by s t r a i n 12  B-1471  Growth and production of phenolic acids and coproporphyrin by s t r a i n  48  W-23  13  Growth and production of phenolic acids and coproporphyrin by s t r a i n WB-746  49  14  E f f e c t on coproporphyrin production of adding iron and DHB(G)  51  15  E f f e c t of ALA supplementation  57  on coproporphyrin and  phenolic acid production by hem-1 16  Growth and coproporphyrin production by dhb-4  60  17  E f f e c t of sideramine supplementation  70  acid excretion i n s t r a i n  B-1471  on phenolic  X  LIST OF FIGURES - continued  Figure  Title  Page  18  Growth of s t r a i n ¥B-jk6 i n iron-deficient medium containing 0.3% glucose  77  19  Oxidation capacities of s t r a i n WB-746 grown i n i r o n -  78  d e f i c i e n t medium containing 0.3% glucose 20  Iron uptake as a function of p h y s i o l o g i c a l age  83  21  Iron uptake as a function of energy  22  E f f e c t of temperature on i r o n uptake  85  23 2k  Incorporation of i r o n into TCA-insoluble material Rate of i r o n uptake as a function of i r o n concentration  87 88  25  Binding of i r o n to c e l l s at 0 C  89  26  E f f e c t of growth without c i t r a t e on iron uptake  91  27  E f f e c t of phenolic acids on i r o n uptake  92  28  E f f e c t of phenolic acids on uptake at lower levels  93  8>k  of i r o n 29  Iron uptake i n mutant strains  95  30  The F e  97  31  Strain differences i n i r o n uptake capacities  100  32  E f f e c t of ferrichrome on i r o n transport  102  33  E f f e c t of ferrichrome on iron uptake i n s t r a i n  3k  Iron uptake by s t r a i n HQ, -l  3 +  (DHBG) complex 3  103  B-1471  r  10k  ACKNOWLEDGEMENTS  To Professor R. A. J . Warren f o r h i s unique and enthusiastic approach to s c i e n t i f i c discovery, and f o r h i s demonstration of the framework on which science must be b u i l t .  To Professor  E. W. Rester f o r allowing me to draw upon h i s experience and knowledge of B a c i l l u s s u b t i l i s .  To Professor F. Gibson f o r  providing many of h i s experimental observations p r i o r to t h e i r publication.  To W. W. Kay f o r valuable assistance with the  planning and i n t e r p r e t a t i o n of transport experiments.  To  A. M. B. Kropinski whose general knowledge of m i c r o b i a l metabolism was continuingly available.  To the other co-workers  of Room 14 f o r constant discussion and c r i t i c i s m which, i n many cases, l e d to experimental elucidation. Morgan f o r generously typing t h i s thesis.  To Mrs. Rosemary To Mrs. R i t a  Rosbergen f o r 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 o f m e t a b o l i s m as a consequence o f 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 o f m i c r o b i a l s p e c 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 m a n i f e s t e d b y the p r o d u c t i o n o f h i g h l e v e l s o f f e r r i c i r o n c o m p l e x i n g agents ( 4 0 ) .  Thus,  sphaerogena ( 1 7 ) p r o d u c e d t h e complex t r i h y d r o x a m a t e s ,  Ustilago ferrichrome  and f e r r i c h r o m a A , w h i l e B a c i l l u s megaterium p r o d u c e d 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 t o e x c r e t e p h e n o l i c a c i d s 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 . produced s a l i c y l i c a c i d ;  M y c o b a c t e r i u m smegmatis  A e r o b a c t e r aerogenes and a s t r a i n o f  E s c h e r i c h i a c o l i p r o d u c e d 2 , 3 - d i h y d r o x y b e n z o i c a c i d (DHB) ( 5 0 ) ; another s t r a i n o f E. (17).  c o l i p r o d u c e d 2 , 3 - d i h y d r o x y b e n z o y l s e r i n e (DHBS)  E x t r a c t s o f t h i s second s t r a i n o f E.  c o l i formed DHBS f r o m  DHB and s e r i n e , and t h e l e v e l o f t h i s enzyme (DHBS s y n t h e t a s e ) was dependent upon t h e l e v e l o f i r o n i n t h e growth medium (17)»  Brot  and,Goodwin (6) have s u g g e s t e d t h a t i r o n may a c t as a c o r e p r e s s o r  in  t h e system c o n t r o l l i n g t h e s y n t h e s i s o f DHBS s y n t h e t a s e . I t o i c a c i d , 2 , 3 - d i h y d r o x y b e n z o y l g l y c i n e (DHBG) was f o u n d t o be excreted by B a c i l l u s s u b t i l i s ( 1 8 ) .  A c c u m u l a t i o n o f t h i s compound  depends upon the l e v e l o f i r o n i n t h e medium: i r o n d i d not grow;  cultures devoid of  h i g h l e v e l s of i r o n prevented i t s production ( 2 7 ) ;  l o w l e v e l s o f i r o n l e a d t o the e x c r e t i o n o f more t h a n 200 mg o f DHBG per l i t r e of c u l t u r e f l u i d  (40).  D u r i n g the p a s t few y e a r s ,  a r e l a t i o n s h i p has been demonstrated  between p h e n o l e x c r e t i o n and a r o m a t i c m e t a b o l i s m .  P i t t a r d et  al  2  (47, 48) showed t h a t washed c e l l s u s p e n s i o n s o f a number o f  aromatic  amino a c i d auxotrophs o f A . aerogenes e x c r e t e d o - d i h y d r o x y p h e n o l s , amongst them DHB, when i n c u b a t e d i n m i n i m a l medium. s t r a i n , NC3, b l o c k e d a t a n t h r a n i l a t e  synthetase,  I n one such  f o r m a t i o n o f o-  d i h y d r o x y p h e n o l s was i n h i b i t e d c o m p l e t e l y b y exogenous t r y p t o p h a n . Furthermore,  i t has been shown t h a t w h i l e a w i l d t y p e s t r a i n o f  A . aerogenes p r o d u c e d DHB o n l y under i r o n d e f i c i e n c y , t h i s s t r a i n blocked after anthranilate  a mutant o f  s y n t h e t a s e p r o d u c e d DHB  r e g a r d l e s s o f t h e 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 o f a r o m a t i c m e t a b o l i s m b r o u g h t about by i r o n d e f i c i e n c y and c a u s i n g the f o r m a t i o n o f p h e n o l i c a c i d s unclear.  R a t l e d g e and Winder  ( 5 2 ) have s u g g e s t e d t h a t M.  may s y n t h e s i z e s a l i c y l i c a c i d f r o m s h i k i m i c a c i d b y s t e p s  is smegmatis  analogous  to 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 formation ( 6 0 ) , but w i t h the i n t r o d u c t i o n o f a h y d r o x y l group i n s t e a d o f an amino g r o u p . iron deficiency,  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 c o u l d be  Under  accentuated,  p o s s i b l y b y a r e q u i r e m e n t f o r i r o n by a competing pathway.  This  s u g g e s t i o n was s u p p o r t e d by t h e o b s e r v a t i o n t h a t e i t h e r magnesium o r i r o n was r e q u i r e d f o r the c o n v e r s i o n o f s h i k i m i c a c i d - 5 - p h o s p h a t e t o a n t h r a n i l i c a c i d b y 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 l i c acid synthesis (53).  In addition,  been shown f o r t h e a n t h r a n i l a t e  ions i n h i b i t  an i r o n r e q u i r e m e n t  s y n t h e t a s e p r e p a r e d f r o m E.  anthranhas.not coli  (5, 5 8 ) .  Cox and G i b s o n ( 1 2 ) showed t h a t DHB was a growth f a c t o r c e r t a i n m u l t i p l e auxotrophs o f E. medium.  for  c o l i , but o n l y i n i r o n - d e f i c i e n t  Young e t a l ( 6 7 ) f o u n d t h a t e x t r a c t s o f E.  c o l i were  3  capable o f f o r m i n g DHB f r o m c h o r i s m a t e , and t h a t t h e a c t i v i t y o f t h e s e p r e p a r a t i o n s was dependent upon t h e l e v e l o f  iron.  I n many m i c r o o r g a n i s m s , growth i n i r o n - d e f i c i e n t media l e a d s to a disturbance of p o r p h y r i n metabolism (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 excreted.  E x t r a c t s prepared from these c e l l s contained l e s s  S-aminolevulinate  dehydratase a c t i v i t y t h a n e x t r a c t s f r o m i r o n  s u f f i c i e n t c e l l s (29).  I n o t h e r o r g a n i s m s , t h e r e was e x c r e t i o n  of high l e v e l s of a p o r p h y r i n . diphtheriae  B.  s u b t i l i s (18), C o r y n e b a c t e r i u m  (23) and M i c r o c o c c u s l y s o d e i k t i c u s (62) p r o d u c e d  coproporphyrin III.  Furthermore,  the p r o d u c t i o n of coproporphyrin I I I  i t has been r e p o r t e d (25) t h a t by B.  s u b t i l i s was always  accompanied b y t h e e x c r e t i o n o f l a r g e amounts o f DHBG.  As DHBG  has a v e r y s t r o n g 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 e x c r e t e d i n t o the medium t o make i r o n a v a i l a b l e to the c e l l (40).  In t h i s regard, i t i s  interesting  t h a t o - d i h y d r o x y phenols- are r e q u i r e d f o r t h e growth o f M. l y s o d e i k t i c u s (56).  Similarly,  low l e v e l s o f t h e s e compounds have been  shown t o r e p l a c e the t y r o s i n e r e q u i r e m e n t o f a s p e c i e s o f  Sarcina  (22). The work d e s c r i b e d i n t h i s t h e s i s was u n d e r t a k e n i n an attempt t o d e f i n e an o v e r a l l approach t o the s t u d y o f i r o n - d e f i c i e n c y i n B.  subtilis. I t was n e c e s s a r y , f i r s t o f a l l , t o determine whether DHB(G) was  p r o d u c e d d u r i n g a c t i v e growth o f t h e o r g a n i s m o r i f i t o n l y d u r i n g t h e s t a t i o n a r y phase.  If  accumulated  i t were p r o d u c e d o n l y d u r i n g  t h e s t a t i o n a r y phase i t w o u l d be much h a r d e r t o d e s i g n and i n t e r p r e t  1+  experiments on the control of i t s production.  Aromatic compounds  which, l i k e DHB, are synthesized by the c e l l from chorismic acid, were examined f o r t h e i r effects on D H B production.  Such studies  would indicate whether or not D H B production was regulated by the control mechanisms known to operate i n the aromatic pathway i n B. s u b t i l i s . The relationship of D H B ( G ) production to production was then examined.  coproporphyrin  Iron i s required f o r the formation  of hemin and of non-heme i r o n proteins.  I t was possible, there-  fore, that the production of coproporphyrin and DHB(G) might be r e l a t e d i n some other way besides a lack of iron. Analogs of D H B were tested f o r t h e i r effects on D H B production. Those analogs which were markedly i n h i b i t o r y might then be u s e f u l i n studying the enzymology of D H B ( G ) synthesis and i t s control. DHB  and D H B G are known to bind i r o n strongly ( 2 6 ) so that a  possible function of these compounds might be to serve as i r o n transport f a c t o r s .  A study was made, therefore, of i r o n uptake  by B . s u b t i l i s and of the effects of D H B ( G ) on t h i s process.  In  addition, the effects of sideramines on D H B ( G ) production and on i r o n uptake were studied.  Sideramines are thought to function as  i r o n transport factors i n other microorganisms (39?  41).  5MATERIALS AND METHODS  I. B a c t e r i a l strains 1.  Wild type strains  The source of each wild-type s t r a i n employed i s shown i n Table I. Strain WB-746 was selected (4-5) as a spontaneous prototrophic revertant of a tryptophan auxotroph s t r a i n (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 r e s i s t a n t to 8-hydroxyquinoline (HQ "), 5-methyltryptophan 1  (MT ), and to various a n t i b i o t i c s were selected by spreading log phase r  c e l l s on minimal medium supplemented with a l e v e l of i n h i b i t o r which prevented the growth of the wild type s t r a i n .  The supplementation  l e v e l required f o r each class of mutants i s presented i n the text.  (b)  NTG-induced mutants  Washed, log phase c e l l s were suspended i n tris-^maleate buffer ( l ) and treated with NTG according to the procedure of Lorence and Nester ( 3 3 ) . Auxotrophs were selected as minute colonies on minimal medium containing a l i m i t i n g l e v e l of the appropriate supplement: glycine auxotrophs, 0 . 3 M-g serine per .ml; i-aminolevulinate  serine(ALA)  6  Table 1 :  B.  s u b t i l i s w i l d type  Strain  strains  Source  B-1471  J . B. N e i l a n d s , U n i v e r s i t y of C a l i f o r n i a , Berkeley.  WB-746 WB-443  E. ¥. N e s t e r , U n i v e r s i t y o f Washington, Seattle.  W-23  J . Spizizen, S c r i p p s C l i n i c and Research La J o l l a , California.  6051 6633  A m e r i c a n Type C u l t u r e C o l l e c t i o n (ATCC) ATCC ATCC ATCC ATCC  6455 I2696 11+807  Foundation,  7 Table I I :  Description of B. s u b t i l i s mutant strains  Strain  Genotype  Enzyme defect  trp-1  trp  anthranilate  synthetase  B-l47i  Source a  trp-l-MT -l  trp~MT  r  anthranilate  synthetase  trp-1  trp-l-MT -2  trp~MT  r  anthranilate  synthetase  trp-1  trp-2  trp  tryptophan synthetase  B-l47l  a  trp-3  trp  InGP synthetase  B-l47i  a  trp-4  trp  anthranilate  B-l471  a  trp-5  trp  tryptophan synthetase  B-l47l  a  trp-6  trp  tryptophan synthetase  B-i47l  a  trp-7  trp  PRA isomerase  B-l47l  a  hem 1  ALA."  ALA. synthetase  B-l47l  a  phe-1  phe  B-i47i  a  phe-2  phe  B-l47l  a  MT -l  MT  r  B-i47l  b  MT -2  MT  r  B-i47i  b  HQ" 1-6  prototroph  B-i47i  b  ser 1-4  ser  B-i47l  a  r  r  r  r  1  synthetase  prototrophic revertants' of DHB auxot r o p h of B-1471  dhb-1-5  3  a  aro-1  shk  SB-168  trp"  InGP synthetase  E.W.  Hester,  WB-746  SB-194  trp  anthranilate  synthetase  WB-746  SB-194-MT -l SB-194-MT -2  trp  anthranilate  synthetase  E.W. Hester, SB-194  trp  anthranilate  synthetase  SB-194  SB-30  tyr t r p  WB  E.W.  Hester,  2102 SB 167  shk"  DAHP synthetase  E.W.  Hester,  shk"  DHQ synthetase  E.W.  Hester,  MT -l  MT r MT  t  r  r  MT -2 r  r  B-i47i  a  b  b  WB-746 WB-746  b  b  WB-746 WB-746 WB-746  8 Table I I  Strain  (continued)  Genotype  Enzyme defect  Source  prototrophs r MT MT'  a: spontaneous derivative;  b: NTG - induced derivative.  Abbreviations used: InGP, indoleglycerol phosphate; PRA, phosphoribos y l a n t h r a n i l i c acid; Shk, shikimic acid; DAHP, 3-deoxy-D-arabino-heptulosonic 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 c i t r a t e being omitted from the medium i n t h i s case because i t has been shown that c i t r a t e can replace DHB as a growth requirement f o r a multiple aromatic auxotroph of Escherichia c o l i (67).  Before p l a t i n g f o r mutant selection,  the mutagenized c e l l s were d i l u t e d and plated on the appropriate s e l e c t i o n medium to score f o r survivors.  To avoid l y s i s of the  treated c e l l s during the incubation of plates f o r scoring survivors, i t was necessary to store the c e l l s at h C i n minimal medium containing 5$ g l y c e r o l (33). about  Under these conditions  90$> of the c e l l s remained viable three days. A l l strains were stored on Difco TAM sporulation agar slants  i n screw-cap v i a l s . II. Media Difco trypticase soy broth supplemented with 1% Difco yeast extract (TSY medium) was used f o r the growth of inocula.  The  minimal medium used f o r the production of porphyrin and phenolic acids was a modification of the medium of Neilands and Garibaldi (42). 3.0;  I t contained, i n g per l i t r e : KgHPO^, 1.0; ammonium acetate, MgSOj^. TH^Oj 0.08;  glucose, 10.0.  I t was supplemented with  5 .ml per l i t r e of a 10% solution of Difco yeast extract i n d i s t i l l e d water, autoclaved with alumina p r i o r to use (10), and with 0.1 .ml per l i t r e of the Neurospora trace elements solution of Vogel (65), from which the i r o n was omitted.  The medium was made up with  g l a s s - d i s t i l l e d water, and was adjusted to pH 7.4 before autoclaving.  10  The glucose was s t e r i l i z e d separately as a  hO'fo  solution i n glass-  d i s t i l l e d water. Extraction of the medium with 8-hydroxyquinoline (HQ,) ( 6 5 ) yielded e r r a t i c r e s u l t s , presumably due to microquantities of HQ, remaining i n the medium after chloroform extraction.  This  procedure was therefore not employed.  I I I . C u l t u r a l conditions  A l l glassware was autoclaved twice with g l a s s - d i s t i l l e d water p r i o r to use.  Erlenmeyer f l a s k s ( 2 5 0 ml), f i t t e d with side arms  and containing 25-ml quantities of the medium, were incubated at 37 C i n a New Brunswick Metabolyte water bath (Hew Brunswick S c i e n t i f i c Co., New Brunswick,  N."J.), rotating at 2 5 0 rev/min.  A l l experiments were performed by use of a 0. 5$> inoculum from a ik-hr culture, which was one transfer away from the stock slant. time of onset of conditions.  DHB(G)*  The  production was constant under these  Additions to f l a s k s were made at the times mentioned  i n the text.  IV. Determinations  1. Growth  Growth was followed t u r b i d i m e t r i c a l l y , 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 d i l u t e the culture with Q complete medium. A c e l l density of 10 per .ml gave a reading of 42 K l e t t u n i t s .  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 i r o n solution  ( 0 . 5 mg per ml) to 2.0 ml of c e l l - f r e e medium adjusted to pH 7.6, centrifuging, and measuring the o p t i c a l density (0.D) of the supernatant at 510 .mu, photometer.  1.0 cm l i g h t path, with a Beckman Model B spectro-  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. 1.0 O.D.  Under these conditions,  u n i t represented a concentration of 3^4 ng DHB(G) per ml  of culture supernatant.  Production of DHB  and DHBG was  confirmed  spectrophotometrically and chromatographically ( 2 7 ) by comparison with authentic specimens.  Descending  performed using two solvent systems; water 4/1/5;  chromatographic  analyses were  ( l ) n-butanol, acetic acid,  ( 2 ) t - b u t y l alcohol, methyl ethyl ketone, water,  d i e t h y l amine 1 0 / l 0 / 5 / l .  The RF values are given i n Table V.  3. Coproporphyrin I I I *  . A 2.0 ml sample of culture supernatant was with acetic acid. volume of ether.  The coproporphyrin was  adjusted to pH 5.0  extracted with a known  The absorption of the ether extract was measured  at 4 0 8 mu. using a Beckman DB spectrophotometer  (15).  Care was  taken to ensure that, at the time of measurement, the volume of  * Coproporphyrin I I I i s referred to as coproporphyrin.  12  the ether was equal to that o r i g i n a l l y added to the a c i d i f i e d culture  supernatant.  4. I d e n t i f i c a t i o n 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 f o r 4 5 min.  Glycine was i d e n t i f i e d 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 A f t e r 12-14 hr growth, cultures were harvested by c e n t r i fugation at 20 C, washed once with complete medium, and the c e l l s resuspended at a concentration of 1 g wet weight per 5 ml i n 0.05 M potassium phosphate - 0.01 M mercaptoethanol, ( 1 0 0 ug per ml), deoxyribonuclease ( 5 x 10  pH 7.5.  ( 1 0 u.g per ml) and MgCl^  M) were added to the suspension and the mixture  f o r 30 min at 37 C.  Lysozyme  The r e s u l t i n g extract was  incubated  sonicated f o r  2 min at a probe i n t e n s i t y of 70'.' using a Biosonik probe o s c i l l a t o r , (Bromwill S c i e n t i f i c , New York, N.Y.), and then centrifuged at 2 5 , 0 0 0 x g f o r 15 min at 4 C. d i r e c t l y f o r enzyme assays.  VI. Enzyme assays  The supernatant f l u i d was used  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 f o r serine i n the reaction mixture.  VII. Respiration studies C e l l s were harvested at 2 0 C at the times indicated i n the text, +2  washed twice with 0.005 M Mg (Tris)-chloride  (pH 7.k),  - 0.1 M tris(hydroxymethyl)amino.methane  and resuspended i n the same buffer at a  concentration of approximately 5 ™g dry weight of c e l l s per ml. Respiration studies were performed at 3 7 C i n Warburg vessels which +2  contained a f i n a l concentration of 0.005 M Mg one .ml of c e l l suspension,  - 0.05 M T r i s pH 7.4,  and 5 umoles of acetate or c i t r a t e .  VIII. Iron transport studies  1, Preparation of c e l l s Cultures were grown to a density of 5.5 x 1 0 c e l l s / m l (unless stated otherwise), then used immediately f o r transport studies. i r o n - s u f f i c i e n t growth, the medium was supplemented with 1 ug of iron/ml.  For  2. Iron uptake  (a)  Iron-deficient  cells  Ten .ml of culture were transferred to a 250 ml Erlenmeyer flask.  The f l a s k was incubated at 3 7 C (unless stated otherwise)  i n a New Brunswick Model G-77 Metabolyte water bath (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.), rotating at 1 0 0 rpm. Additions were made after 1 0 min.  (b)  Iron-sufficient  cells  Ten ml of culture were f i l t e r e d through a 0.45 n M i l l i p o r e membrane.  The c e l l s were washed with 1 0 volumes of prewarmed,  i r o n - d e f i c i e n t medium, and the membrane transferred to 1 0 .ml prewarmed i r o n - d e f i c i e n t medium i n a 250 ml Erlenmeyer f l a s k . The  c e l l s were resuspended by gently blowing medium over the  membrane with a pipette.  The f l a s k was incubated as f o r i r o n -  deficient c e l l s , with additions again being made after 1 0 min. In both cases, additions were made i n a t o t a l volume of 0.5 ml g l a s s - d i s t i l l e d water.  When uptake was measured at 0 C, g l y c e r o l  was added to a concentration of 5 $ to prevent l y s i s of the c e l l s . Unless stated otherwise, i r o n was used at a concentration of 5.0 mug/ml.  3. Assay of i r o n uptake  Samples of 1.0 ml were withdrawn with hypodermic syringes, f i l t e r e d through 0.45 LI M i l l i p o r e membranes and the c e l l s washed with 2.0 ml prewarmed, iron-deficient medium.  The membranes were  dried, placed i n v i a l s containing 1 0 ml s c i n t i l l a t i o n f l u i d ( L i q u i f l u o r , New England Nuclear Corp.) and assayed f o r radioa c t i v i t y i n a l i q 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 i n t e r v a l 3 min to 8 min after i r o n addition. Radioactivity i n the cold TCA-insoluble f r a c t i o n of c e l l s was measured by the method of Roberts et a l ( 5 3 ) . was added to 1 . 0 ml of 10% TCA at 0 C.  A 1 . 0 ml sample  After 3 0 min at 0 C,  the suspension was f i l t e r e d through a 0,1*5 u M i l l i p o r e membrane, and the membrane washed with two volumes of i r o n - d e f i c i e n t medium.  The membrane was dried and assayed f o r r a d i o a c t i v i t y .  IX. Chemicals  Chorismic acid was p u r i f i e d from the c u l t u r a l supernatant of A. aerogenes 6 2 - 1  according to the method of Gibson  (19)  except that the acid was p r e c i p i t a t e d from the Dowex effluent as the barium s a l t ( l 8 ) .  Free chorismic acid was obtained by  sedimenting most of the barium as the phosphate s a l t , 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 C a l i f o r n i a , Berkeley.  The iron was removed  from t h i s compound by the method of Emery and Neilands  (ik).  Ferrioxamine was obtained from W. K e l l e r - S c h i e r l e i n , of Eldg.  Technische Hochschule, Zurich,  Switzerland.  The following chemicals were obtained from commercial sources: o- and m-tyrosine, ^-aminolevulinic 2-  a c i d (Sigma Chemical Co.);  and 3 - f l u o r o , and 2,3-dimethoxybenzoic acid, DHB,  NTG  Chemical Co.); m-hydroxybenzoic acid, a - p i c o l i n i c acid, hippuric a c i d ( j . T. Baker Chemical Co.); MT,  ^Fe  and  3-hydroxyanthranilic  acid, hemin, s e r i n e - l - ^ C ( s p e c i f i c a c t i v i t y 1 0 . 3 (Calbiochem Co.);  (Aldrich  nc per umole)  s a l i c y l i c acid (The B r i t i s h Drug Houses, Ltd.);  as FeCl^, s p e c i f i c a c t i v i t y 2 5 - 5  Chemical and Huclear Co.); per umole) (Merck &  Co.).  M-c/ng ( i n t e r n a t i o n a l  glycine-1-^C  ( s p e c i f i c a c t i v i t y 2 u.c  RESULTS  Section I: - General properties of phenolic acid excretion  1. Excretion of phenolic acids by w i l d type strains  A number of w i l d type strains of B. s u b t i l i s were grown i n i r o n d e f i c i e n t medium to determine the compounds produced under these conditions.  The strains f e l l into three groups according to t h e i r  patterns of phenolic acid production:  strains of group I produced  only DHBG, those of group I I only DHB, and those of group I I I both of these compounds (Table I I I ) . was selected f o r further study,  A s t r a i n representing each group i . e . B-1471 (group I ) , WB-746  (group II) and W-23 (group I I I ) .  2. Influence of i r o n on DHBG production  Production of DHBG by s t r a i n B-1471, i n the absence of added iron, started after about 8 hrs of growth and continued logarithmically, p a r a l l e l l i n g growth, u n t i l the early stationary phase was reached. S i g n i f i c a n t production of DHBG d i d not occur i n the stationary phase. I f the medium was supplemented with 1 mg of i r o n per l i t r e , of DHBG was i n h i b i t e d completely 150 to  (Fig. l ) .  production  At concentrations below  ug of i r o n per l i t r e , DHBG production was i n v e r s e l y proportional the l e v e l of i r o n i n the medium.  As the l e v e l of i r o n added was  increased from 0 to 1 5 0 ug per l i t r e of medium, the production of DHBG began proportionately l a t e r . i n the growth period ( F i g . 2 ) . DHBG production was i n h i b i t e d completely by the addition of 1 5 0 ug of  i r o n per l i t r e .  Table I I I . Excretion of phenolic acids by w i l d type strains  Group  I  Strain  B - 1 4 7 1  _  l e v e l produced (mg/l) 3 0 0  +  5 0  +  6 0  WB-746  +  S B - 4 4 3  +  -  w-23  +  +  2 5 0  6 0 5 1  +  +  2 0 0  1 4 8 0 7  +  +  1 0 0  6455 1 2 6 9 6  III  DHBG  -  6 6 3 3  II  DHB  1 5 0  1 0 0 0  1 0 0  HOURS  •Figure 1. . Growth and p r o d u c t i o n o f DHBG b y s t r a i n B-1471. C e l l s were grown i n t h e absence (©) and p r e s e n c e (0) o f 1 mg o f added i r o n p e r l i t r e . DHBG was produced under c o n d i t i o n s o f i r o n d e f i c i e n c y (X) . • ..- b u t was n o t produced i n t h e p r e s e n c e o f added i r o n .  3.  Fe  : (DHBG)^ complex and the control of DHBG production  When added to an i r o n - d e f i c i e n t culture at any time "between 5 and 9 hrs a f t e r inoculation, 1 mg of i r o n per l i t r e did not completely i n h i b i t DHBG production.  The addition of DHB or DHBG to an iron-'  d e f i c i e n t medium at zero time had no e f f e c t on subsequent DHBG production. (Fig. 3 ) . - 4  The. simultaneous addition of 3 . 5 x 1 0 M DHB and 1 mg of i r o n per l i t r e , 5 hrs after inoculation, resulted i n the complete i n h i b i t i o n of DHBG production (Fig. 4 ) .  In f a c t , 8 0 ug of i r o n per l i t r e was - 4  equally e f f e c t i v e 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 i n h i b i t o r y e f f e c t on DHBG production was not f u l l y manifested f o r about 3 hrs (Fig. 4 ) .  I t appeared therefore,  that between 5 and 7  hrs i n the growth cycle, an i r o n - d e f i c i e n t culture became committed to the production of some DHBG, regardless  of subsequent addition of  an iron-DHB mixture. 4.  E f f e c t of aromatic amino acids on DHBG production  The  excretion of DHB by c e r t a i n 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. s u b t i l i s . None of the supplements, at the l e v e l s used, affected the growth rate of cultures. Supplementation of media with p-hydroxybenzoic acid or p-aminobenzoic acid, at concentrations up to subsequent DHBG production.  10~^M,  had no e f f e c t on  Addition of tryptophan at a concentration  - 4  of 1 0 M caused e a r l i e r and s i g n i f i c a n t l y higher production of DHBG  21  8  9  10  II  12  13  14  HOURS  Figure 2.  E f f e c t of i r o n added at zero time on the production of DHBG by s t r a i n 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 i r o n was added to the control f l a s k (X).  0.8  HOURS Figure 3.  E f f e c t on-DHBG production of adding i r o n or D H B to s t r a i n B-1471. D H B (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 i r o n or D H B was added to the control f l a s k (0).  2 3  i  Figure 4 .  -  4  E f f e c t of- simultaneous addition of 3 . 5 x 1 0 M DHB or DHBG and 1 mg of i r o n per l i t r e on DHBG production "by strain B - 1 4 7 1 . The additions were made at hr (X), 7 hr (©), and 9 hr (A). Wo phenolic acids or i r o n was added to the control f l a s k (.0). 5  2k than i n the control f l a s k .  As the l e v e l of tryptophan was increased  beyond 10 ^M, DHBG accumulation was i n h i b i t e d ( F i g . 5 ) .  Phenyl-  -k alanine and tyrosine reduced DHBG accumulation at 10 M, and to a greater extent at higher concentrations.  Tyrosine was a more  e f f e c t i v e i n h i b i t o r than phenylalanine ( F i g . 5 ) . effects were not additive.  These i n h i b i t o r y  The percentages of i n h i b i t i o n observed  f o r each amino acid at 10 "^M,.were tryptophan, 25; phenylalanine, 2 7 ; and tyrosine, 52. at 10  When a l l three amino acids were present together  the i n h i b i t i o n was 50$.  I n h i b i t i o n of DHBG production, comparable to that seen with tyrosine, was produced by a n t h r a n i l i c acid. was  The production of DHBG  inversely proportional to the l e v e l of a n t h r a n i l i c acid i n the  medium ( F i g . 6).  This i n h i b i t i o n occurred i n the presence of  tryptophan, so that the e f f e c t of a n t h r a n i l i c 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  i n i t i a t i o n of DHBG synthesis, 50 umoles of g l y c i n e - l - ^ C a c t i v i t y , 2 uc per umole) were added.  (specific  Four hours l a t e r , the c e l l s  were removed by centrifugation and the DHBG was p u r i f i e d according to the method of Ito and Neilands ( 2 9 ) .  A 30-mg sample (wet  weight) of c e l l s was fractionated according to the procedure of Roberts et a l (14).  Radioactivity of the various f r a c t i o n s was  measured by use of a Nuclear Chicago model l 8 l A planchet counter (Table IV).  The p u r i f i e d DHBG gave a single spot when chromato-  0.6  Figure .5.  E f f e c t of adding end product amino acids on DHBG production by s t r a i n 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  0.10 8  9  10  II  12  13  HOURS  Figure 6 . E f f e c t of adding a n t r h a n i l i c acid on the production of DHBG by.strain B - 1 4 7 1 . A n t h r a n i l i c acid was added at zero time at l e v e l s ' o f 1 0 M ( 0 ) , 1 0 . - 3 M ( A ) , and 2 x 1 0 " 3 M (©). I d e n t i c a l curves were obtained when 1 0 - ^ - M tryptophan was added to the a n t h r a n i l i c acid supplemented flasks. .No aromatic addition was made to the control f l a s k (.X).'  2 7  graphed on paper with three different solvent systems ( 2 9 ) .  A  sample of the p u r i f i e d DHBG was hydrolysed i n 6 N HC1 for 1 6 hrs i n a sealed, evacuated tube at 1 2 0 C.  The hydrolysate was chromato-  graphed i n the above systems, and also was subjected to high voltage paper electrophoresis.  Labelled DHB was not detected.  The l a b e l  was found only i n the glycine moiety.  6.  Variations among strains of B. s u b t i l i s  By use of chromatographic and spectrophotometric methods (see Materials and Methods), two other wild-type strains of B. s u b t i l i s were examined f o r compounds produced during i r o n - d e f i c i e n t 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, a n t h r a n i l i c acid, and aromatic amino acids i n the medium as s t r a i n B-1V71.  Strain WB-746, however, exhibited s i g n i f i c a n t l y  d i f f e r e n t responses.  In t h i s s t r a i n , DHB synthesis was independent  of the l e v e l of added i r o n up to a concentration of 1 2 0 ug per l i t r e . At 1 5 0 |ig of added i r o n 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 i r o n (Fig. 7 ) .  At higher levels of iron, DHB  production by s t r a i n WB-746 was i n h i b i t e d completely. B - 1 4 7 1  and  W-23,  Unlike strains  production of DHB by s t r a i n WB-746 was not stimulated  by low levels of tryptophan.  In addition, the i n h i b i t i o n of pro-  duction by high levels of tryptophan and by anthranilic acid was  Table IV.  D i s t r i b u t i o n of g l y c i n e - 1 -  Fraction T o t a l culture C e l l supernatant f l u i d  10  C  count s/min 5 . 0 2  2 . 0 3  Coproporphyrin I I I  0 . 5 9 0  P u r i f i e d DHBG  0 . 6 0 0  Cells  2 . 9 6  Cold t r i c h l o r o a c e t i c acid-soluble  0 . 0 4 5  Alcoho1-soluble  0 . 0 0 5  Ether-alcohol  0 . 0 0 0  Hot t r i c h l o r o a c e t i c acid-soluble  0 . 8 9 7  Residual  2 . 0 6  Table V. Characteristics of phenolic acid excretion by strains of B . s u b t i l i s i n i r o n deficiency  Characteristics of phenolic acid excretion  •Strain •B-1471  WB-746  Phenolic a c i d produced  DHBG  DHB  Rp i n 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.69  100$  1 0 0 $  1 0 0 $  1 2 4 $  1 0 0 $  1 2 0 $  w-23  DHBG and  E f f e c t of aromatic supplementation on excretion: Control 10 10 10 10 10  - 4  M tryptophan _ 3  M tryptophan - 3  M tyrosine  7 5 $  48$  8 9 $  ' 7 5 $  7 4 $  5 0 $  9 0 $  7 5 $  84$  5 5 $  7 8 $  2 5 $  - 3  M  phenylalanine  7 3 $  _ 3  M anthranilate  2 x 10  5 8 $  - 3  M anthranilate  2 1 $  DHB  30  Figure "7.  only about 5 0 $ of that observed i n the two other strains (Table V). 7« E f f e c t of h i s t i d i n e on phenolic acid excretion When 2 0 u,g h i s t i d i n e per .ml was added at zero time to each of the wild-type strains, phenolic acid excretion was decreased about 2 5 $ i n B-11+71 and W-23,  and about 5 0 $ i n WB-746 (Table VI).  Wester has  demonstrated a regulative involvement of h i s t i d i n e i n aromatic b i o synthesis (1+3) J  i n a l l cases, the e f f e c t of h i s t i d i n e supplementation  was overcome by tyrosine supplementation at 2 0 ug per ml (1+3). H i s t i d i n e i n h i b i t i o n of phenolic acid excretion was not r e l i e v e d by tyrosine;  instead, the i n h i b i t o r y e f f e c t was additive (Table VI,  Fig. 5 ) .  These results may r e f l e c t an additional unknown involvement  of h i s t i d i n e i n aromatic metabolism.  8.  In v i t r o syntheses  (a)  Formation of DHB  C e l l - f r e e 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). twice as active as B-1471 extracts.  WB-746 extracts were  The addition of 10 %  anthran-  i l a t e completely i n h i b i t e d the formation of DHB (Table V I I ) .  (b)  Formation of DHBG  Extracts of B-ll+71 formed DHBG from DHB or from chorismate i n 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 f o r glycine.  The addition of F e  + 3  had  Table VI. E f f e c t of h i s t i d i n e on phenolic acid production i n wild type strains  Strain  Supplement (20 ug/ml)  % Phenolic acids excreted  100  B-1471 B-1471  histidine  75  B-1471  h i s t i d i n e & tyrosine  55  W-23  -  W-23  histidine  70  W-23  h i s t i d i n e & tyrosine  50  100  WB-746  -  WB-746  histidine  50  WB-746  h i s t i d i n e & tyrosine  4o  100  33  Table VII.  Extract  Source  In v i t r o synthesis of  Reaction Mixture  DHB  DHB Formed (umoles)  Complete  0.32  2.  - chorismate  0.02  3.  Complete, at 0 C  4.  Complete + 1 0 ilate  1.  5-  6.  WB-746  _3  ,  "  B-1471  M anthran-3  0.03 0.01  Complete + 10 M of each of tryptophan, tyrosine and phenylalanine  0.13  Complete  0.13  Synthesis of DHB from chorismate. The complete reaction mixture c o n t a i n e d : c r u d e e x t r a c t ( 3 . 0 mg protein), 50 umoles T r i s - C l buffer (pH 8 . 0 ) , 1.0 umole chorismate, 1.0 umole NAD, and 5.0 umoles MgCl^ i n a t o t a l volume of 1.0 ml.  Table VIII.  Extract 1.  Source  B-1471  2. 3.  In v i t r o synthesis of DHBG  Reaction Mixture  10  4 counts/min / i n DHBG  Unsupplemented  0.08  + chorismate  3.1 +3  3.0  4.  + chorismate + Fe + 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), 1 0 umoles T r i s - C l buffer (pH 7 . 4 ) , 0.1 umoles - ^ C - l - g l y ( 0 . 2 u.c), and 1.0 umole ATP. Additions were made as indicated: 0.1 umole DHB, 0.5 umole chorismate, F e 3 0.05 umole. The t o t a l volume of each reaction mixture was 1.0 ml. The incubation time was 1 hr at 3 7 C +  no e f f e c t on DHBG formation from chorismate.  .  WB-746  extracts were  incapable of DHBG formation (Table VIII).  9 .  Metabolism of DHB(G) by s t r a i n  B - 1 4 7 1  When DHB was added to cultures of s t r a i n of i t disappeared from the medium.  B - 1 4 7 1  at zero time, some  In the absence of added iron,  this disappearance continued u n t i l the culture started to produce DHBG ( F i g . 3 ) .  When iron, to a concentration of 1 mg per l i t r e ,  added with the DHB, 1 2  hrs (the OD at  was  a similar rate of disappearance was observed f o r  5 1 0  mu dropped from  change occurred during the next 8 hrs.  0 . 5 5  to  0 . 3 5 ) .  Ho further  I f i r o n 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 t h i s 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) i n Warburg experiments.  Washed c e l l s were tested after growth with and without  added i r o n 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 i n s t r a i n did  B - 1 4 7 1 .  Strain WB-746, however,  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  c e l l s of either of these strains.  0.5n  F i g u r e 8.  M e t a b o l i s m o f DHBG b y 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 o f i r o n p e r l i t r e (&) a n d . 1 mg o f i r o n p e r l i t r e (0) were added t o 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  11.  Properties of .mutant strains  (a)  5-methyltryptophan  Resistance to MT ( 3 7 )  r e s i s t a n t (MT ) strains r  i n B. s u b t i l i s ( 4 5 )  of the tryptophan biosynthetic enzymes ( 4 3 ) . B - 1 4 7 1  causes derepression  When MT  mutants of  were grown i n i r o n - d e f i c i e n t medium, a l l produced lower levels  of phenolic acids than the corresponding parent strains (Fig. 9 ) » MT of  mutants selected from anthranilate synthetase-less (ant ) strains B - 1 4 7 1  (Table I I ) , produced the same l e v e l of phenolic acids as  MT-sensitive ant strains ( F i g . 9 ) patterns were observed i n MT  (b)  r  Similar DHB(G) excretion  and ant  mutants of W B - 7 4 6 and  W-23.  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 I I ) , however, produced no phenolic acids i n i r o n - d e f i c i e n t medium supplemented with the required aromatic end-products.  (20  ug per ml tryptophan,  tyrosine and phenylalanine;  2 ug per ml p-aminobenzoic acid and p-hydroxybenzoic acid; and 20  ug per ml shikrmic acid).  (c)  HQ, r e s i s t a n t mutants ( H Q ) R  H Q i s a powerful iron-binding compound ( 6 6 ) .  Growth of the  three wild-type strains was i n h i b i t e d by H Q at a concentration of 0 . 0 1  ug per .ml.  Spontaneous mutants of  B - 1 4 7 1  were obtained, however,  which were r e s i s t a n t to 1 0 ug H Q per ml (Table I I ) .  Five days were  9  Figure 9»  10  II 12 HOURS  13  Production of DHBG by MT strains of B-147J,. DHBG excretion was measured by: B-1471 (0); MT -1 and MT -2 (X); Trp-1 (•).; and T r p - l - M T - l (®). r  r  r  required f o r colony formation under these conditions, although the growth rate of these mutants i n the absence of HQ, was the same as the parent s t r a i n .  Mutants of W - 2 3  or W B - 7 4 6 r e s i s t a n t to  1 0 ug HQ per ml were not obtained, but mutants r e s i s t a n t to 0 . 1 ug HQ, per ml were obtained (Table I I ) .  In a l l strains, one mutant  8  7  colony was obtained f o r every 1 0 When HQ mutants of W - 2 3 r  to 1 0  c e l l s plated.  or W B - 7 4 6 were grown i n i r o n - d e f i c i e n t  medium i n the absence of HQ, normal levels of phenolic acids were produced.  Under these conditions HQ mutants of B - 1 4 7 1  only very low levels of DHBG.  produced  Supplementation of media with  0.1  ug HQ/ml restored DHBG excretion i n these mutants to the l e v e l of the parent s t r a i n . Three series of spontaneous derivatives of B - 1 4 7 1 were selected which were r e s i s t a n t to: and (c) catechol.  (a) albomycin ( 4 9 ) ,  (b) actinomycin  (49)  These strains produced DHBG at the same l e v e l  as the parent s t r a i n .  40  DISCUSSION  Under conditions of iron-deficiency, D H B ( G ) production by B. s u b t i l i s i s associated with active growth of the organism. I t i s not a metabolic by-product accumulating during the stationary phase.  The c o n t r o l of D H B ( G ) production by iron appears to  3+ involve the Fe  : (DRT^G)).^ complex, and a c r i t i c a l l e v e l of t h i s  effector complex i s required to stop production.  The complex may  function d i r e c t l y , as a corepressor, a feedback i n h i b i t o r , or as both, or i t may  function i n d i r e c t l y , by f a c i l i t a t i n g the transport  of iron into the c e l l . The r e s u l t s of adding i r o n to i r o n - d e f i c i e n t cultures showed that a f t e r a c e r t a i n time the c e l l s are committed to the production of D H B ( G ) ,  and the addition of i r o n does not reverse t h i s commitment.  3+ These r e s u l t s also support the suggestion that the Fe  : (DHE^G))^  complex i s involved i n the control of D H B ( G ) production.  I f iron  i s not added u n t i l the culture has started to produce D H B ( G ) , production i s stopped more e f f e c t i v e l y than by the addition of i r o n p r i o r to the onset of production  (Fig. 3).  DHB  and D H B G  +3 were equally e f f e c t i v e as the Fe  :(phenolic a c i d ) ^ complex.  B. s u b t i l i s u t i l i z e d glycine as a nitrogen source, but was unable to cleave the glycine from D H B G for i t s nitrogen requirement. Therefore D H B G was  not being converted to D H B under the conditions  described. From the levels of D H B ( G ) accumulated i n the medium, i t i s obvious that an iron deficiency either ( i ) causes a severe d i s t o r t i o n of aromatic biosynthesis i n B . s u b t i l i s , or ( i i ) allows the c e l l  1+1  to u t i l i z e a metabolic pathway which i s not f u n c t i o n a l i n the presence of s u f f i c i e n t iron. the shutoff of  DHB(G)  The length of time required f o r  production,  i n the presence of the F e : 3 +  (DKE^G))^ complex, suggests that repression i s involved (see F i g . 3 and 1 + ) ,  thereby favouring the second a l t e r n a t i v e .  This alternative  agrees with the r e s u l t s obtained with E. c o l i , where the a c t i v i t i e s of extracts f o r the synthesis of DHB  from chorismate were dependent  on the l e v e l of i r o n i n the growth medium The production of DHB(G) was  (67),  influenced markedly by the exogenous  l e v e l s of aromatic amino acids.  These e f f e c t s could be direct,  the amino acids acting on the enzymes s p e c i f i c to DHB  synthesis,  or they could be indirect, decreasing the amount of chorismate available f o r DHB  synthesis.  A d i r e c t effect i s u n l i k e l y because  these amino acids do not i n h i b i t DHB  synthesis by extracts. (Table VII).  This suggests that the production of DHB  i n v i t r o i s regulated  by the mechanisms which form part of the general system of control  kh,  of aromatic biosynthesis i n B. s u b t i l i s ( 2 8 ,  1+5).  That portion  of aromatic biosynthesis relevant to the present study i s presented in Fig. 1 0 .  At low levels of exogenous tryptophan (Fig. 5 ) DHBG  synthesis was  i n i t i a t e d e a r l i e r and occurred to a higher extent than  i n the control f l a s k .  This phenomenon i s expected, since low  levels of tryptophan would i n h i b i t anthranilate synthetase prephenate (PPA)  dehydratase  ( 1 + 4 ) .  The increased l e v e l s of  chorismate would then be available f o r DHB enzyme(s) responsible f o r DHB  and  synthesis;  synthesis ( 6 7 )  was  and,  i f the  derepressed,  e f f i c i e n t u t i l i z a t i o n of chorismate might prevent PPA  accumulation.  High exogenous l e v e l s of tryptophan and the other aromatic amino  k2  SCHEME O F A R O M A T I C BIOSYNTHESIS  E-4-P PEP  DAHP D A H P SYNTHETASE  \  SHK D H B • C H O R S IM A T E ANT  ANT SYNTHETASE  PPA £ P P A D E H Y D R A / X D E H Y D R O G E N A S E TASE P  PHENYb ALANINE  A  TYROSINE  TRYPTOPHAN *  F i g u r e 1 0 . A b b r e v i a t i o n s : E-4-P, e r y t h r o s e - 4 - p h o s p h a t e ; PEP, p h o s p h e n o l • . p y r u v a t e ; DAHP, d - a r a b i n o h e p t u l o s o n i c a c i d - 7 - p h o s p h a t e ; SHK, s h i k i m a t e ; PPA, prephenate;. P P , ' p h e n y l p y r u v a t e ; HPP, 4-hyd r o x y p h e n y l p y r u v a t e ; ANT, a n t h r a n i l a t e ; PRT, phosphor i b o ' 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, phosphori b o s y l i s o m e r a s e ; CAR, l - ( o - c a r b o x y p h e n y l a m i n o ) - 1 - d - D - r i b u l o s e - 5 - p h o s p h a t e ; INGP S; i n d o l e g l y c e r o l phosphate s y n t h e t a s e ; . . T. S., t r y p t o p h a n s y n t h e t a s e .  ^3 acids may have caused direct feedback i n h i b i t i o n and repression at the l e v e l of DAHP synthetase ( 2 8 ) .  The greater i n h i b i t i o n of  DHBG- accumulation by phenylalanine and tyrosine, when compared to tryptophan may r e f l e c t the higher s e n s i t i v i t y of DAHP synthetase to i n h i b i t i o n by PPA than by chorismate ( 2 8 ) . The i n h i b i t i o n of DHB(G) production by a n t h r a n i l i c acid was not caused by an i n d i r e c t effect on tryptophan biosynthesis, since the i n h i b i t i o n was s t i l l observed i n the presence of t r y ptophan.  In addition, the i n h i b i t i o n of DHB(G) production was  not caused by the conversion of anthranilate to tryptophan, because the i n h i b i t i o n has been observed i n mutants blocked at any step between anthranilate and tryptophan. (Table I I ) .  Anthranilate,  however, i n h i b i t e d 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. no e f f e c t on DHBG formation.  The addition of coenzyme A had Extensive studies were not done on  these i n v i t r o syntheses because they were being conducted i n other laboratories  (6,7,21).  The formation of DHB but not DHBG, by s t r a i n WB-746 may r e s u l t from the r i g i d control of glycine synthesis i n t h i s s t r a i n .  The  observation that WB-746 d i d not form appreciable amounts of coproporphyrin I I I under iron deficiency provides further evidence f o r t h i s s t r i c t control.  Strains B-1471 and W-23 did excrete copro-  porphyrin I I I under these conditions.  I t i s also s i g n i f i c a n t that  s t r a i n WB-746 consistently produced greater quantities of DHB(G)  than strains  B - 1 4 7 1  or W - 2 3  (Fig.  8  )  .  Evidence i s presented i n  Sections I I and IV that glycine production may be r e l a t e d to an o x i d a t i v e l y f u n c t i o n a l t r i c a r b o x y l i c acid (TCA) The  cycle.  disappearance of DHB(G) from cultures remains to be explained.  An enzyme which decarboxylates DHB to catechol was p u r i f i e d from Aspergillus niger.  Oxygen was not required f o r t h i s reaction  (6l).  A pseudomonad, capable of using DHB as the sole carbon source, cleaved the aromatic r i n g of DHB with the uptake of 1 mole of oxygen. Decarboxylation did not occur p r i o r to oxidation DHB(G) was metabolized by strains  W-23  and B - 1 4 7 1 ,  (55).  Although  i t was not  possible to demonstrate either decarboxylation or oxidation of DHB by whole c e l l s of these strains after growth under a v a r i e t y of conditions.  Cultures appear to be able to metabolize DHB(G) during  the e a r l y log phase and the e a r l y stationary phase, but not during the l a t e log phase, the time during which DHB(G) i s produced i n i r o n - d e f i c i e n t cultures  (Fig. 3 , 4 and 8 ) .  DHB was present i n a s t r a i n 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. present after  2 4  Catechol, on the other hand, was  hrs but not after  1 1  hrs  (  5  l  )  .  A series of  B - 1 4 7 1  mutants (Table I I ) , which were blocked at each of the steps of tryptophan biosynthesis,  were examined f o r the compounds produced  under iron-deficiency.  Although each of these auxotrophs produced  normal l e v e l s of DHBG and showed the usual responses to iron, none of them produced catechol  during the 20-hr growth and incubation  period. The  r i n a b i l i t y of MT strains to produce normal l e v e l s of phenolic  acids may  r e f l e c t i n t r a c e l l u l a r chorismate depletion by.the  tryptophan biosynthetic enzymes.  derepressed  In addition, excessive tryptophan  production would tend to i n h i b i t DAHP synthetase ( 2 8 ) further decreasing the chorismate available f o r DHB was  synthesis ( 6 7 ) .  This interpretation  supported by the observation (Fig. 9) that a l l ant  strains of  B-1471 produced the same l e v e l of phenolic acids whether they were r e s i s t a n t to or sensitive to MT. The excretion properties of HQ,  strains of B-1471 i n the presence  absence of HQ indicated that these mutants may have a lower f o r iron.  This theory was  (Section IV).  and  requirement  supported by subsequent iron-uptake studies  46 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.  Introduction  Strains B-1471 and ¥-23 produced DHBG and coproporphyrin during logarithmic growth (Table I I I , F i g . 1, p . 4 3 ) . accumulated only i n i r o n d e f i c i e n t medium.  Both products Since glycine i s  required f o r the synthesis of both compounds, strains auxotrophic for to  glycine were selected and were grown i n i r o n d e f i c i e n t medium determine alterations i n t h e i r excretion capacities. Since an i r o n requirement has been demonstrated i n porphyrin b i o -  synthesis ( 3 0 , 3 2 ) , a s t r a i n blocked at the f i r s t step of porphyrin biosynthesis (£-aminolevulinate synthetase) was  selected.  t h i s mutant i n the presence and absence of o*-aminolevulinate the e f f e c t of heme i r o n requirement  By growing (ALA),  on phenolic acid excretion was  studied. Strains lacking the a b i l i t y to synthesize normal l e v e l s of phenolic acids were selected to further elucidate the r e l a t i o n s h i p 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 1 0  v i a b l e c e l l s per ml, and continued logarithmically,  p a r a l l e l l i n g growth ( F i g . 1 1 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 p r o d u c t i o n o f p h e n o l i c a c i d s and C o p r o p o r p h y r i n b y s t r a i n B-1471. C e l l number (0); p h e n o l i c a c i d s ( A ) ; and c o p r o p o r p h y r i n (X).  kQ  0  2  4  6  8  10  12  14  16  18  HOURS  Figure  12.  Growth and p r o d u c t i o n o f p h e n o l i c a c i d s and c o p r o p o r p h y r i n by s t r a i n W-23. C e l l number (0); p h e n o l i c a c i d s ( A ) ; and c o p r o p o r p h y r i n ( X ) .  ^9  F i g u r e 13.  Growth and p r o d u c t i o n o f p h e n o l i c a c i d s and coproporphyrin by s t r a i n WB-746. C e l l number (X); p h e n o l i c a c i d s (A); and c o p r o p o r p h y r i n ( X ) .  a l s o c o n t i n u e d . l o g a r i t h m i c a l l y ( F i g . . 1 1 and 1 2 ) .  Phenolic  acid  p r o d u c t i o n 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 c o n t i n u e d at a f a s t e r r a t e t h a n i n the two coproporphyrin and  other s t r a i n s  (Fig. 13).  p r o d u c t i o n s t a r t e d l a t e r , o c c u r r e d at a slower r a t e ,  d i d not p a r a l l e l growth ( F i g . 1 3 ) .  2. I n h i b i t i o n o f c o p r o p o r p h y r i n  The  p r o d u c t i o n by  iron  a d d i t i o n o f v a r i o u s l e v e l s o f i r o n at zero time t o . s t r a i n  B-1471 showed t h a t w h i l e 1 5 0 ug per l i t r e phenolic acid production l i t r e was of  Conversely,  completely  inhibited  ( F i g . 2 ) , a l e v e l i n excess  o f 200 ug  required to inhibit  growth was  coproporphyrin production.  The  not s i g n i f i c a n t l y a l t e r e d by t h e a d d i t i o n o f  As t h e l e v e l o f added i r o n was  per rate  iron.  i n c r e a s e d above 200' yg p e r l i t r e  time o f appearance o f c o p r o p o r p h y r i n was  d e l a y e d , and at t h e  the  higher  c o n c e n t r a t i o n s o c c u r r e d i n the s t a t i o n a r y phase. The  a d d i t i o n o f 1 mg  i r o n p e r . l i t r e a f t e r 7 hrs i n c u b a t i o n  r e s u l t e d i n an 8 5 % decrease strain B-l471 (Fig. 14).  i n t o t a l coproporphyrin  I f t h i s amount o f i r o n was  8 h r s i n c u b a t i o n , p r o d u c t i o n was ; a f t e r . 9 h r s , by 30%  ( F i g . 14).  decreased The  I f 1 mg  a c i d , however, t h e r e was production  .3.  The  added a f t e r  a d d i t i o n o f 3.5 x 10~^M  i r o n per l i t r e was  by  by 4 3 % , and i f added  or DHBG alone d i d not cause a s i g n i f i c a n t decrease production.  production  in  DHB  coproporphyrin  added w i t h the p h e n o l i c  a r a p i d cessation of  coproporphyrin  (Fig. 14).  I n h i b i t i o n o f . c o p r o p o r p h y r i n p r o d u c t i o n by.hemin  a d d i t i o n of low. l e v e l s o f hemin.to i r o n - r d e f i c i e n t c u l t u r e s  51  HOURS  F i g u r e lh. • E f f e c t on c o p r o p o r p h y r i n p r o d u c t i o n o f adding i r o n and , DHB(G"> • One mg i r o n p e r l i t r e was added a t 7 h r ( ® ) ; . 8hr.(*0> or 9 h r (0). DHB(G) a t 50 mg p e r l i t r e and 1 mg i r o n p e r l i t r e were added a t 8 h r (A). • No •• ' a d d i t i o n s were made t o t h e c o n t r o l f l a s k ( X ) . The a d d i t i o n o f 50 mg o f DHB(G) a l o n e h a d no e f f e c t ( X ) .  52  of s t r a i n B-1471 at zero time r e s u l t e d i n l a t e r production of coproporphyrin and phenolic acid (Table IX).  Under these conditions,  coproporphyrin was eventually produced at the normal l e v e l but the l e v e l of phenolic acid accumulated was markedly reduced  (Table IX).  There was no i n h i b i t i o n of growth at 0.5 - 1.0 ug hemin per ml. Growth, however, was i n h i b i t e d above 1.0 ug of hemin per ml.  4. I n h i b i t i o n of coproporphyrin production by aromatic amino acids  Whereas accumulation of phenolic acids was i n h i b i t e d by adding aromatic amino acids and a n t h r a n i l i c acid to the medium at zero time (Table V), coproporphyrin production was not influenced by supplementation of the medium with these acids at concentrations up to 2 x 10  Similarly, h i s t i d i n e supplementation  decreased the  excretion of phenolic acids (Table VI) but had no e f f e c t on coproporphyrin production.  5. Coproporphyrin  and phenolic acid production by mutants of  s t r a i n B-1471  (a)  Glycine auxotrophs  Repeated attempts to i s o l a t e glycine auxotrophs were unsuccessful.  (b)  Serine auxotrophs  Glycine i s known to be synthesized v i a serine i n E. c o l i (64). Therefore, attempts were made to i s o l a t e serine auxotrophs so that t h e i r capacity f o r coproporphyrin and phenolic acid production could be examined.  Three absolute serine auxotrophs, ser-1, ser-2 and  Table IX.  I n h i b i t i o n of the production of phenolic•acids and coproporphyrin by hemin i n s t r a i n B-1471.  O.D. 510  O.D. 4o8  14 hr  18 hr  24 hr  16 hr  20 hr  24 hr  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  1. Control  4. + 2.0 ug hemin per ml  growth i n h i b i t e d  Hemin was added to cultures at zero time. The growth rate was unaffected by hemin supplementation at 1.0 ug per ml.  5 ^  ser-3,  and one leaky serine auxotroph,  ser-4,  were isolated.  Glycine  would not replace serine as a growth.requirement f o r any of the strains. None of the strains produced phenolic acids, as determined by colorimetric assay, when grown under conditions of iron deficiency i n minimal medium supplemented with 3 0 ug serine per ml. porphyrin was produced by  ser-1,  ser-2  and  at about  ser-3  Copro1 0 $  and by  ser -k at about 1 5 $ of the l e v e l produced by the wild-type (Table X). When the culture supernatants from these experiments were concentrated and the phenolic acids extracted into e t h y l acetate, chromatographic examination of the extracts revealed low l e v e l s of DHB but no DHBG (Table X). These experiments were repeated using 5 0 i n addition to the serine supplement.  - 100  ug glycine per ml  Under these conditions, the  r e s u l t s were q u a l i t a t i v e l y 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 f o r 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 several attempts.  In unsupplemented  doubling time of 1 9 0 min.  hem-1,  was i s o l a t e d i n  glucose medium, hem-1 had a  When the medium was supplemented with  Table X.  Production of coproporphyrin and phenolic acids by serine auxotrophs derived from s t r a i n B - 1 4 7 1 .  Per Cent Production Cop  Phenolic acids  B - 1 4 7 1  1 0 0  1 0 0  0  1 0 0  1 0  5  1 0 0  0  1 5  5  1 0 0  0  1 0 0  1 0 0  Ser Ser  1 - 3  4  DHB  *  Strain  DHBG*  Revertants of Ser  1 - 4  7 0 - 8 0  2 0 - 3 0  *• Expressed as per cent of t o t a l phenolic•acids produced  5.0 ug ALA. per ml, the doubling time was 1 3 0 min.  This s t r a i n would  not grow i n unsupplemented medium containing c i t r a t e 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 l e d to the production of both  coproporphyrin and phenolic acids.  However, the responses to  d i f f e r e n t l e v e l s of ALA showed that low l e v e l s l e d to the production of phenolic acids but not coproporphyrin, and that higher l e v e l s l e d to the production of coproporphyrin and a decreased quantity of phenolic acids (Fig. 1 5 ) .  Furthermore, when glycine or serine ( 5 0 -  1 0 0 ug per ml) was added to unsupplemented medium, phenolic acid excretion by hem-1 was restored to 1 0 $ of the l e v e l i n the parent s t r a i n , 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 c i t r a t e as sole carbon source, even i n the presence of 1 ug of i r o n per l i t r e .  Similarly, supplementation  of the glucose medium with  0.5 - 1.0 ug protoporphyrin IX per ml d i d not increase the growth rate, as seen with ALA, nor d i d i t allow the production of phenolic acids and coproporphyrin during i r o n - d e f i c i e n t growth.  I t should  be pointed out that the growth of s t r a i n B-1471 was i n h i b i t e d by 0.5 ug per ml of protoporphyrin IX. Ten spontaneous revertants of hem-1 were selected on c i t r a t e medium.  A l l showed normal production of phenolic acids and copro-  porphyrin under iron-deficiency.  57  r2.4  CO  o < d d  5  10 jig  F i g u r e 15.  AL  per  15 ml.  E f f e c t o f ALA s u p p l e m e n t a t i o n on p o r p h y r i n and p h e n o l i c a c i d p r o d u c t i o n b y hem-1. The. l e v e l s o f c o p r o p o r p h y r i n (0) and p h e n o l i c a c i d s ( X ) ' . were measured a f t e r 2k h r s growth.  5 8  (d)  DHB auxotrophs  Only f i v e strains, dhb-1,  dhb-2,  dhb-3,  dhb-4  and d h b - 5 , which  required DHB f o r growth were i s o l a t e d i n many attempts.  When f i r s t  isolated, supplementation of the minimal medium with 0 . 2 ml resulted i n the appearance of normal colonies.  ug DHB per  However, a l l  f i v e strains r a p i d l y l o s t t h e i r dependence on DHB f o r growth.  The  designations dhb-1, etc. were retained f o r these revertant strains because they produced low levels of phenolic acids.  A "revertant"  from each s t r a i n was screened f o r coproporphyrin and phenolic acid production under i r o n 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 X i ) . from s t r a i n  dhb-4,  A "revertant"  however, 'showed very interesting properties.  Under conditions of iron-deficiency i t produced a several-fold higher l e v e l of coproporphyrin than the wild-type .(Fig. l 6 ) . Subsequently,  i t was found to produce coproporphyrin i n the presence  of iron, i r o n plus  DHB(G),  or ferrichrome (Table XTl).  The w i l d -  type s t r a i n did not produce coproporphyrin under any of these conditions.  Strain  dhb-4  also had an extended lag period when  inoculated into i r o n - d e f i c i e n t medium ( F i g . 14).  Supplementation  of the medium with 2 mg i r o n 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 t h i s lag. The lag was shortened by the addition of 0 . 5 $ yeast extract. I t should be pointed out that a l l the dhb-less strains grew more slowly than the wild-type s t r a i n i n i r o n - d e f i c i e n t medium.  Table XI.  Products excreted by "revertants" of DHB auxotrophs derived from s t r a i n B - 1 4 7 1 .  Excretion l e v e l (per cent of parent strain)  Strain  DHBG  Dhb-1  5  Dhb-2  2 0  Dhb-3  1 0  Coproporphyrin  2 0  8 .  1 5  Dhb-4  5  3 0 0  Dhb-5  5  5  6o  3.0-  w  4.0  2.0-1  -3.0  o  0_  -2.0 bJ O  e CO  f-  <  •  1.0-  d -1.0  0 > _  Q-fi—Q—  F i g u r e l6..  Growth and c o p r o p o r p h y r i n p r o d u c t i o n b y dhb-U. C e l l number ( 0 ) ; c o p r o p o r p h y r i n ( X ) . Note t h a t : t h e s c a l e f o r O.D. a t k08 mu does not c o r r e s p o n d to that given i n Figure 11-13.  Table XII.  Coproporphyrin production i n Dhb-4.  Per Cent Control  100 3+  + 2 mg Fe  100  per l i t r e  + 50 mg per l i t r e DHB(G) + 2 mg Fe  + k mg per l i t r e ferrichrome  per l i t r e  100 100  Discussion The r e s u l t s indicate that when cultures of B. s u b t i l i s strains B - 1 4 7 1  and  ¥ - 2 3  became iron-deficient, the c e l l s f i r s t started to  produce phenolic acids. coproporphyrin was  About an hour l a t e r the production of  started.  Phenolic acids have very strong  a f f i n i t i e s f o r f e r r i c iron ( 2 6 ) ,  and i t has been suggested that  they serve as i r o n s o l u b i l i z a t i o n factors ( 4 0 ) . of  DHB(G)  may  The production  thus f a c i l i t a t e scavenging of the l a s t traces of  i r o n from the medium f o r the synthesis of heme and non-heme i r o n proteins.  Once the available i r o n was taken up by the c e l l ,  hemin would not have been formed anymore, r e s u l t i n g i n a loss of control over porphyrin biosynthesis, which was manifested by the production of high levels of coproporphyrin. hemin was  shown to i n h i b i t ALA synthetase  the same organism ( 3 0 ,  In R.  (9).  3 2 ) and i n T. vorax ( 3 0 ) ,  In addition, i n i r o n appeared to  be required f o r the conversion of coproporphyrinogen porphyrin IX ( 9 ) .  spheroides,  I I I to proto-  Loss of c o n t r o l over porphyrin synthesis i n  B. s u b t i l i s would be expected to lead to the accumulation  of  coproporphyrinogen  to  I I I , which would oxidize spontaneously  coproporphyrin I I I , either before or after excretion.  This  i n t e r p r e t a t i o n i s supported by the observation that phenolic acid production was  i n i t i a t e d before coproporphyrin production, by  the effect of hemin, and by the e f f e c t of iron at 1 5 0 ug per on phenolic acid and coproporphyrin production. also by the properties of with  hem-1;  litre  I t i s supported  only phenolic acids were produced  low levels of added ALA, whereas both phenolic acids and  coproporphyrin were produced with higher l e v e l s .  Only the higher  l e v e l s of ALA would have allowed porphyrin synthesis i n excess of iron. Phenolic acid production appeared also t o be r e l a t e d to the demand f o r i r o n .  In the absence of added ALA, hem-1 did not  produce phenolic acids, suggesting that the i r o n available i n the medium was s u f f i c i e n t to s a t i s f y the non-heme i r o n of the culture.  In the presence of ALA,  requirements  a d d i t i o n a l i r o n was  required f o r heme synthesis, and phenolic acid production was initiated. Compared with strains B-1471 and W-23,  s t r a i n WB-746 showed an  e a r l i e r onset of phenolic acid production, and a l a t e r onset of coproporphyrin production.  I t has been shown (Section IV) that  the rate of uptake of i r o n by c e l l s of s t r a i n WB-746 i s much slower than the rates i n strains B-1471 and W-23.  This suggests  that under the experimental conditions, i r o n was not made a v a i l able to c e l l s of s t r a i n WB-746 at a rate f a s t enough to s a t i s f y non-heme i r o n requirements.  In addition, the t r i c a r b o x y l i c acid  (TCA) cycle i n s t r a i n WB-746 appears to be o x i d a t i v e l y inoperative when high l e v e l s of glucose are present (Section I I I ) .  This could  lead to a reduced demand f o r heme iron, and a l a t e i n i t i a t i o n of porphyrin production. The patterns of i n h i b i t i o n of coproporphyrin production by 1.0 .mg i r o n per l i t r e were i n good agreement with those obtained f o r i n h i b i t i o n of phenolic acid production ( F i g . 3 ) .  The r e s u l t s  obtained by adding i r o n during growth showed that once a culture became i r o n - d e f i c i e n t , i t was committed to the synthesis of some phenolic acid and coproporphyrin, even after the addition of iron.  64 The much more rapid i n h i b i t i o n obtained by the addition of i r o n and  Biffin)  the c e l l .  suggested that D H B ( G ) might help i n carrying i r o n into Neilands ( 4 2 )  suggested some time ago that the addition  of DHBG to the medium could make i r o n more available to cultures of B. s u b t i l i s . The  This has now been demonstrated (Section V).  serine auxotrophs made no DHBG when grown i n medium  supplemented with serine, suggesting that the conversion of serine to glycine may from DHB  have been the l i m i t i n g step i n the synthesis of DHBG  (Table V I I I ) .  However, when these auxotrophs were grown  i n the presence of both serine and glycine, again no DHBG was  formed.  I t has been shown that l a b e l l e d glycine added to the medium was incorporated into the glycine moiety of DHBG (Table IV), that B. s u b t i l i s can take up glycine from the medium.  implying The  r e s u l t s obtained with the serine auxotrophs are d i f f i c u l t to interpret i n l i g h t of t h i s observation.  However, i t i s possible that B.  has a common active transport system f o r glycine and serine.  subtilis If  t h i s system i s i n e f f i c i e n t , and transports serine i n preference  to  glycine, the r e s u l t s could be explained by the c e l l being unable to transport glycine at a rate f a s t enough to support growth, and serine only at a rate f a s t enough to support growth but not DHBG production. The f a c t that the i n a b i l i t y to synthesize serine prevents the production of the normal l e v e l of phenolic acids and reduces the production of coproporphyrin  suggests that there may  be a linked  system of c o n t r o l involving glycine-serine, phenolic acids coproporphyrin.  Since coproporphyrin  and  i s produced by the serine  auxotrophs, although at low levels, i t appears that glycine i s used p r e f e r e n t i a l l y f o r porphyrin biosynthesis.  The f a c t that  supple-  mentation of the medium with glycine or serine l e d to the production of some phenolic acid by hem-1 The  i s of significance i n t h i s regard.  low l e v e l of coproporphyrin production by some strains  defective  i n phenolic acid production again suggests a relationship  between these compounds.  The properties  of s t r a i n dhb-4 cannot  be explained at present, but they also support the existence of such a r e l a t i o n s h i p .  The f a c t that the extended lag period  observed with t h i s mutant i n i r o n - d e f i c i e n t medium could not reduced by supplementation of the medium with i r o n or compounds suggested that s o l u b i l i z a t i o n of i r o n was  be  iron-binding  not the factor  l i m i t i n g growth. Genetic analyses were attempted, to further explain the r e l a t i o n ship between the excretion extracted  capacities of mutant s t r a i n s .  from strains B-1471 and WB-746 was  DNA  used to transform  the auxotrophs derived from B-1471, but none of the strains  (35)  was  competent, even after addition of the "competence inducing f a c t o r " i s o l a t e d from B. s u b t i l i s l68 I ( 2 ) .  66 Section I I I : - I n h i b i t i o n of phenolic acid production  i n B. s u b t i l i s  Introduction In bacteria, certain compounds i n h i b i t growth by acting as f a l s e regulatory metabolites of biosynthetic pathways (37).  Thus, i n  E. c o l i (37) and B. s u b t i l i s (46), 5-methyltryptophan has been shown to mimic tryptophan as a feedback i n h i b i t o r of anthranilate synthetase (37) and as a repressor of synthesis of the enzymes of the tryptophan pathway.  Mutants r e s i s t a n t to t h i s compound are either ( i ) i n s e n s i t i v e  to feedback i n h i b i t i o n or ( i i ) derepressed f o r the tryptophan b i o synthetic enzymes (37> 46).  Various compounds s t r u c t u r a l l y r e l a t e d  to DHB(G) were therefore tested f o r t h e i r capacity to i n h i b i t acid production.  phenolic  Such i n h i b i t o r s might be u s e f u l i n the i s o l a t i o n of  mutants defective i n the control of phenolic acid synthesis. DHB(G) production was r e l a t e d not only to the l e v e l of iron i n the medium, but also to the l e v e l of i r o n required by the c e l l f o r heme biosynthesis.  Because heme biosynthesis requires an operative TCA  cycle, compounds known to i n h i b i t the operation of t h i s cycle i n B. s u b t i l i s were tested f o r t h e i r effects on phenolic acid production.  1.  Analogs of DHB  The compounds tested as analogs of DHB(G) varied widely i n t h e i r effects on phenolic acid and coproporphyrin production.  Table XIII  l i s t s the analogs i n order of t h e i r effectiveness as i n h i b i t o r s .  2. Compounds a f f e c t i n g the functioning of the TCA cycle  Some of the compounds tested are known to i n h i b i t sporulation i n  Table XIII.  E f f e c t of analogs of DHB on phenolic acid and coproporphyrin production.  Per cent Supplement ( 2 0 0 ug/ml)  Strain DHBG  B - 1 4 7 1  Cop*  accumulation  Strain W - 2 3 DHB(G) Cop  Strain WB-746 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  5 0  1 0 0  5 0  1 0 0  55  1 0 0  6 5  1 0 0  5 0  1 0 0  75  1 0 0  6 5  1 0 0  4 0  1 0 0  6 5  1 0 0  1 0 0  3-hydroxybenzoate 2,3-dimethoxy-  benzoate 2-fluorobenzoate 3-fluorobenzoate Dipicolinic acid 3 -hydr oxyanth ranilate  0  40  0  8 0  1 0  0  55  0  ^5  2 0  0  1 0 0  * Cop refers to coproporphyrin  0  1 0 0  0  4o 1 0 0  B a c i l l i , p o s s i b l y because they i n h i b i t the functioning of the TCA Glutamic acid ( 6 2 )  cycle.  and a-ketoglutaric acid (24) i n h i b i t e d  sporulation i n B. s u b t i l i s by repressing the synthesis of aconitase. a - p i c o l i n i c acid i s an i r o n chelating agent which has been shown to i n h i b i t sporulation i n B. cereus ( 1 6 ) of aconitase.  by preventing derepression  m-Tyrosine i n h i b i t s sporulation i n B. s u b t i l i s  but i t s mechanism of action i s unknown.  (4),  The compounds tested, and  t h e i r effects on phenolic acid and coproporphyrin production are shown'in Table XIV.  3. E f f e c t of sideramines on phenolic acid and porphyrin production by s t r a i n  B - 1 4 7 1 .  Sideramines are f e r r i c trihydroxyamates factors f o r c e r t a i n b a c t e r i a ( 3 9 ) 5 into the c e l l ( 4 0 ) .  Micrococcus  medium i f a dihydroxyphenol, acid ( 5 6 ) ,  or a sideramine,  which can act as growth  possibly by transporting iron lysodeikticus w i l l grow i n minimal  such as catechol ( 5 6 )  or protocatechuic  such as ferrichrome ( 4 l ) ,  i s present.  Therefore, the effects of sideramines on phenolic acid and coproporphyrin production were examined i n s t r a i n  B - 1 4 7 1 .  types of sideramine  (49),  chosen f o r study.  When added at zero time, 0 . 4  ml or 0 . 2  There are two  and a representative of each group was ug ferrichrome per  ug ferrioxamine per ml i n h i b i t e d phenolic acid production  completely,  and delayed and reduced coproporphyrin production (Fig. 1 7 ,  Table XV).  The l e v e l of ferrichrome used was equivalent to the  addition of 2 0 ug i r o n per l i t r e .  The addition of t h i s much iron  alone did not i n h i b i t phenolic acid or coproporphyrin production by strain  B - 1 4 7 1  (Fig.  2  and Section I I ) .  When the same levels of the  6  9  Table XIV. E f f e c t of i n h i b i t o r s acting at the l e v e l of the TCA cycle on phenolic acid and coproporphyrin production.  Per cent accumulation  Strain Inhibitors None  DHBG  Cop  1 0 0  1 0 0  7 0  a-ketoglutaric or glutamic acid  acid  7 5  0 . 1 0 $ cn-ketoglutaric or glutamic acid  acid  5 5  0 . 0 5 $  B - 1 4 7 1  Strain W - 2 3  Strain WB-746  Cop  DHB  Cop  1 0 0  1 0 0  1 0 0  1 0 0  8 0  7 0  1 0 0  8 0  6 0  5 0  1 0 0  5 0  DHB(G)  a - p i c o l i n i c acid  0  0  1 0  3 0  1 0 0  8 0  m - tyrosine  0  0  0  0  0  0  6 0  0  2 0  8 0  6 0  8 5  0  - tyrosine  A l l TCA cycle i n h i b i t o r s were added at zero time. None of the supplements s i g n i f i c a n t l y affected growth except m - tyrosine, which retarded growth s l i g h t l y i n W B - 7 4 6 . Corrections were made f o r t h i s growth impairment (see t e x t ) .  0.6^ 0.5-  9  10  II  12  13  14  15  HOURS F i g u r e 17.  E f f e c t o f s i d e r a m i n e s u p p l e m e n t a t i o n on p h e n o l i c a c i d e x c r e t i o n i n s t r a i n B-1471. F e r r i c h r o m e (0.4 ug p e r ml) o r f e r r i o x a m i n e (0.2 :' ' Ug p e r ml) was added a t zero time (A) o r a f t e r 8 h r growth ( X ) . N o . a d d i t i o n s were made t o t h e c o n t r o l f l a s k (0).  16  Table XV.  I n h i b i t i o n of the production of coproporphyrin by sideramines i n s t r a i n B-1471  O.D.  at  408 mu  12 hr  14 hr  18 hr  24 hr  0.45  1.1  1.5  1.6  0  0  0.45  0.60  3. 0.4 ug ferrichrome per ml added  0  0  0.4o  0.50  4. 0.2 ug ferrioxamine per ml added  0  0  0.65  O.90  0  0  0.80  1.2  1. Control 2. 0.4 ug ferrichrome per ml added at zero time  at 8 hours  at zero time  5. 0.2 ug ferrioxamine per ml added at 8 hours  The growth rate was or ferrioxamine.  not affected by supplementation with ferrichrome  7 2  sideramines were.added to cultures after 8 hr growth under i r o n d e f i c i e n t conditions, phenolic acid production was stopped r a p i d l y (Fig. 1 7 )  and coproporphyrin production was delayed and reduced  (Table XV).  Discussion I t appears that there are two classes of compounds a f f e c t i n g phenolic acid production i n i r o n - d e f i c i e n t c e l l s of B. s u b t i l i s . Compounds of Class 1 i n h i b i t both coproporphyrin and phenolic acid production.  Those of Class 1 1 i n h i b i t only phenolic acid  production. The production of phenolic acids by B. s u b t i l i s i s dependent upon the l e v e l of i r o n i n the medium. i r o n requirements  of the c e l l .  blocked by mutation,  I t i s dependent also on the  I f &-aminolevulinate  synthesis i s  phenolic acids are not produced under i r o n -  deficiency, presumably because the non-heme i r o n requirements are met by the r e s i d u a l i r o n i n the medium (Fig. 1 5 ) .  Therefore, i t  i s not surprising that compounds a f f e c t i n g the functioning of the TCA cycle i n h i b i t both coproporphyrin and phenolic acid production. None of the compounds of Class 1 1 i n h i b i t e d the growth of B. subtilis.  This suggests that phenolic acids are not e s s e n t i a l f o r  growth under the experimental conditions used. from B. s u b t i l i s s t r a i n  B - 1 4 7 1  Mutants i s o l a t e d  as DHB auxotrophs l o s t t h e i r dependence  on DHB within one or two transfers.  When examined, however, such  "revertants" were found to produce very low l e v e l s of phenolic acid under iron-deficiency.  Some of these strains also produced low  l e v e l s of coproporphyrin, whereas one of them produced increased  levels (Table X l ) .  Phenolic acids, therefore, are not  for growth or coproporphyrin production, under the  essential  experimental  conditions employed. The most e f f e c t i v e i n h i b i t o r s i n Class II are meta-substituted benzoic acids.  D i p i c o l i n i c acid i s , i n effect, such a compound.  I t i s i n t e r e s t i n g that anthranilate (Table V) and m-hydroxybenzoic acid i n h i b i t e d phenolic acid production about 50 per cent, whereas 3-hydroxyanthranilic acid i n h i b i t e d i t 100 per cent (Table XIV). The effects of these i n h i b i t o r s on DHB  synthesis by c e l l - f r e e  extracts are being investigated. The i n h i b i t o r y effect of sideramines i s probably due to t h e i r f a c i l i t a t i n g the uptake of i r o n by the c e l l , a conclusion (Section VI) which i s supported by the f a i l u r e of desferri-ferrichrome to 3+ produce the i n h i b i t i o n seen with ferrichrome.  Since Fe  :(DHB(G)).  i s a more e f f e c t i v e i n h i b i t o r of phenolic acid production than i r o n 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 i n h i b i t phenolic acid production.  This affords an experimental approach  to the study of the s p e c i f i c i t y of the i r o n uptake mechanism(s) of B. s u b t i l i s (Section V).  Section IV: - E f f e c t s of aeration and glucose concentration on growth and phenolic acid excretion  Introduction  A l l three w i l d type strains which were studied extensively produced phenolic acids during logarithmic growth i n i r o n - d e f i c i e n t 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-7^-6 was not affected by i n h i b i t i o n of the TCA cycle (Table XIV).  I t was found sub-  sequently that WB-746 excreted coproporphyrin at lower glucose l e v e l s , and that excretion was not i n i t i a t e d u n t i l the late s t a t i o n 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 f u n c t i o n a l under the conditions employed.  Because high l e v e l s of glucose have been shown to  repress the operation of t h i s cycle i n B. s u b t i l i s ( 1 1 ,  l6, 1 7 ) ,  phenolic acid and coproporphyrin production i n WB-746 were studied i n medium containing lower levels ( 0 . 3 per cent) of glucose.  In  addition, the e f f e c t of aeration on phenolic acid production was studied i n the three strains.  1.  E f f e c t of aeration on phenolic acid production  When strains  B - 1 4 7 1  and  W-23  were grown i n 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 r e s u l t i n g over a 2 4 hr period was only Q 7.5 x 1 0 c e l l s per ml (Table XVl). Under these conditions, no  7 5  Table XVI.  Strains  E f f e c t of aeration on growth and phenolic acid production i n wild-type s t r a i n s .  Aeration  B - 1 4 7 1  maximum  B - 1 4 7 1  s t i l l culture  W-23  W-23  maximum s t i l l culture  WB-746  maximum  WB-746  s t i l l culture  Generation time (min)  Max. c e l l density ( c e l l s per ml) x  1  0  O.D. at O'.D. at mu mu 5 1 0  4 0 8  1 . 6  1 3 0  2 . 8  1 9 0  7.5.x  1 3 0  2 . 8  x  1  0  9  1 9 0  8 . 0  x  1  0  8  0  0 . 3  1 9 0  2 . 8  x  1  0  9  1 . 2  1 . 3  1 9 0  2 . 7  x  1  0  9  1 . 1  0 . 0 5  9  0 . 5 5  Q 0  1 0  0 . 4 5  0 . 2  1 . 6  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 l e v e l 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  coproporphyrin was produced;  WB-746  was grown i n s t i l l culture, no  but the generation time, maximum c e l l  density and production of DHB were i d e n t i c a l to those obtained under aerobic growth conditions (Table XVI).  2.  E f f e c t of glucose concentration on phenolic acid excretion  When  WB-746  was grown i n medium containing  a diauxic growth curve was observed (Fig. 1 7 ) .  0  .  3  per cent glucose,  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;  t h i s was followed by a second period  of logarithmic growth. . DHB production was i n i t i a t e d after 8 hr incubation, and continued logarithmically f o r 3 hr.  The DHB con-  centration then decreased l i n e a r l y 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 $  c i t r a t e and 0 . 3 $  the oxidation of these substrates by WB-746 was studied.  acetate,  Cells  harvested and washed (see Materials and Methods) after 1 0 hr growth(Fig.  1 8 ) showed a l a g of 1 . 5 hr before acetate and c i t r a t e were  oxidized (Fig. 1 9 ) .  Even after t h i s period, oxidation of these  substrates proceeded very slowly.  Extracts prepared from these  LU Q_ 00 LU O  0  Figure 18.  5  10 15 HOURS  20  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 p e r i o d .  78  Figure  19.  O x i d a t i o n c a p a c i t i e s o f 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 c o n t a i n i n g .0.3% g l u c o s e . • C e l l s h a r v e s t e d a f t e r 10 h r o x i d i z e d a c e t a t e (©) • and c i t r a t e (&) a f t e r a $0 min l a g . Cells h a r v e s t e d after.17 h r growth o x i d i z e d a c e t a t e (0) and c i t r a t e w i t h o u t a l a g (A).  1 0 hr c e l l s were also unable to oxidize acetate or c i t r a t e .  In  contrast, c e l l s harvested after 1 7 hr growth (Fig. 1 8 ) oxidized acetate and c i t r a t e without a lag.  At no time during growth were  c e l l s able to oxidize DHB(G) i n the presence or absence of 1 mg per l i t r e .  Fe  The extended plateau (Fig. 1 8 ) suggested that i r o n  deficiency may have prevented the formation or functioning of TCA cycle enzymes.  The addition of 1 mg i r o n per l i t r e at 9 hr,  however, did not reduce t h i s plateau period. Unlike s t r a i n  WB-746,  strains  B - 1 4 7 1  and  W-23  did not display  diauxic growth when grown i n medium containing 0 . 3 per cent glucose, and were able to oxidize acetate and c i t r a t e without a lag when harvested at any time after 7 hr incubation. Discussion  The i n a b i l i t y of s t r a i n  WB-746  to produce coproporphyrin during  logarithmic growth i n i r o n - d e f i c i e n t 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 c i t r a t e i n the presence of glucose.  Prior to and during subsequent acetate and  c i t r a t e oxidation, the l e v e l of DHB decreased; the medium.  t h i s decrease was  i n the culture supernatant  independent of the l e v e l of i r o n i n  Ho oxidation of DHB was observed by c e l l s harvested  at any time during growth.  I t i s possible that during the plateau  preceding acetate and c i t r a t e oxidation (Fig. 1 7 ) DHB the quinone form, as an electron transport by-pass,  served, v i a 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 g l y c o l y s i s was grown with adequate  ' glucose, enough ATP would become available from the Embden-Meyerhof pathway to minimize the r o l e 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 i n the synthesis of (a) t r i c a r b o x y l i c 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 i n  WB-746.  Glucose was much less i n h i b i -  tory to the oxidative functioning of the TCA cycle i n strains and  B - 1 4 7 1  W-23.  The production (Table XIV)  of high l e v e l s of DHB but no DHBG i n s t i l l cultures  again r e f l e c t s the r e l a t i o n s h i p of DHBG production  cycle a c t i v i t y .  to TCA  81  Section V: - Iron transport and phenolic  acids  Introduction  Phenolic acids have very strong a f f i n i t i e s f o r f e r r i c iron ( 2 6 ) , and i t has been suggested that they may be involved i n i r o n transport i n B. s u b t i l i s  (40).  The production of phenolic acids was r e l a t e d  not only to the l e v e l of i r o n i n the medium (Fig. 2 ) , but also to the i r o n requirements of the c e l l (see Section I i ) .  Therefore, an  examination was made of the uptake of i r o n by B. s u b t i l i s , and the effects of phenolic acids on t h i s  1  process.  Preliminary experiments  Labelled i r o n was added to s t e r i l e medium and the mixture incubated as described i n Materials and Methods.  Samples were  removed at i n t e r v a l s over a 50 min period and passed-through 0.45 U M i l l i p o r e membranes. Therefore,  No r a d i o a c t i v i t y was retained on the membranes.  insoluble i r o n 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 i r o n from the membranes than washing with 2.0 ml.  The smaller volume was used i n a l l  experiments.  2. Experiments with i r o n - d e f i c i e n t cultures.  Iron transport  as a function of culture age  The rate of i r o n uptake was proportional to c e l l concentration,  g i . e . culture age, up to a density of 2.0 x 1 0 /ml.  At higher  densities, the curve became sigmoid (Fig. 2 0 ) .  I t should be  emphasized that these experiments employed growing c e l l s , the culture medium contained traces of iron.  and that  I t was possible  that the increased rate of transport observed above a c e l l density of 2.0 x 1 0 /ml r e f l e c t e d the development of an increased capacity to take up i r o n i n response to the approach to an i r o n - d e f i c i e n t state.  A l l subsequent experiments were performed using a c e l l  density of 5«5  x  1 0 /ml.  I n the absence of added iron, cultures  started to produce DHBG at a c e l l density of about 7-0 x 1 0 /ml (see (Fig. l l ) .  3. Iron transport as a function of energy  The addition of i r o n l e d to an i n i t i a l rapid binding of l a b e l to the c e l l s ( F i g . 2 l ) .  This was followed by a slower, l i n e a r  rate of uptake u n t i l about 30 min, after which time the rate decreased ( F i g . 2 1 ) .  A mixture of sodium azide and iodoacetamide  i n h i b i t e d the slower phase of uptake, but was without e f f e c t on the i n i t i a l r a p i d binding of i r o n (Fig. 2 l ) .  The gradual decrease  i n bound i r o n i n the presence of the i n h i b i t o r s was the r e s u l t of cell  lysis.  k. The e f f e c t of temperature on i r o n transport  The binding of i r o n to the c e l l s was temperature independent (Fig.  21).  The slower rate of uptake was temperature dependent,  with an optimum at 3 7 C (Fig. 2 2 ) .  Measurements at temperatures  above 1+5 C were not made because of c e l l  lysis.  83  F i g u r e 20.  I r o n u p t a k e 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 density, i n d i c a t e d . Each p o i n t r e p r e s e n t s t h e r a t e o f uptake measured o v e r t h e i n t e r v a l 3-8 m i n a f t e r i r o n a d d i t i o n .  Qk  cn LU Q.  2.0 CL 3 IX.  CD lO  0>  10  F i g u r e 21.  20 30 MINUTES  40  I r o n uptake as a f u n c t i o n o f energy. The r a t e o f i r o n uptake was measured a t 37 C (0), o- C (A), a t 37 C a f t e r p r e - i n c u b a t i o n f o r 30 min w i t h 30 mM sodium a z i d e and 1 mM iodoacetamide (X), and .at 37 C a f t e r t h e a d d i t i o n o f 0.1$ c i t r a t e (A).  0.3i  20  2'5 3'0 35* 40 TEMPERATURE •  ~45  F i g u r e 2 2 . • E f f e c t o f t e m p e r a t u r e on i r o n u p t a k e . C u l t u r e . samples were p r e - i n c u b a t e d a t t h e a p p r o p r i a t e t e m p e r a t u r e f o r 10 m i n b e f o r e t h e a d d i t i o n o f iron.  5.  Incorporation of i r o n into TCA-insoluble material  A s i g n i f i c a n t f r a c t i o n of the i r o n bound to the c e l l s , and of that subsequently  taken up, was insoluble i n cold 5 $ TCA (Fig. 2 3 ) .  This was true also of the i r o n bound at 0 C. 6.  Iron transport as a function of i r o n concentration  The rate of i r o n uptake at 3 7 C was proportional to the i r o n concentration, up to a l e v e l 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 i r o n concentrations showed that a period of very rapid uptake, l a s t i n g about 6 - 7 min, was followed by a period during which iron was l o s t very r a p i d l y from the c e l l s .  This loss of i r o n was not  observed at lower i r o n concentrations ( i . e . at 5 mug/ml), not even when c e l l s were allowed to take up i r o n f o r 3 0 min, were f i l t e r e d , washed with iron-free medium, and then incubated i n i r o n free medium f o r a further 3 0 min. were saturated ( i . e .  The binding s i t e s on the c e l l s  Fe uptake at 0 C) at an i r o n 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 e f f e c t of c i t r a t e on i r o n uptake  The medium used contained sodium c i t r a t e .  I t has been reported  that c i t r a t e could replace the DHB requirement of a multiple aromatic auxotroph of Escherichia c o l i ( 6 7 ) .  When c i t r a t e was omitted  from the medium, the rate of i r o n uptake by c e l l s was increased about 4-fold, and the amount of i r o n taken up was increased about  3.0i cc u  CL  LJ  2.0  /  0.  /  / 1.0  ID  .X, .0.  0  T  10  T  20 30 MINUTES  F i g u r e . 2 3 . I n c o r p o r a t i o n of i r o n material. Whole c e l l s .insoluble f r a c t i o n at ' c e l l s a t 0 C ( X ) , and f r a c t i o n a t O.C ( « ) . .  .0  40  50  into TCA-insoluble a t 37 C (A), TCA37 C ( 0 ) , w h o l e . TCA-insoluble  88  ug  Figure 2h.  Fe  PER  LITRE  Rate of i r o n uptake as a f u n c t i o n of i r o n concentration. I r o n - 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 rates were measured over the i n t e r v a l 2-6 min after iron addition.  89  0  itiO ^ig  F i g u r e 25.  2b0 Fe  300  PER  .400  500  LITRE  B i n d i n g o f i r o n t o c e l l s a t 0 C as a f u n c t i o n o f i r o n concentration. I r o n 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 o f i r o n .  40$  (Fig. 2 6 ) .  There was also a 2 - 3 f o l d increase i n the amount  of i r o n bound by the c e l l s at 0 C (Fig. 2 6 ) . 5 - 3 0 0  The addition of  ug DHB(G)/ml to cultures grown i n the absence of c i t r a t e  caused a 2 0 $ increase i n the l e v e l of i r o n uptake (Fig. 2.6). addition of 0 . 1 $  The  c i t r a t e at zero time or after 3 0 min incubation,  however, d i d not a f f e c t i r o n uptake by c e l l s growing i n i r o n deficient, citrate-containing 8.  The  medium (Fig.  21).  The e f f e c t of phenolic acids on iron uptake 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 i r o n uptake (Fig. 2 7 ) . to 3 0 0 uptake.  Increased concentrations of phenolic acid up  ug/ml d i d not give any further  stimulation of the rate of  I t should be pointed out that the phenolic acids were  mixed with the i r o n and l e f t at room temperature to e q u i l i b r i a t e f o r 1 5 min ( 2 6 )  p r i o r to t h e i r addition to the c e l l s .  In the  absence of a phenolic acid about 5 5 $ of the added iron was taken up by the c e l l s .  In the presence of a phenolic acid about  9°$  was taken up (Fig.  2 7 ) .  Phenolic acid i n the range  ug/ml  decreased the binding of i r o n at 0 C by about 5 0 $ .  5 - 3 0 0  I f less than  5 ug/ml phenolic acid was added, the rate of uptake was  still  elevated, but there was some decrease i n the amount of i r o n taken up by the c e l l s (Fig. 2 7 ) .  The addition  of 5 ug/ml of phenolic  acid with 0 . 5 mug iron/ml d i d not a f f e c t the rate of uptake, but i t d i d increase the amount of i r o n taken up from 3 0 $ to 7 0 $ 28).  (Fig.  The binding of i r o n at 0 C again was reduced i n the presence  of phenolic acid (Fig.  28).  on LU Q. LU  in  °6"  F i g u r e 26.  10  20  30  MINUTES  40-  50  E f f e c t o f growth w i t h o u t c i t r a t e on i r o n uptake. Uptake was measured a t 37 C w i t h 5 Ug DHB(G)/ml ( X ) , and w i t h no a d d i t i o n (A). B i n d i n g was a t 0 C w i t h 5 ug DHB(G)/ml (O), and w i t h no a d d i t i o n ( A ) .  MINUTES  .Figure 2 7 . E f f e c t of phenolic acids on iron uptake. Iron was added to a l l f l a s k s at 5 mug/ml. Uptake was measured at 3 7 C with 5 - 3 0 0 ug • • . ' DHB(G)/ml ( A ) , 1 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 ) . u  g  0  A  A  A  A  A  10  20  30  40  50  A  60  MINUTES  F i g u r e 28.  E f f e c t o f p h e n o l i c a c i d s on uptake a t lower levels of iron. I r o n was added t o a l l f l a s k s at 0.5 mug/ml. Uptake was measured a t 37 C w i t h 5 ug DHB(G)/ml (0), and w i t h no a d d i t i o n (X). B i n d i n g was measured a t 0 C'with 5 g DHB(G)/ml (A), and w i t h no a d d i t i o n (•). :  9.  Experiments with i r o n - s u f f i c i e n t cultures  C e l l s grown i n i r o n - s u f f i c i e n t medium bound less than 5 $ of the l e v e l of i r o n bound by i r o n - d e f i c i e n t c e l l s at 0 C ( F i g . 2 5 ) .  In  addition, the rate of uptake was greatly reduced as a consequence of growth i n i r o n - s u f f i c i e n t medium ( F i g . 2 4 ) .  The addition of  phenolic acids to i r o n - s u f f i c i e n t c e l l s did not a f f e c t either the binding or the rate of uptake of i r o n . 10.  Experiments with mutant strains  A f t e r growth i n i r o n - d e f i c i e n t medium, the rates of i r o n uptake i n strains  dhb-1,  dhb-4  observed i n s t r a i n  and d h b - 5 were less than  B - 1 4 7 1  (Fig.  2  9  )  .  2  0  $  of the rate  The binding of i r o n at  was reduced by 8 0 $ i n these strains ( F i g . 2 9 ) .  0  S  In the presence of  5 0 'ug DHB(G)/ml, the rate of uptake was increased 1 0 - f o l d i n s t r a i n but the rates i n strains  dhb-4,  (Fig.  2  9  )  dhb-1  and d h b - 5 were unchanged  .  Discussion  The r e s u l t s indicate that B. s u b t i l i s possesses a system f o r the transport of iron, which has the properties of an active transport system ( 3 ) ,  being temperature and energy-dependent.  Growth  under conditions of iron-deficiency r e s u l t s i n a considerable increase i n the capacity of the transport system.  I n f a c t , the  i r o n - d e f i c i e n t c e l l develops t h i s capacity to such an extent that eventually i t can no longer r e t a i n the high levels of i r o n taken up when increased quantities are added to the medium.  I n addition  to the transport system, c e l l s of B. s u b t i l i s can bind i r o n at 0 C,  95  3.0-  rr LU  2.0-  LU  3  C7>  0  10  20  MINUTES  Figure  29.  I r o n uptake i n mutant s t r a i n s . S t r a i n dhb-1, dhb-U,. and dhb-5: w i t h o u t a d d i t i o n a t 37 C ( © ) , and a t 0 C ( X ) . S t r a i n s dhb-1 and dhb-5 w i t h 50 ug DHB(G)/ml a t 37 C (0). S t r a i n dhb-k w i t h 50 ug DHB(G)/ml a t 37 C (•).  and t h i s binding also i s increased considerably by growth under iron-deficiency. The addition of a phenolic a c i d to i r o n - d e f i c i e n t c e l l s stimulated the rate and increased the l e v e l of iron transported. At lower i r o n concentrations, only the l e v e l of iron transported was a l t e r e d 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 d i r e c t l y i n i r o n transport. At  3+ neutral pH, the 3:1 complex of DHBG:Fe (ho),  i s favoured (Fig. 3 0 )  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 i r o n to the c e l l . The transport system may be inducible because the capacity f o r transport increases during iron-deficiency.  I f i t i s inducible,  the question of the nature of the inducer becomes important.  It  3+ i s u n l i k e l y to be the Fe  (DKLXG)).^ complex because the increased  capacity i s the consequence of a lack of iron.  An a t t r a c t i v e  alternative i s that the system i s repressible, with the corepressor  3+ being an i r o n complex, possibly Fe  (DHE^G)).^  C i t r a t e can bind iron, and could serve to make l i m i t i n g i r o n more available to the c e l l , thereby postponing deficiency.  the onset of i r o n -  In the present case, the omission of c i t r a t e from  the medium l e d to an increased capacity f o r iron transport. Almost h a l f of the iron taken up by the c e l l s was insoluble i n cold TCA.  This was true also of the iron bound to the c e l l s .  97  The nature of t h i s "insoluble" i r o n i s not clear at present, i t i s being investigated.  but  Attempts are being made to i s o l a t e  mutants t o t a l l y unable to synthesize phenolic acids so that t h e i r capacities f o r i r o n transport and binding may  be  studied.  99  Section VI: - Control of i r o n transport Introduction  Phenolic a c i d production started e a r l i e r and continued f a s t e r rate i n coproporphyrin  WB-746  than i n B - 1 4 7 1  (Fig.  1 1 ,  at a  Conversely,  . 1 3 ) .  production started l a t e r and occurred at a slower  rate i n WB-746 than i n B - 1 4 7 1  (Fig.  1 1 ,  1 3 ) .  Oxidative functioning  of the TCA cycle i n WB-746 was i n h i b i t e d by 0 . 3 $ glucose (Fig. 1 8 ) , while that of B - 1 4 7 1 was not.  Hem-1  had no oxidative TCA cycle  i n unsupplemented media, but the normal functioning of t h i s cycle was restored following supplementation with ALA (Fig. l 4 ,  p. ).  These properties prompted an i n v e s t i g a t i o n of the r e l a t i o n s h i p between i r o n transport capacity and heme-iron requirement.  1.  E f f e c t of heme-iron requirement on transport capacity  When uptake studies were conducted, the rate and extent of i r o n uptake i n B - 1 4 7 1 greatly exceeded that i n WB-746 or of unsupplemented medium (Fig. 3 1 ) .  hem-1  in  When hem-1 was grown i n medium  supplemented with 5 Hg ALA per ml, I t s i r o n uptake capacity approached that of the parent s t r a i n ( F i g . 3 1 ) . 2.  E f f e c t of ferrichrome + 3  Addition of the phenolic a c i d Fe acid (Fig.  4 )  and coproporphyrin  complex i n h i b i t e d phenolic  production  (Fig.  l  4  )  by s t r a i n  B - 1 4 7 1 .  This was probably the r e s u l t of phenolic acids increasing i r o n uptake (Fig. 2 7 ) .  The addition of low l e v e l s ( 0 . 4 ug per ml) of ferrichrome  to i r o n - d e f i c i e n t cultures of B - 1 4 7 1 at zero time or a f t e r  1 0 0  0  10  20 MINUTES  30  40  Figure 3 1 * - • .Strain differences i n i r o n uptake capacities. Iron uptake was measured i n strains B - 1 4 7 1 and W B - 7 4 6 (^). Hem-1 growing i n the absence of ALA had a low i r o n 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 s t r a i n ( 0 ) . ( 0 )  8 hr growth, i n h i b i t e d DHBG (Fig. 1 7 ) (Table XIV).  and coproporphyrin  production  The l e v e l of i r o n provided by t h i s supplementation  i s about 2 0 ug per l i t r e , a l e v e l which alone caused no i n h i b i t i o n of excretion ( F i g . 2 ) .  Ferrichrome has been implicated as an i r o n  s o l u b i l i z a t i o n f a c t o r f o r several microorganisms (39? 41).  Ferri-  chrome and desferri-ferrichrome were tested therefore f o r t h e i r effects on i r o n transport. ^%e  When c e l l s were incubated with 5 mug  per ml, 5 5 $ of t h i s i r o n was available to the c e l l over a 4 0  min incubation period (Fig. 3 2 ) . was  When 0 . 4  ug ferrichrome per ml  added simultaneously with the radioactive iron, there was a 3 0 $  decrease i n the amount of l a b e l incorporated into the c e l l s .  Pre-  59 incubation of the ferrichrome with  Fe for one hr, which would  allow about 2 2 $ of the ferrichrome-bound unlabelled i r o n to exchange with  Fe ( 3 4 )  resulted i n only an 1 8 $ decrease i n the amount- of  radioactive l a b e l incorporated compared to c e l l s i n the absence of ferrichrome  (Fig. 3 2 ) .  The addition of desferri-ferrichrome  simultaneously with ^ % , made 99$ °f the l a b e l l e d iron available to e  the c e l l s within 1 5 min incubation ( F i g . 3 3 ) . r 3. HQ, mutants The properties of strains HQ -l-6 (Section I) suggested that r  these strains required less available i r o n than the parent  strain.  To check t h i s theory, i r o n transport studies were conducted.  When  these strains were grown i n the absence of HQ, the rate and extent of i r o n uptake were s i g n i f i c a n t l y increased (Fig. 3 4 ) . mentation of media with 0 . 1  Supple-  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 2.0 LU  CL  £  CD LO  -1.0-  10  Figure 32. .'•  20 MINUTES  30  T  40  - E f f e c t o f f e r r i c h r o m e on i r o n transport.- • F e r r i c h r o m e (0.4 ug/ml) was p r e - i n c u b a t e d f o r 1 h r with' i r o n ( A ) , added s i m u l t a n e o u s l y w i t h i r o n (0), or ferrichrome , (0.8 ug/ml) was added s i m u l t a n e o u s l y w i t h i r o n ( X ) . Wo f e r r i c h r o m e was added t o t h e c o n t r o l f l a s k ( 8 ) .  103  if)  LU O  4.0 LU Q_ LU Q_ 3 0) Li-  CD ID  en  10  20  30  40  MINUTES Figure  E f f e c t o f f e r r i c h r o m e on i r o n uptake i n s t r a i n B-1471. C e l l s were grown t o a v i a b l e d e n s i t y o f 5. 5 x 10" p e r m l p r i o r t o study. 59Fe (5 mug p e r ml) -and d e s f e r r i . f e r r i c h r o m e (0.4 ug p e r ml) were t h e n added ( a t zero . time ( © ) . F e r r i c h r o m e was- o m i t t e d f r o m t h e c o n t r o l flask (0).  33•  CO  o  5,0  -—©  4.0LLI  UJ  3.0  < Q_ 3  LL.  8'  2..0"  1.01  1-  o  10  20  30  40  MINUTES Figure 34.  I r o n u p t a k e b y s t r a i n H Q - l . Uptake o f ^^Fe'oy s t r a i n B-1471 (0) was compared t o u p t a k e b y s t r a i n H Q - l grown i n . t h e absence (X) and p r e s e n c e •'(©) o f 0 . 1 u-g/ml HQ. r  r  'Discussion  A requirement f o r heme-iron appeared to cause an increase i n the transport capacity of c e l l s .  The i n h i b i t o r y e f f e c t of low  levels of ferrichrome on DHBG and coproporphyrin  excretion i n  B - 1 4 7 1  appeared to be r e l a t e d to i t s a b i l i t y to d r a s t i c a l l y increase both the rate and extent of i r o n 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 s i t e f o r free  i r o n transport, or ( i i ) upon entering the c e l l , ferrichrome caused i n h i b i t i o n of free iron uptake. I t has been suggested ( 4 l )  that ferrichrome i s active i n the  i n s e r t i o n of i r o n into porphyrin.  Thus, ferrichrome may have  provided i r o n d i r e c t l y f o r heme synthesis i n  B - 1 4 7 1 ,  allowing normal  i r o n s u f f i c i e n t porphyrin biosynthesis, thereby eliminating the requirement f o r DHBG synthesis.  Results with  B - 1 4 7 1  (Section II)  indicated that i f i r o n were available f o r heme synthesis, neither coproporphyrin  nor DHBG would accumulate.  The extent of iron transport under the experimental  conditions  employed allows speculation concerning the l e v e l of e x t r a c e l l u l a r i r o n remaining p r i o r to the excretion of DHBG and coproporphyrin (Fig.  l l ) by t h i s s t r a i n .  Transport  studies were conducted under  conditions i d e n t i c a l to those used f o r studying excretion products, +3  except that 5 Mg Fe  per l i t r e were added to c e l l s approximately  one hour p r i o r to the onset of phenolic acid excretion (Fig. l l ) . Of t h i s l e v e l of added iron, 4 5 $ was unavailable to c e l l s i n the absence of phenolic acids (Fig. 2 6 ) ,  so that the e x t r a c e l l u l a r iron  concentration p r i o r to DHBG excretion could be estimated to be 5 ug per l i t r e .  Excretion of DHBG by c e l l s would then f a c i l i t a t e  s o l u b i l i z a t i o n of about 9 0 $ of t h i s i r o n (Fig. 2.6) preceding the onset of coproporphyrin production ( F i g . 1 1 ) .  Only about 0.35 Hg v  per l i t r e of the remaining i r o n would subsequently be available to the c e l l (Fig. 27), which accounts f o r the excretion of very high levels of coproporphyrin (Fig. l l ) . r HQ  mutants had an increased iron uptake capacity compared to  the parent s t r a i n .  When these mutants were grown i n the presence  of 0.1 ug HQ per ml, t h e i r i r o n 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 d i d accumulate i n the medium (Section I ) .  I t remains to be determined i f HQ forms a  complex with i r o n 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 i r o n cannot be released.  GENERAL DISCUSSION  Although the f a c t o r exerting the greatest e f f e c t on phenolic acid production by B. s u b t i l i s  was the l e v e l of i r o n i n the medium,  production was influenced also by other aromatic compounds synthesized from chorismic acid and by the requirements of the  cell  for i r o n . The e f f e c t s of the aromatic amino acids i n reducing  phenolic  acid production were to be expected because i t i s known that the aromatic biosynthetic pathway i n B. s u b t i l i s i s subjected to feedback i n h i b i t i o n by these amino acids ( 2 8 ,  33)-  Repression of the  enzymes of the pathway has been demonstrated recently ( 4 3 )  and i t  i s s i g n i f i c a n t that i t appears to be mediated most strongly by tyrosine, the amino a c i d exerting the greatest degree of i n h i b i t i o n of phenolic a c i d production.  The aromatic amino acids would reduce  the l e v e l of chorismic a c i d available for DHB synthesis.  The  marked i n h i b i t o r y e f f e c t of a n t h r a n i l i c a c i d appeared to be d i r e c t l y on DHB synthesis from chorismic acid. That the production of phenolic acids was r e l a t e d to the actual i r o n requirements of the c e l l was shown by the properties of s t r a i n 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  produce phenolic acids under iron-deficiency.  and  W-23  did not  Such cultures  would have had a low capacity f o r porphyrin biosynthesis.  The  production of DHB by s t r a i n WB-746 under these conditions may have r e f l e c t e d the decreased i r o n uptake capacity of t h i s s t r a i n .  It  108  was possible that, i n spite of these observations, the r e l a t i o n s h i p between phenolic acid production and a requirement f o r i r o n f o r hemin biosynthesis was more apparent than r e a l , with phenolic acid and coproporphyrin production occurring independently to an i r o n deficiency. production.  i n response  However, hemin i n h i b i t e d phenolic acid  I t has been shown that hemin i s probably unable to  s a t i s f y the non-heme i r o n requirements of certain b a c t e r i a (39)> suggesting that i t does not r e a d i l y release i t s i r o n inside the cells.  I t was  s i g n i f i c a n t , also, that phenolic acid production  started before coproporphyrin production.  . This aspect of the  problem i s to be extended by examining mutants blocked after f o r t h e i r capacities f o r phenolic acid production. also, to use s t r a i n B-1471 hem-1  ALA  I t i s intended  to determine the heme-iron, the  -non-heme i r o n and the t o t a l i r o n requirements of s t r a i n B-1471 under various conditions.  The r e l a t i v e importance of i r o n f o r  hemin biosynthesis can then be assessed and the r e l a t i o n s h i p of phenolic a c i d production to porphyrin biosynthesis worked out i n detail) DHB  and DHBG have strong a f f i n i t i e s f o r f e r r i c i r o n (2.6),  that they could chelate low l e v e l s of i r o n i n the medium.  so The  development of an increased capacity f o r i r o n uptake by c e l l s grown i n an i r o n - d e f i c i e n t medium, and the enhancement of t h i s capacity by phenolic acids, supported the idea that the acids were serving to scavenge the l a s t traces of i r o n from the medium.  The  production  of phenolic acids could have been i n i t i a t e d when the l e v e l of i r o n inside the c e l l f e l l below a c r i t i c a l value.  I t remains to be  determined what t h i s value might be and whether i t represents t o t a l ,  free or bound iron. Perhaps the most i n t e r e s t i n g aspect of t h i s problem i s the question of how  the l e v e l of i r o n i n the medium controls the  production of phenolic acids.  There are two major p o s s i b i l i t i e s :  the enzymes f o r D H B synthesis are either repressible or inducible. The nature of the corepressor or inducer i s not clear at t h i s point.  I t might be that i r o n i t s e l f i s a corepressor, with a  c r i t i c a l l e v e l being required to activate the repressor.  The  : ( D H B ( G ) ) 2 complex might be the corepressor;  i n t h i s case i t  would be necessary to postulate that sideramines  i n h i b i t phenolic  Fe  acid production by carrying i r o n into the c e l l to allow formation of the Fe  : ( D H B ( G ) ) 2 ^^P- - -' 1  625  be induced by free D H B ( G ) .  A l t e r n a t i v e l y , the system might  In the presence of s u f f i c i e n t iron,  a l l the D H B ( G ) could be i n the F e ^ : ( D H B ( G ) ) +  complex;  as the  l e v e l of i r o n f a l l s below the c r i t i c a l l e v e l , free DHB(G) could appear to induce the system.  I t i s u n l i k e l y that the  m-substi-  tuted benzoic acids i n h i b i t e d 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 D H B ( G ) to prevent induction. The increased capacity f o r i r o n uptake seen i n i r o n - d e f i c i e n t c e l l s could have r e s u l t e d from the appearance of a s p e c i f i c permease i n 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 i s under the same system(s) of control as the enzymes involved i n D H B synthesis.  The system  can be subjected to genetic analysis, using transformation  and  110  or transduction.  Attempts w i l l be made to i s o l a t e mutants i n which  the production of phenolic acids i s no longer controlled by iron. Ferrichrome and ferrioxamine served as e f f e c t i v e " i n h i b i t o r s " of phenolic acid production, and ferrichrome served very e f f e c t i v e l y to carry i r o n into the c e l l .  I f a s p e c i f i c permease was involved  i n carrying f e r r i c i r o n complexes into the c e l l , i t might be characterized by being r e l a t i v e l y non-specific with regard to the ligands binding the iron.  The s p e c i f i c i t y of the i r o n uptake mechanism seen i n i r o n -  d e f i c i e n t c e l l s could be examined using phenolic compounds c l o s e l y and d i s t a n t l y related to DHB and DHBG.  Ill  LITERATURE CITED  1.  Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Conditions optimal f o r mutagenesis by N-methyl-N'-nitro'N-N-nitrosoguanidine i n Escherichia c o l i K-12. Biochem. Biophys. Res. Commun. 18: 788-795.  2.  Afcrigg, A., S. R.. Ayad, and G. R. Baker. 1967. The nature of a competence-inducing factor i n B a c i l l u s s u b t i l i s . Biochem. Biophys. Res. Commun. 28: IO62-IO67.  3.  Albers, R. W. 1967. Biochemical aspects of active transport. Ann. Rev. Biochem. 36: 727-756.  4.  Aronson, J. N., and G. R. Wermus. 1965. Effects of m-tyrosine on growth and sporulation i n B a c i l l u s species. J. B a c t e r i o l .  9p_:  38-46.  5.  Baker, T. I., and I. P. Crawford. 1966. Anthranilate synthetase. P a r t i a l p u r i f i c a t i o n and some k i n e t i c studies on t h i s enzyme from Escherichia c o l i . J. B i o l . Chem. 241: 5577-5584.  6.  Brot, N., and J. Goodwin. 1968. Regulation of 2,3-dihydroxybenzoylserine synthetase by iron. J. B i o l . Chem. 243: 510-513.  7.  Brot, N., J. Goodwin, and H. Fales. 1966. In vivo and i n v i t r o formation of 2,3-dihydroxybenzoylserine by Escherichia c o l i K-12. Biochem. Biophys. Res. Commun. 25_: 454-461.  8.  Burnham, B. F. 1961. Doctoral dissertation. C a l i f o r n i a , Berkeley, Cal.  9.  Burnham, B. F., and J. Lascelles. 1963. Control of porphyrin biosynthesis through a negative-feedback mechanism. Biochem. J 8J_: 462-472.  University of  10. Byers, B. R., M. V. Powell, and C. E. Lankford. 1967. Iron-chelating hydroxamic acid (schizokinen) active i n i n i t i a t i o n of c e l l d i v i s i o n i n B a c i l l u s megaterium. J. B a c t e r i o l . 9 3 : 286-294. 11. Chasin, L. A., and J. Szulmajster. 1967. Biosynthesis of d i p i c o l i n i c acid i n B a c i l l u s s u b t i l i s . Proc. N.A.S. 29: 648-654. 12.  Cox, G. B., and F. Gibson. I 9 6 7 . 2,3-dihydroxybenzoic acid, a new growth factor f o r multiple aromatic auxotrophs. J. B a c t e r i o l . 9 3 : 502-503.  13.  Doy, C. H. 1966. Chemical synthesis of the tryptophan pathway intermediate l-(O-carboxyphenylamino)-l-deoxy-D-ribulose-5phosphate. Nature 211: 736-737.  112  14. Emery, T., and J. B. Neilands. i960. Contribution to the structure of the ferrichrome compounds; Characterization of the a c y l moieties of the hydroxamate functions. J. Am. Chem. Soc. 82:  3658-3662.  15. Falk, J . E. 1964. Porphyrins and metalloporphyrins. Publishing Company, New York.  Elsevier  16. Gallokata, K. G., and H. 0. Halvorson.  i960. Biochemical changes occurring during sporulation by B a c i l l u s cereus. Inhibitions of sporulation by a - p i c o l i n i c acid. J. B a c t e r i o l . 79j 1-8.  17. Garibaldi, J . A., and J. B. Neilands. 1955I s o l a t i o n and propert i e s of ferrichrome A. J . Am. Chem. Soc. 7J_: 2429-2430. 18. Garibaldi, J . A., and J . B. Neilands, 1956. Formation of i r o n binding compounds by microorganisms. Nature 177: 526-527. 19. Gibson, F.  1964.  Chorismic acid:  and p h y s i c a l studies. 20. Gibson, F.  1968.  p u r i f i c a t i o n and some chemical  Biochem. J . 9£  Chorismic acid.  :  256-261.  Biochem. Prepn. 12:  94-97.  21. Gibson, F. Personal communication. 22. Gorini, L., and R. Lord. 1956. Necessite des arthodiphenols pour l a croissance de Coccus (Sarcina sp.). Biochim. Biophys. Acta.  19_: 84-90.  23. Gray, C. H., and L. B. Holt. 1948. The i s o l a t i o n of coproporphyrin I I I from Corynebacterium diphtheriae culture f i l t r a t e s . Biochem.  J. 43: 191-193. 24. Hanson, R. S., and D. P. Cox. 1967. E f f e c t of d i f f e r e n t n u t r i t i o n a l conditions on the synthesis of t r i c a r b o x y l i c acid cycle enzymes. J. B a c t e r i o l . 93: 1777-I787. 25. Hanson, R. S., J . Blicharska, M. Arnaud, and J . SzuLmajster. 1964. Observations on the regulation of the synthesis of the tricarboxyl i c acid enzymes i n B a c i l l u s s u b t i l i s , Marburg: Biochem. Biophys. Res. Commun. 1J_: 690-695. 26. Ito, T. 1954. Doctoral dissertation. Berkeley, Cal.  University of C a l i f o r n i a ,  27. Ito, T., and J . B. Neilands. 1958. Products of "low-iron ferment a t i o n " with B a c i l l u s s u b t i l i s : i s o l a t i o n , characterization and synthesis of 2,3-dihydroxybenzoylglycine. J . Am. Chem. Soc. 80:  4645-4647.  28. Jensen, R. A., and E. W. Nester. 1965. The regulatory significance of intermediary metabolites:, control of aromatic biosynthesis by feedback i n h i b i t i o n i n B a c i l l u s s u b t i l i s . J . Mol. B i o l . 12:  468-481.  ~~  1 1 3  29.  Lascelles, J . 1 9 5 6 . An assay of i r o n protoporphyrin based on the reduction of n i t r a t e by a variant s t r a i n of Staphylococcus aureus: synthesis of i r o n protoporphyrin by suspensions of Khodopseudomonas spheroides. J . Gen. M i c r o b i o l . 1 5 : 4 o 4 - 4 l 6  .  30.  Lascelles, J . 1 9 5 7 . Synthesis of porphyrins by c e l l suspensions of Tetrahymena vorax: e f f e c t of members of the vitamin B group. Biochem. J . 6 6 : 5 j p 7 2 .  31.  Lascelles, J . 1 9 6 1 . Synthesis of tetrapyrroles by microorganisms. Physiol. Rev. 4 l : 1+17-441.  32.  Lascelles, J . 1 9 6 5 . The synthesis of porphyrins and b a c t e r i o chlorophyll by c e l l suspensions of Rhodopseudomonas spheroides. Biochem. J . 6 2 : 7 8 - 9 3 .  33-  Lorence, J . H., and E. W. Nester. 1 9 6 7 . M u l t i p l e molecular forms of chorismate mutase i n B a c i l l u s s u b t i l i s . Biochemistry 6 : 1 5 4 1 - 1 5 5 2 .  34.  Lowenburg, W., B. B. Buchanan, and J . C. Rabinowitz. 1963. Studies on the chemical nature of C l o s t r i d i a l ferredoxin. J . B i o l . Chem. 2 3 8 :  35«  Lowry, 0 . H., N. J . Rosebrough, A. L. Farr, and R. J . Randall. 1 9 5 L Protein measurement with the phenol reagent. J . B i o l . Chem. 1 9 3 :  36.  3 7 .  3 8 9 9 - 3 9 1 3 .  2 6 5 - 2 7 5 .  McCarthy, C., and E. W. Nester. c e l l s of B a c i l l u s s u b t i l i s .  1 9 6 7 . Synthesis i n newly transformed J . Bacteriol. 9 _ 4 : 1 3 1 - 1 4 0 .  Moyed, H. S. i 9 6 0 . False feedback i n h i b i t i o n : i n h i b i t i o n of tryptophan biosynthesis by 5-methyltryptophan. J . B i o l . Chem. 2 3 5 :  1 0 9 8 - 1 1 0 2 .  38.  Neilands, J . B. 1 9 5 2 . A c r y s t a l l i n e organo i r o n compound from the fungus Ustilago sphaerogena. J . Am. Chem. S o c . ' 7 4 : 4846-4847.  39«  Neilands, J . B. 1 9 5 7 . Some aspects of m i c r o b i a l i r o n metabolism. B a c t e r i o l . Rev. 2 1 : 1 0 1 - 1 1 1 .  40.  Neilands, J . B. 1 9 6 1 . E a r l y stages i n the metabolism of iron, p. 1 9 4 - 2 0 6 . In J . E. Falk, R. Lemberg, and R. K. Morton (ed.)., Haematin Enzymes. Pergamon Press, Inc., New York, N.Y.  41.  Neilands, J . B.  1967.  Hydroxamic acids i n nature.  Science  156:  1 4 4 3 - 1 4 4 7 .  4 2 .  43.  Neilands, J . B., and J . A. Garibaldi, i 9 6 0 tetramethyl ester. Biochem. Prepn. ]_: Nester, E. W.  Personal communication.  .  Coproporphyrin I I I 3 6 - 3 8 .  1 1 4  44. Nester, E. W., and R. A. Jensen. 1966. Control of aromatic biosynthesis i n B a c i l l u s s u b t i l i s : sequential feedback  inhibition.  J. B a c t e r i o l . 91:  1594-1598.  45. Nester, E. W., J . H. Lorence, and D. S. Nasser. 19°7. An enzyme aggregate involved i n the biosynthesis of aromatic amino acids i n B a c i l l u s s u b t i l i s . I t s possible function i n feedback regulation. Biochemistry 6: 1553-1562. 46. Nester, E. ¥., M. Schafer, and J . Lederburg. 1963. Genetic linkage i n DNA transfer: a cluster of genes concerned with aromatic biosynthesis i n B a c i l l u s s u b t i l i s . Genetics 48:  529-551.  47. P i t t a r d , A. J., F. Gibson, and C. H. Doy. 1961. Phenolic compounds accumulated by washed c e l l suspensions of a tryptophan auxotroph of Aerobacter aerogenes. Biochim. Biophys. Acta 49:  485-494.  48. P i t t a r d , A. J . , F. Gibson, and C. H. Doy. 1962. A possible r e l a t i o n s h i p between the formation of o-dihydric phenols and tryptophan biosynthesis by Aerobacter aerogenes. Biochim. Biophys. Acta 5J_: 29O-298. 49. Prelog, V. 1963. Iron-containing a n t i b i o t i c s and microbial growth factors. Pure and Appl. Chem. 6: 327-338. 50. Ratledge, C. 1964. Relationship between the products of aromatic biosynthesis i n Mycobacterium smegmatis and Aerobacter aerogenes.  Nature 203:  428-429.  51. Ratledge, C.  I967. The production of N-acylanthranilic acid from shikimic acid and the e f f e c t of iron-deficiency on the b i o synthesis of other aromatic compounds by Aerobacter aerogenes. Biochim. Biophys. Acta l 4 l : 55-63.  52. Ratledge, C , and F. G. Winder. 1962. The accumulation of s a l i c y l i c acid by Mycobacteria during growth i n an i r o n - d e f i c i e n t medium. Biochem. J . 84: 501-506. 53. Ratledge, C , and F. G. Winder. 1966. Biosynthesis and u t i l i s a t i o n of aromatic compounds by Micobacterium smegmatis with p a r t i c u l a r reference to the o r i g i n of s a l i c y l i c acid. Biochem. J . 101:  274-283.  54. Roberts, R. B., D. B. Cowie, P. H. Abelson, E. T. Bolton, and R. Y. B r i t t e n . 1957. Studies of biosynthesis i n Escherichia c o l i . Carnegie Inst. Wash. Publ. 607: 13-14. 55. Ronald, W. P.  Personal  communication.  56. Salton, M. R. J . 1964. Requirement of dihydroxyphenols f o r the growth of Micrococcus lysodeikticus i n synthetic medium. Biochim. Biophys. Acta 86: 421-422.  1 1 5  57.  Schaeffer, P., J. M i l l e t , and J . P. Aubert. 1 9 6 5 . Catabolite repression of b a c t e r i a l sporulation. Proc. W.A. S. 5 3 _ : 7 0 4  - 7 1 1 .  58.  Somerville, R. L., and R. ELford. 1967. Hydroxamate formation by anthranilate synthetase of Escherichia c o l i . Biochem. Biophys. Res. Commun. 2 8 : 437-W+.  59-  Srinivasan, P. R., and A. R i v i e r a J r . 1 9 6 3 . The enzymatic synthesis of anthranilate from shikimate-5-phosphate and 1-glutamine. Biochemistry 2 : 1 0 5 9 - 1 0 6 2 .  60.  Srinivasan, P. R., and B. Weiss. 1 9 6 1 . The biosynthesis of p-aminobenzoic acid: studies on the o r i g i n of the amino group. Biochim. Biophys. Acta 5 1 : 5 9 7 - 5 9 9 .  61.  Subba Rao, P. V., K. Moore, and G. H. H. Towers. 1 9 6 7 . 0-pyrocatechuic acid carboxylase from Aspergillus niger. Arch. Biochem. Biophys. 1 2 2 : 466-473.  62.  Szulmajster, J., and R. S. Hanson. 1 9 6 5 . P h y s i o l o g i c a l control of sporulation i n B a c i l l u s s u b t i l i s , p. 1 6 2 - 1 7 3 . In W. W. Campbell and H. 0 . Halvorson (ed.77^ Spores I I I . American Society f o r Microbiology.  63.  Townsley, P. M., and J . B. Heilands. 1957. The i r o n and porphyrin metabolism of Micrococcus lysodeikticus. J . B i o l . Chem. 2 2 4 : 6 9 5 - 7 0 5 .  64. Umbarger, H. E., M. A. Umbarger, and P. M. L. Siu. 1 9 6 3 . Biosynthesis of serine i n Escherichia c o l i and Salmonella typhimurium. J. B a c t e r i o l . 8 5 : 1431-1439. 65.  Vogel, H. J . 1 9 5 6 . A convenient growth medium f o r Heurospora (medium H). M i c r o b i o l . Gen. B u l l . 1 3 : 4 2 - 4 3 .  66.  Waring, W. S., and C. H. Werkman. 1 9 4 2 . Growth of b a c t e r i a i n an iron-free medium. Arch. Biochem. 1 : 3 0 3 - 3 1 0 . Also, M a r t e l l , A. E. 1 9 6 4 . S t a b i l i t y constants of metal ion complexes - Section I I . Organic Ligands Chemical Society, London.  67.  Young, I. G., G. B. Cox, and F. Gibson. 1 9 6 7 . 2,3-Dihydroxybenzoate as a b a c t e r i a l growth f a c t o r and i t s route of biosynthesis. Biochim. Biophys. Acta l 4 l : 3 1 9 - 3 3 1 .  68.  Young, I. G., L. M. Jackman, and F. Gibson. I 9 6 8 . 2 , 3 - D i h y d r o 2,3-dihydroxybenzoic acid as an intermediate i n the biosynthesis of 2,3-dihydroxybenzoic acid. Biochim. Biophys. Acta 1 4 8 : 3 1 3 - 3 1 5 .  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0104612/manifest

Comment

Related Items