Regulation of metabolism in facultative bacteria

22

BIOCHIMICA ET BIOPHYSICAACTA

BBA 25495 REGULATION OF METABOLISM IN FACULTATIVE BACTERIA I. STRUCTURAL AND FUNCTIONAL CHANGES IN E S C H E R I C H I A

COLI

ASSOCIATED W I T H SHIFTS B E T W E E N T H E AEROBIC AND ANAEROBIC STATES C. T. GRAY,

J. W. T. WIMPENNY*,

D. E. HUGHES*

AND

MARY

R, MOSSMAN

Department of Microbiology, Dartmouth Medical School, Hanover, N.H. (U.S.A.) (Received March 2nd, 1965) (Revised manuscript received October 25th, 1965)

SUMMARY The nature and regulation of facultative metabolism in Escherichia coli has been investigated in terms of structural and functional changes mediated by 0 9. Cells were cultivated under controlled conditions of aerobiosis and anaerobiosis. The membrane containing and soluble fractions of these cells are compared with those from obligate aerobes and anaerobes, in terms of quantity, the localization, and the function of enzymes involved in formate metabolism, the Krebs cycle and the terminal respira t o r y apparatus. E. coli grown aerobically corresponds closely to a strict aerobe in that it has an intact Krebs cycle and a similar array of terminal respiratory enzymes. The level of Krebs cycle enzymes is greatly reduced in anaerobically grown E. coli. The membrane-bound cytochromes are maintained at a high level, and at the same time a series of enzymes which function in anaerobic electron transport are derepressed. In contrast to strict anaerobes, anaerobically grown E. coli contains many membranebound enzymes. The membrane-bound cytochromes are maintained, and even certain of the derepressed enzymes, such as hydrogenase and fumaric reductase, are membrane bound; whereas both the latter are soluble in anaerobes. In certain instances E. coli can show complete structural and functional correspondence with both strict aerobes and anaerobes. For example, it possesses a membrane-bound formate dehydrogenase aerobically and in addition a soluble formate dehydrogenase anaerobically. It is concluded that when E. coli is grown anaerobically it differs from the true anaerobe in that the membrane remains a maior site of enzyme activity. INTRODUCTION Escherichia coli and other facultative anaerobes are distinguished from strictly aerobic or anaerobic bacteria by their ability to grow in the presence or absence of O z. Oz mediated changes in enzyme levels of facultative bacteria have been observed 1-~ * Present address: MicrobiologyDepartment, University College, Cardiff (Great Britain). Biochim. Biophys. Acta, Ix 7 (1966) 22-32

E N Z Y M E LOCALIZATION I N F A C U L T A T I V E B A C T E R I A

23

and there have been many studies on enzyme localization in various organisms 1°-14. However, there has been no attempt to relate these facultative changes in enzyme content to possible changes in enzyme localization and function. Certain enzymic and structural changes are known to take place during aerobic-anaerobic transitions in yeast, but these occur in a cell containing mitochondria and a discrete nucleus and may not necessarily hold true for bacteria. Very little is known about the membrane system in E. coli and even less about changes in its character which might be imposed by shifts between the aerobic and anaerobic states. E. coli and other Enterobacteriaceae normally do not possess the extensive membrane systems of Azotobacter 15 or the compact membranous organeUes found in the genus Bacillus and elsewhere, which may be mitochondrial equivalents 16. The protoplast membrane and/or small, irregular, rod-like structures adjacent to this membrane are at present thought to bear such activities in the gram-negative enteric bacteria17, TM. It is known that strictly aerobic microorganisms n-14 have many more membrane-bound systems than are found in obligate anaerobic bacteria 10. This indicates a tendency to localize in membranes enzymic activities that link with molecular oxygen. On the other hand, anaerobic electron transport and the associated energy yielding reactions, such as glycolysis are mediated by soluble enzymes. Complete correspondence of structure and function with respect to oxygen utilisation would mean that aerobically grown facultative cells would resemble strict aerobes whilst anaerobically grown cells would resemble strict anaerobes. This would demand large alterations and expenditure of energy when adjusting from one type of growth to the other. This question is studied by examining changes in enzyme localization and function in E. coli grown aerobically or anaerobically under different nutrient conditions and by relating these changes to the normal pattern in strictly aerobic or anaerobic bacteria. Three categories of enzymes have been investigated: those associated with aerobic terminal oxidation, those involved in formate metabolism, and enzymes of the Krebs cycle. Enzymes have been separated into those contained in the solid or cell-wall membrane fraction and soluble fractions. The cell-wall membrane fraction has previously been shown to consist of the cell-wall to which the major portion of the cytoplasmic membrane is attached to its inner surface. It is washed to remove cytoplasmic contents which constitute the bulk of the soluble fraction. This latter also contains some activity associated with membrane which can be removed by further prolonged centrifugation and is therefore assumed to be due to comminuted membrane. It is also possible that enzymes normally associated with the membrane but which are easily removed by dilution will also appear in the soluble fraction. The method has usually given clear cut results but where this is not so, for instance with hydrogenase, the question is discussed further. M A T E R I A L S A N D METHODS

Organism and media E. coli strain KI, was maintained on nutrient agar slants. Inocula for large fer-

menters were grown in I00 ml quantities of Trypticase soy broth shaken for I0 h at 37 °. Biochim. Biophys. Acta, 1I 7 (I966) 22-32

24

c.T. GRAYet al.

The synthetic medium, before adding a carbon source, contained per liter, I o o m l 0.5 M NH4C1, 4 m l I M KH~P04, 36ml I M K z H P O 4, and 5 ml mineral salts. The stock solution of mineral salts contained per liter, IO g MgSO 4. 7H~O, I g MnCI~. 4H~O, 0.4 g FeSO4"7HzO, and o.I g CaCI~. I t was stabilized b y adjusting the p H to 2.0. The complex medium contained per liter, 14 g Casamino acids (Difco), I ml M KH~PO4, 4 ml M K z H P O 4, and 5 ml mineral salts. Sterile glucose was added to either medium when desired, to give a concentration of 0. 4 % (w/v).

Growth conditions The organisms were grown in a 15-1 fermenter (New Brunswick model CF 14) containing IO 1 of medium and 1% inoculum. The temperature was controlled at 37* b y partial immersion of the fermenters in a water bath. The culture was agitated mechanically and gassed with air or a mixture of CO~-N 2 (4: 96) • The rate of flow of air through aerobic cultures was often increased as the cell density increased in order to avoid oxygen deficiency. An oxygen electrode (Beckman 39 065) with an amplifier (Beckman model 777) was used to monitor oxygen in the fermenter. The rate of flow of the CO~-Nz gas in anaerobic cultures was not critical. The p H was maintained at 7.00 :~ 0.o2 by the use of a p H stat (New Brunswick model pH 162). The progress of growth was followed by measuring the turbidity of culture sampled at 42o mt~ in a colorimeter (Lumitron model 4olA). The growth constant, k, was defined as the number of generations/h. Cells were harvested during the exponential phase of growth at an absorbancy of o.5 with a Szent-Gy6rgyi and Blum continuous flow system in a Servall RC2 refrigerated centrifuge. The packed cells were resuspended in o.oi M phosphate buffer (pH 7.4) and recentrifuged. Preparation of cell-wall membrane and soluble fractions The cell-wall membrane and soluble fractions were prepared b y modifications of the method of HUNT, RODGERS AND HUGHESTM. Washed cells were suspended in o.oi M Tris buffer (pH 7.5) (6 volumes cells/volume Tris) and crushed at --26 ° in a HUGHES' press19, z° activated by a Carver Laboratory Press. The crushed preparation was transferred to a glass homogenizer (Konte) containing 30 ml of chilled o.oi M Tris buffer (pH 7.5), and 0.04 mg deoxyribonuclease (EC 3.1.4.5) and stirred every 5 min for 20 min. The deoxyribonuclease treated homogenate was centrifuged at 23500 × g for 20 rain. The superuatant fluid was then centrifuged at 144o00 × g (Spinco centrifuge model L-5o) for 60 rain. The resulting supernatant fluid was named the soluble fraction. The residue from the initial centrifugation had two layers. The upper layer consisted principally of cell walls, with the cytoplasmic membrane adherent on the inside of the walls, and some whole cells. The lower layer contained unbroken whole ceils with some cell-wall membrane fraction. The upper layer was removed with a curved spatula, suspended in 35 ml o.oi M Tris buffer (pH 7.5) and washed in a glass homogenizer. This process, centrifugation and removal and washing of the upper layer, was repeated two more times. After the final wash the cell-wall membrane fraction was resuspended in 15 ml of the buffer and centrifuged at 80o × g for 15 min and the residue, if any, was discarded. The validity of the process was established b y examination of osmium tetroxide fixed preparations in the electron microscope. Electron micrographs of the osmium tetroxide fixed E. coli cell-wall Biochim. Biophys. Acta, xz7 (x966) 22-32

ENZYME LOCALIZATION IN FACULTATIVE BACTERIA

25

membrane fraction had the same general appearance of those published of the Pseudomonas fluorescens cell-wall membrane fraction TM.

Enzyme assays Condensing enzyme, aconitase, isocitrate dehydrogenase, fumarase, malate dehydrogenase, and NADH oxidase were determined spectrophotometrically with a Cary 14 recording spectrophotometer using the O.l-O.2 slide wire. Specific activities are expressed as/,moles of substrate transformed/mg protein/h at 25 °. Succinate dehydrogenase, formate oxidase, hydrogenase, formate dehydrogenase, and formate hydrogenlyase were determined manometrically in the Warburg apparatus and specific activities are expressed in terms of ~1 of gas exchanged/rag protein/h at 30 °. All figures reported are an average figure from at least two batches of cells. Condensing enzyme (citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7) was measured by determining the disappearance of acetyl CoA at 233 m/* in the presence of oxaloacetate ~1. Aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) was measured by observing the increase in absorbance at 24 ° m/~ in the presence of citrate z~. Because of its lability and loss in activity upon dilution, aconitase was measured before other enzymes and the enzyme was added directly to the test system in small quantities. Dilution of the original enzyme before use in the test system was avoided. Isocitrate dehydrogenase (NADP +) ELs-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42 ] was measured-by following the reduction of NADP + at 34 ° m/, in the presence of DL-isocitrate~3. Fumarase (L-malate hydro-lyase, EC 4.2.1.2) was measured by observing the increase in absorbance at 240 m/* in the presence of malate ~4. Malate dehydrogenase (L-malate: NAD + oxidoreductase, EC 1.1.1.37 ) was determined by measuring the oxidation of NADH at 340 m/* in the presence of oxaloacetate zS. Because the malate dehydrogenase of E. coli is abnormally sensitive to inhibition by oxaloacetate, the concentration of the latter was reduced to o.I raM. NADH oxidase was measured by following the decrease in absorbancy at 340 m/*26. Succinate dehydrogenase (succinate: phenazine methosulphate oxidoreductase, EC 1.3.99.1) was determined manometrically using phenazine methosulfate as an electron carrier 27. Formate oxidation was measured manometrically. Flasks contained 20/,moles of substrate, in a total volume of 2 ml, which was 0.05 M with respect to phosphate buffer (pH 7.4). Hydrogenase (EC 1.98.1.1 ) was determined by measuring the rate of hydrogen uptake anaerobically in the presence of methylene blue 2s. Formate dehydrogenase was measured by observing the rate of CO 2 evolution from formate anaerobically with benzyl viologen or methylene blue as electron acceptors zg. Formate hydrogenlyase was determined by measuring the rate of hydrogen formation from formate anaerobically in the presence of a CO n adsorbant 3°. Cytochromes in the cell-wall membrane and soluble preparations were identified and measured using the Cary 14 spectrophotometer. Difference spectra, ferricyanide Biochirn. Biophys. Acta, xI 7 (I966) 22-32

26

C.T. GRAY etal.

oxidized vs. dithionate reduced, were obtained and peak heights were compared on a protein basis. Proteins were estimated with Folins reagent using the method described by LOWRY et al. 31.

Coenzymes and enzymes NAD +, NADP + and deoxyribonuclease were purchased from Sigma Chemical Company, N A D H and acetyl-CoA were purchased from Pabst Laboratories. RESULTS

Location and function of enzymes metabolizing formate Formate oxidase. This enzyme system which oxidizes formate to CO S and H~O comprises a formate dehydrogenase (see below) and the remainder of the terminal respiratory pathway with oxygen as an acceptor. It is formed in large amounts in complex medium containing glucose in aerobic cells and is considerably reduced anaerobically. I t is essentially a membrane-bound enzyme system (Table I). Formate hydrogenlyase. This enzyme is believed to be a composite one consisting of hydrogenase and a soluble formate dehydrogenase (FD II) and probably one or more intermediate electron carriers. Its reactions result in the overall formation of CO S and Hz from formate~gm. As shown in Table I, it is formed only in anaerobically grown cells on a complex medium at an acid pH. Formate dehydrogenase (cytochrome b 1 oxidoreductase: EC 1.2.2.I) referred to in Table I as F D I. I t is distinguished from the dehydrogenase FD I I b y its reactions to benzyl viologen and methylene blue. The two enzymes m a y also be distinguished in haemin-less mutants of E. coli which fail to produce FD I but produce FD I I in the absence of haemin. Other mutants are known which produce FD I I but not FD I (unpublished observations). In Table I it is apparent that there is considerable overlapping in dye specificity. The results, however, support the idea that FD I I is a soluble enzyme formed anaerobically as is the case in Desulfovibrio desulfuricans3*, ss and that FD I is membrane bound, and formed mainly aerobically (cf. Table III) but in significant quantities anaerobically. Some interesting aspects of enzyme regulation are also demonstrated b y the two formate dehydrogenases. Both enzymes are quantitatively much reduced when the organism is grown on a synthetic medium, and even when grown on a complex medium FD I I requires both anaerobiosis and an acid p H to induce its formation. Hydrogenase. This enzyme is found in anaerobic cells grown either on a synthetic medium or in greater amounts from cells grown in a complex medium (Table I). When formed in obligate aerobes such as Azotobacter and Hydrogenomonas TM it is probably bound to the membrane but it can be solubilised b y sonication 34. In Clostridia and other obligate anaerobes this enzyme is soluble 35. The question of the location of the hydrogenase in facultative organisms is complex. Reference to Table I indicates the high levels of hydrogenase present in the soluble fraction. In absolute units of activity it is estimated that there is about as much hydrogenase in the soluble fraction as there is in the membrane fraction. I t is possible to remove some of this soluble activity b y prolonged high speed centrifugation, and it seems likely that this activity resides on small particles. The situation is also made more complex b y the possibility Biochim. Biophys. Aaa, i17 (i966) 22-32

~D

.q

t~

OF GROWTH

CONDITIONS

ON ENZYMES

CONCERNED

Formate hydrogenlyase

Hydrogenase

Soluble + cell-wall m e m b r a n e

734 3020

Solu bl e Cell-wall m e m b r a n e

2. 7 89

S ol ubl e C ell -w al l m e m b r a n e

1.8 6.1

S ol ubl e •

Formate dehydrogenase: b e n z y l v i o l o g e n (FD I I ) C ell -w al l m e m b r a n e

o 7.o

S ol ubl e Cell-wall m e m b r a n e

0.34

4.2

21oo 6900

225

16

25

4. 2

o I 1o

o.71

92. 5

2265 7680

146

--

--

64

2.8 90

0.43

o

5. I 59

794

43

28

lO. 5

3.8 855

1.15

Complex glucose

Synthetic glucose

Complex glucose (pH 6.2)

coli

Aerobic

Cell fraction

Formate dehydrogenase : m e t h y l e n e blue ( F D I)

IN E.

Complex glucose (pH 7.o)

METABOLISM

Anaerobic

FORMATE

Formate oxidase

WITH

Enzyme

Growth k

A c t i v i t i e s , e x p r e s s e d i n / , 1 gas e x c h a n g e d / m g p r o t e i n p e r h.

THE EFFECT

TABLE I

o

4.0 o

97 °

lO9

25

7.8

2.7 896

0.62

Complex

o

4.8 8I

33

1.2

5. i

1.o

o I I. 7

o.87

Synthetic glucose

tO "O

~0

~O

c~

©

t~

N

c . T . GRAY et al.

28

of a c t i v a t i o n which m a y occur on c o m m i n u t i o n of p a r t i c u l a t e m a t e r i a l s a n d to synergistic effects between the p a r t i c u l a t e a n d soluble fractions3L Differences in the Knallgass reaction between E. cull a n d other Enterobacteriaceae35, ss suggest also t h a t t h e function of h y d r o g e n a s e in this organism m a y be more complex t h a n in obligate anaerobes a n d o t h e r f a c u l t a t i v e organisms.

Location and function of Krebs cycle enzymes The effect of oxygen on the K r e b s cycle enzymes is c o m p l i c a t e d b y n u t r i t i o n a l a n d g r o w t h conditions. F o r the sake of simplicity the results for six enzymes are given in T a b l e I I for cells grown with a n d w i t h o u t oxygen, a n d w i t h a s y n t h e t i c m e d i u m with glucose. F u r t h e r results are to be given in g r e a t e r detail in a subsequent paper s . Succinate dehydrogenase and fumarase. The a c t i v i t y of b o t h enzymes is r e d u c e d b y anaerobiosis b u t their location is u n c h a n g e d (Table II). Succinate d e h y d r o g e n a s e r e m a i n e d as a m e m b r a n e b o u n d enzyme whereas fumarase was soluble. These results agree w i t h previous o b s e r v a t i o n s (cf. refs. 40-42). The a d d i t i o n a l soluble enzyme f u m a r a t e r e d u c t a s e formed in anaerobes is t h o u g h t to be m e m b r a n e b o u n d in E. coli 43 grown anaerobically. This p o i n t has n o t been e x a m i n e d in this work. Malate dehydrogenase. The m a l a t e d e h y d r o g e n a s e formed in E. coli u n d e r all conditions e x a m i n e d was soluble (Table II). T h u s this organism does not form the m e m b r a n e b o u n d m a l a t e dehydrogenases formed b y m a n y obligate aerobes11,n, 44. Two m a l a t e d e h y d r o g e n a s e s are k n o w n to occur in E. coli: A, which is found highest TABLE II THE KREBS

EFFECT CYCLE

OF

GROWTH

ENZYMES

CONDITIONS IN

ON

SOLUBLE

AND

MEMBRANE

BOUND

E. coli

Cells were grown on the salt-glucose m e d i u m with or without oxygen. After growth the washed cells were crushed, separated into cell-wallm e m b r a n e and soluble fractions and enzymic activities estimated as described in the text. Activities are in/~moles substrate converted/rag protein per h. Suecinate dehydrogenase activity is expressed as/~i Oz/mg protein per h.

Anaerobic

Growth k

0.34

Enzyme

Cell fraction

Succinate dehydrogenase

Soluble Cell-wall membrane Soluble Cell-wall membrane Soluble Cell-wall membrane Soluble Cell-wall membrane Soluble Cell-wall membrane Soluble Cell-wall membrane

Fumarase Malate dehydrogenase (NAD+) Condensing enzyme Aconitase Isocitrate dehydrogenase (NADP +)

Biochi,,n. Biophys. Acta, 117 (1966) 22-32

3.6 52 20.6 o 11 o 0.63 o 0.97 o 8.32 o

Aerobic

Ratio aerobic~anaerobic

0.87 7.5 231 50.8

z.i 4.4 2.5

O

225

20

O

3.08

4-9

0

19.1

2o

O

85. 3 O

io

29

ENZYME LOCALIZATION IN FACULTATIVEBACTERIA

anaerobically, a n d B, which is found aerobically4s. Malate dehydrogenase does n o t n o r m a l l y occur a m o n g strict anaerobes b u t m a y be replaced b y a malate-lactate t r a n s h y d r o g e n a s e 45, or a similar enzyme. Condensing enzyme, aconitase and isocitrate dehydrogenase. The a c t i v i t y of these enzymes is reduced in anaerobically grown cells b u t t h e y remain as soluble e n z y m e s (Table II) as is the case in most obligate aerobes 12.

Location and function of the terminal oxidase system N A D H oxidase The a c t i v i t y of the N A D H oxidase was considerably reduced b y anaerobiosis b u t most of the enzyme was m e m b r a n e b o u n d in b o t h aerobic a n d anaerobic cells. I n this respect, therefore, the anaerobic cells resemble obligate aerobes n-13 a n d other facultative bacteria 7. I n contrast to these the N A D H oxidising a c t i v i t y of obligate anaerobes is soluble 1° a n d is linked with flavo proteins when oxygen is used as a final electron acceptor.

(Table III),

TABLE III THE

EFFECT

OF GROWTH

CONDITIONS

ON THE

TERMINAL

RESPIRATORY

APPARATUS

IN

E. Coli

Activities are expressed in /*moles NADH oxidised/mg protein per h. Figures for cytochromes are derived from dithionite-reduced versus ferricyanide-oxidised difference spectra, and are expressed as absorbance/g protein at the following wavelengths: cytochrome bI, 559 m/~; cytochrome a2, 63o m#; cytochrome c, 552 m/~. Cytochrome a I is present but not in sufficient quantities to measure reliably. Cytochrome o was identified by its carbon monoxide spectrum which was derived from each cell-wall membrane preparation.

Cell fraction

NADH oxidase Cytochrome bI Cytochrome a 1 Cytochrome a 2 Cytochrome c Cytochrome o

Soluble Cell-wall membrane Cell-wall membrane Cell-wall membrane Cell-wall membrane Soluble Cell-wall membrane

Anaerobic

Aerobic

Synthetic Complex glucose glucose

Synthetic Complex glucose glucose

0.49 5-33 7.9 Present 2.3 1.4 Present

0.75 38.2 18.1 Present 1.7 o Present

0.43 6.28 5.6 Present 1.7 o.3 Present

0.95 48.4 12.6 Present 3.4 o Present

Average raNo aerobic/anaerobic

I-8 5

7.5° 2.28 1-3 --

Heine containing oxidases As previously reported b y other workers E. coli was found to c o n t a i n the same c o m b i n a t i o n of al, a s a n d 0 oxidases as other Enterobacteriacee (Table 111)14. Other p a t t e r n s of a, a 1, as, a3 a n d o occur in obligate aerobes where t h e y are always m e m b r a n e bound. Typical anaerobes with the exception of Bacteroides ruminocola 4~ c o n t a i n none of these oxidases. The levels of a 1 a n d a 2 oxidases in E. coli r e m a i n e d essentially u n c h a n g e d d u r i n g anaerobic growth (Table III) (cf. refs. 7, 48) a n d r e m a i n e d m e m b r a n e b o u n d u n d e r all conditions tested.

Type b cytochromes E. coli like other facultative anaerobes contains b 1 as the principal b type cytochrome although typical obligate aerobes m a y c o n t a i n b a n d b114. The b 1 cytochrome r e m a i n e d m e m b r a n e b o u n d in anaerobically grown cells a n d was decreased signifi-

Biochira. Biophys. Acta, 117 (1966) 22-32

l

O~ 0~

5

4

A b s e n t 14 (o in one species47) Absent (b1 in one species47) Membrane b 1 (50 % aerobic)

Soluble eaSX,5z Soluble TM

Soluble cs° Membrane (very low)

A b s e n O° Membrane 7

M e m b r a n e al-14 A b s e n t 14 M e m b r a n e xx-la

Cytoehrome e and c x (regular)

Cytochrome c or c a (low redox)

N A D H oxidase

* See Table I.

A b s e n t 14 AbsenO °

A b s e n t 14

M e m b r a n e la b a n d b1

Cytochrome b or b1

a , a l , a 2, a a, o

Membrane al, a2, a n d o

Cytochrome oxidase

M e m b r a n e 14 al, a 2 , o Membrane 7 bl

Condensing enzyme, aconitase and isocitrate dehydrogenase M e m b r a n e 14

Malate dehydrogenase isoenzymes A and B

Not reported ( ? absent)

N o t r e p o r t e d (? A) Soluble A ae

Soluble A + B 4°

N o t r e p o r t e d (? B)

Malate dehydrogenase

Soluble

Absent ? (see t e x t 45) Soluble (low)

Soluble

Soluble a n d m e m b r a n e n-la,44

Soluble or easily solubilized ll-la

Present ?

Soluble (low)

Soluble

Solubleli-la

Fumarase

Soluble (low)

Soluble 40

M e m b r a n e (low) 4a

Not reported

F u m a r a t e reductase (suecinate dehydrogenase)

9

A b s e n t a°

M e m b r a n e (high) 4a

Soluble 32

M e m b r a n e (low)

Soluble (high)

M e m b r a n e 7 (high)

Soluble (low)

M e m b r a n e ll-la

Not reported ( ? absent)

Suecinate dehydrogenase

II)

M e m b r a n e (low)

M e m b r a n e ~ (high)

Membrane

F o r m a t e dehydrogenase* (FD I)

F o r m a t e dehydrogenase* (FD

Soluble 3~ Not reported ( ? absent)

Membrane n-Is

Obligate anaerobes

A b s e n t as

Anaerobic

Aerobic

Facultative E. coli

Membrane n-la

Obligate aerobes

Hydrogenase

Enzyme

DISTRIBUTION OF ENZYMES IN AEROBIC, FACULTATIVE AND ANAEROBIC BACTERIA

TABLE IV

O

ENZYME LOCALIZATION IN FACULTAIIVE BACTERIA

31

c a n t l y in a m o u n t (Table In). These cytochromes, with few exceptions 47, are not found in anaerobes b u t do occur in some microaerophiles 49. Type c cytochromes Type c cytochromes have not been n o t e d in aerobically grown E. coli b u t do occur in other facultative organisms '4. Both c a n d c I occur b o u n d with v a r y i n g degrees of firmness to the m e m b r a n e of obligate aerobes b u t do not occur in anaerobes. As shown previously5° a p r e d o m i n a n t l y soluble low redox c t y p e cytochrome is formed in anaerobically b u t not aerobically grown E. coli (Table In). This is similar to the c 3 type formed b y the obligate anaerobe D. desulfuricans a n d which is t h o u g h t to function as an electron carrier in the formate hydrogenlyaseS~,51,5~. DISCUSSION The scattered literature on the s t r u c t u r a l a n d enzymic composition of aerobic, anaerobic a n d facultative bacteria is s u m m a r i s e d in Table IV, together with the results of the present experiments. I t is clear from this s u m m a r y t h a t although no simple generalisation can be made, nevertheless two m a i n points emerge as to the changes which occur in facultative organisms grown aerobically a n d anaerobically. Firstly, aerobically grown E. coli have m a n y of the s t r u c t u r a l a n d enzymic properties of obligate aerobes although the p a t t e r n of the cytochromes is not typical. Secondly, the anaerobically grown cells still retain m a n y of the characteristics of aerobes, especially the m e m b r a n e b o u n d systems, b u t form in addition soluble enzymes similar to those of typical anaerobes. This m a y afford to the cells a considerable saving in biosynthetic a c t i v i t y a n d enable rapid changes to take place u n d e r the influence of changing oxygen tension. REFERENCES

I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 I6 17 18 19 20 2I

E. F. GALE,Bacteriol. Rev., 7 (1943) 139. P. SCHAEFFER,Biochim. Biophys. Acta, 9 (1952) 261. P- SCHAEFFER,Biochim. Biophys. Acta, 9 (1952) 362. H. E. UMBARGER,J. Bacteriol., 68 (1954) 14o. E. ENGLESBERG,A. GIBORAND J. B. LEVY,J. Bacteriol., 68 (I954) 146. E. ENGLESBERG,J. B. LEvy AND A. GIBOR,J. Bacteriol., 68 (19541 178. R. E. AsNis, V. G. VELY AND M. C. GLICK,J. Bacteriol., 72 (1956) 314• F. M. COLLINSAND J. LASCELLES,J. Gen. Microbiol., 29 (I962) 531. P- MCPHEDEAN, t3. SOMMERAND E. C. C. LIN, J. Bacteriol., 81 (1961) 852. i . I. DOLIN, in I. C. GUNSALUSAND R. Y. STANIER, The Bacteria, Vol. 2, Academic New York, 1961, p. 425 . M. ALEXANDER,Bacteriol. Rev., 20 (1955) 67, A. G. 1V[ARR,Ann. Rev. Microbiol., 14 (196o) 241. A. G. MARR, in I. C. GUNSALUSAND R. Y. STANIER, The Bacteria, Vol. I, Academic New York, 196o, p. 443L. SMITH, in I. C. GUNSALUSAND R. Y. STANIER, The Bacteria, Vol. 2, Academic New York, 1961, p. 365. S. A. ROBRISHAND A. G. MARR,J. Bacteriol., 83 (1962) 158. R, G. E. MURRAY, Microbial Classification, Cambridge University Press, Cambridge, p. 119. W. VAN ITERSONAND W. LEENE, J. Cell Biol., 20 (1964) 361. W. VAN ITERSONAND W. LEENE, J. Cell Biol., 20 (1964) 377, A. L. HUNT, A. RODGERSAND D. E. HUGHES,Biochim. Biophys. Acta, 34 (1959) 354D. E. HUGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. P. A. SRERE AND G. W. KOSlCKI,J. Biol. Chem., 236 (1961) 2557.

Press,

Press, Press, 1962,

Biochim. Biophys. Acta, II 7 (1966) 22-32

32

C . T . GRAY e.~ al.

22 C. B. ANFINSEN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Acad e m i c Press, N e w York, 1955, p. 695. 23 S. OCHOA, in S. P. COLOWICK AND 1~. O. KAPLAN,Methods in Enzymology, Vol. i, A c a d e m i c Press, N e w York, 1955, P. 699. 24 E. RACKER, Biochim. Biophys. Acta, 4 (195o) 2o. 25 S. OCHOA, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, A c a d e m i c Press, N e w York, 1955, p. 735. 26 E. C. SLATER, Bioehem. J., 46 (I95 o) 484 . 27 P. BERNATH AND T. P. SINGER, in S. P. COLOWICK AND N. O. KAPLAN,Methods in Emymology, Vol. 5, A c a d e m i c Press, N e w York, 1962, p. 597. 28 A. SAN PIETRO, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 2, Acad e m i c Press, N e w York, 1955, P. 861. 29 H. GEST AND H. D. PECK, JR., J. Bacteriol., 7 ° (1955) 326. 3 ° M. BOVARNICK, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 1, Acad e m i c Press, N e w York, 1955, P. 539. 31 O. H. LowRY, lq'. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265. 32 J. P. WILLIAMS, J. T. DAVIDSON AND H. D. PECK, JR., Bacteriol. Proc., (1964) IiO. 33 C. T. GRAY, Biochem. J., 9o (1964) 23 P. 34 C. L WILLENBERGER AND R. REPASKE, Biochim. Biophys. Acta, 47 (1961) 542. 35 M. I. DOLIN, in I. C. GUNSALUS AND R. Y. STANIER, The Bacteria, Vol. 2, A c a d e m i c Press, N e w York, 1961, p. 319 . 36 D. E. ATKINSON AND B. A. MCFADDEN,J. Biol. Chem., 21o (1954) 885. 37 H. E. SwIM AND H. GEST, J. Baeteriol., 68 (1954) 755. 38 F. PICHINOTY, Biochim. Biophys. Aeta, 64 (1962) I I I . 39 C . T . GRAY, J. W. T. WIMPENNY AND M. R. MOSSMAN, Biochim. Biophys. Aeta, 117 (1966) 33. 4 ° H. D. PECK, JR., O. H. SMITH AND H. GEST, Bioehim. Biophys. Acta, 25 (1957) 142. 41 A. T. JOHNS, J. Gen. Mierobiol., 5 (1951) 326. 42 J. P. GROSSMAN AND J. R. POSTGATE, J. Gen. Microbiol., 12 (1955) 429 • 43 C. A. HIRSCH, M. RASMINSKY, B. D. DAVIS AND E. C. C. LIN, J. Biol. Chem., 238 (1963) 377 o. 44 M. J. o . FRANCIS, D. E. HUGHES, H. L. KORNBERG AND P. J. R. PHIZACKERLEY, Biochem. J., 89 (1963) 43 ° • 45 M. I. DOLIN, H. F. PHARES AND M. V. LONG, Federation Proc., 23 (1964) 427 . 46 C. R. AMARASINGI-IAM,Federation Proc., 23 (1964) 487. 47 D. C. WHITE, M . P. BRYANT AND D. R. CALDWELL, J. Bacteriol., 84 (1962) 822. 48 F. M o s s , Australian J. Exptl. Biol. Med. Sci., 3 ° (1952) 531. 49 N. ft. JACOBS AND M. J. WOLIN, Biochim. Biophys. Acta, 69 (1963) 18. 5 ° C. T. GRAY, J. W. T. WIMPENNY, D. E. HUGHES AND M. RANLETT, Biochim. Biophys. Acta, 67 (1963) 157. 51 J. POSTGATE, in J. E. FALK, R. LEMBERG AND R. K. MORTON, The Haematin Enzymes, Part 2, P e r g a m o n , O x f o r d 1961, p. 4o7 . 52 F. EGAMI, M. ISHIMOTO AND S. TANIGUCHI, in J. E. FALK, R. LEMBERG AND R. K. MORTON, The Haematin Enzymes, P a r t 2, P e r g a m o n , Oxford, 1961, p. 392.

Bioehim. Biophys. Aeta, 117 (1966) 22-32