FERMENTATION OF AMINO ACIDS BY MICROCOCCUS AEROGENES'

H. R. WHITELEY

Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington

Received for publication March 11, 1957

The anaerobic micrococci described by Foubertand Douglas (1948a) may be divided into 3groups on the basis of ability to degrade organiccompounds: (1) those capable of fermenting onlyglycine (Douglas, 1951), (2) those able to degradepurines (Whiteley and Douglas, 1951) and manyorganic acids (Foubert and Douglas, 1948b), and(3) those able to ferment purines and a limitednumber of amino acids and organic acids. To thefirst group belong the species Micrococcus vari-abilis and Micrococcus anaerobius, to the second,Micrococcus lactilyticus (also called Veillonellagazogenes), and to the third, Micrococcus aero-genes, Micrococcus prevotii, Micrococcus activus,and Micrococcus asaccharolyticus. A survey ofnine strains belonging to the third group hasshown that serine, threonine, histidine, glutamicacid (and glutamine), pyruvate, a-ketobutyrateand urocanate are fermented. Although thestrains vary considerably, each compound isdegraded by at least one strain of each species,and all are fermented by strain 228 of M. aero-genes. The fermentation of purines by this strainhas been described (Whiteley, 1952). This com-munication presents the results of studies on thefermentation of amino acids and a discussion ofthe mechanisms of degradation by M. aerogenes.

METHODS

Cells were grown either in the peptone-yeastextract medium ("peptone medium") describedpreviously (Whiteley, 1952) or in the samemedium supplemented with 0.4 per cent sodiumglutamate ("glutamate medium"). The gluta-mate medium supports a more rapid rate ofgrowth, and yields cells capable of fermenting allsubstrates at a high rate. Since such cells have aconsiderably higher rate of endogenous metabo-ism, both media were used depending on experi-mental needs. Conditions of growth, harvesting,

1 This work was supported in part by an AtomicEnergy Commission Postdoctoral Fellowship atthe Hopkins Marine Station and in part by agrant from the Atomic Energy Commission, Con-tract No. AT(45-1)-173.

and preparation of cell suspensions have beendescribed (Whiteley, 1952). Dried cell prepara-tions were made by allowing a cell paste to standfor 12 to 16 hr under vacuum over severalchanges of P205 at room temperature. Cell freeextracts were obtained by grinding with aluminaaccording to the method of Mcllwain (1948).The alumina-cell paste mixture was eluted with0.01 M phosphate buffer at pH 6.8 and centrifugedfor 20 min at 15,000 X G. Cell free extracts werealso prepared by sonic disintegration in a Ray-theon 10 KC oscillator. In the latter procedure,6 to 10 g (wet wt) of cell paste were suspended inapprox 25 ml of 0.01 M phosphate buffer at pH6.8, subjected to sonic disintegration for 20 to 30min and centrifuged for 20 min at 15,000 X G.All preparations contained a reducing agent in afinal concentration of 0.001 per cent-0.05 percent. Aged preparations are those which havebeen stored frozen at -4 C for 5 to 25 days.Dialysis of extracts was performed in the coldagainst 0.005 M cysteine (pH 7.0). The proteincontent of the cell free extracts was estimated bythe method of Stadtman et al. (1951).Manometric experiments were performed in an

atmosphere of oxygen-free nitrogen. TheWarburgvessels contained 10 to 30 mg cell protein, 50,umoles phosphate buffer at pH 7.0, 10 to 50,umoles of substrate, and other additions as noted.The agar modification of the Thunberg technique(Umbreit et al., 1949) was used for measurementof dehydrogenase activity.

Qualitative identification of volatile acidsresulting from the fermentation of 100 to 500,umoles of substrate in 250 ml Warburg vesselswas made by partition chromatography usingeither silica gel columns (Elsden, 1946) or paper(Kennedy and Barker, 1951). Paper chromatog-raphy was also used for the identification ofcompounds containing imidazole (Ames andMitchell, 1952), lactate (Buch et al, 1952),formamide (Wachsman and Barker, 1955a),hydroxamic acids (Stadtman and Barker, 1950),amino acids (Levy and Chung, 1953) anda-keto acids as the 2 ,4-dinitrophenylhydrazones

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(Crane and Ball, 1951). Preparations wereboiled and treated with Dowex-50 before chroma-tography for amino acids.Methods used for the quantitative determina-

tion of volatile acids, lactate, and ammonia andfor the calculation of O/R indices have beendescribed previously (Whiteley, 1952). Formatewas determined according to Grant (1948),hydroxamic acids by the method of Lipmann andTuttle (1945), and urocanic acid was estimatedfrom its absorption at 277 m,4 (Mehler andTabor, 1953).

Adenosine triphosphate (ATP) and coenzymeA (CoA) were obtained from Pabst and Co.Mesaconic acid was obtained from AldrichChemical Company, Inc. I am indebted to Dr.H. Tabor for urocanic and formiminoglutamicacids, to Dr. F. M. Huennekens for pyridoxalphosphate, and to Dr. H. A. Barker for citra-malate.

RESULTS

Fermentable substrates. Serine, threonine, gluta-mate, and histidine are readily fermented by cellsuspensions and dried cell preparations of M.aerogenes. Cell free extracts rapidly degradeserine and threonine but do not ferment gluta-mate. Histidine is fermented by some extractsbut not by others.

Neither M. aerogenes nor the other speciesbelonging to the third metabolic group of an-

aerobic micrococci produce gas or ammonia fromthe following amino acids: glycine, alanine,cysteine, cystine, valine, norvaline, methionine,leucine, norleucine, isoleucine, citrulline, arginine,phenylalanine, tyrosine, proline, hydroxyproline,tryptophan, lysine, aspartic acid, a-aminobutyric acid, or y-amino butyric acid.The possibility of the occurrence of a "Stick-

land reaction" (the oxidation of one amino acidwith the concomitant reduction of another) was

investigated with all available strains of Micro-coccus variabilis, M. anaerobius, M. aerogenes, M.prevotii, M. activus, and M. asaccharolyticus. Twomixtures of amino acids (Stickland, 1934) weretested separately, together, and in the presenceof a fermentable substrate (serine). Mixture Icontained glycine, proline, and arginine; mixtureII contained alanine, cysteine, and aspartic acid.When these mixtures were incubated with cellsuspensions, neither gas nor ammonia was pro-duced and dehydrogenation could not be de-tected. When serine was added to either mixture,

TABLE 1

End products from the fermentation of serine,threonine, glutamate and histidine by

Micrococcus aerogenes

End Products* Serine Threo- Gluta- Histi-nine mate dine

CO2 ..................... 9.5 10.0 9.8 10.3H29.6. ..6 ...6 9.8 0.5 0.0Formate ................. 0.0 0.0 0.0 0.5Acetate .................. 9.6 0.0 10.1 19.1Propionate .............. 0.0 10.2 0.0 0.0Butyrate ................ 0.0 0.0 4.5 0.5Lactate.................. 0.0 0.0 0.0 3.5NH3................... 10.2 10.110.3 29.0% Carbon recovery...... 96 101 96 101O/R index...... 1.02 1.0 0.96 0.96

*,gMoles per 20 ,umoles DL-serine, 10 ,umolesL-threonine, 10 ,moles L-glutamate, 10 /AmolesL-histidine. Reaction mixtures contained 25 mgprotein, 50,moles phosphate buffer pH 7.0, andsubstrate in a total volume of 2.2 ml. A dried cellpreparation was used for serine and threoninefermentations and suspensions of cells grown in"peptone medium" were used for histidine andglutamate degradations.

the quantities of end products formed and therate of dye decolorization indicated that onlyserine was degraded.

Studies with other microorganisms have shownthat fermentable amino acids may be deaminated.Thus, pyruvate and a-ketobutyrate arise fromserine and threonine, respectively (reviewed bySnell, 1953), urocanate from histidine (reviewedby Tabor, 1954), and a-ketoglutarate fromglutamate. Pyruvate, a-ketobutyrate, and uro-canate are degraded by M. aerogenes, but a-keto-glutarate is neither formed from glutamate noris it fermented by any type of preparation.

Serine and threonine. The end products result-ing from the fermentation of serine and threonineby dried cell preparations are given in columns 1and 2 of table 1. Judging from the quantities ofammonia and other end products, only L-serinedehydrase is present in M. aerogenes. A test forresidual D-serine was not performed.During the fermentation of serine and thre-

onine traces of pyruvic and a-ketobutyric acid,respectively, have been detected. If the fermenta-tions are carried out in an alkaline medium, or inthe presence of inhibitors such as fluoride,arsenate, or arsenite (table 2), larger amounts ofthese keto acids are found. This suggests that the

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TABLE 2Effec,t of inhibitors on the fermentation of serine,threonine, glutamate and histidine by dried cell

preparations of Micrococcus aerogenes

Compound

Fluoride .........

Cyanide .........

Arsenite .........

Arsenate.........

FinalConcen-tration

-Af

0.040.0080.0080.008

Serine*

53t1298136

Threo- Gluta-nine* mate

551508545

1524104

7

* Essentially identical values were obtainedfrom serine and pyruvate, threonine and a-keto-butyrate, and histidine and urocanate.

t Total gas as per cent of control withoutinhibitor. Reaction mixture: 20,umoles substrate,25 mg protein, 50 Amoles phosphate buffer (pH 6.5for a-keto acids and pH 7.0 for others) in 2.2 ml.Inhibitors mixed with reaction mixture 30 minbefore addition of substrate.

TABLE 3Rates of fermentation of serine, pyruvate, threonineand a-ketobutyrate by dried cell preparations of

Micrococcus aerogenes

Substrate TotalGas/10 Min

AL1. Serine........................... 1792. Pyruvate........................ 2253. Serine and pyruvate.............. 2244. Threonine....................... 1305. a-Ketobutyrate.................. 1626. Threonine and a-ketobutyrate... 169

Same reaction mixture as for table 1 with 40/umoles of pyruvate and a-ketobutyrate and 80umoles of DL-serine and DL-threonine.

primary conversion of the amino acids involves adeamination. No attempt has been made todetermine whether one single deaminase acts onboth serine and threonine, or whether two sepa-rate deaminases are required.An initial deamination of these amino acids is

further supported by the finding that the fermen-tation of pyruvate and a-ketobutyrate yields,except for ammonia, the same end products, andin virtually identical quantities, as that of serineand threonine, respectively. In addition, mix-tures of serine and pyruvate, and of threonineand a-ketobutyrate, are decomposed at the samerates as the keto acids alone (table 3). The lower

rate of gas formation from the amino acids thanfrom the corresponding keto acids indicates thatit is the initial deamination that controls the rateof decomposition. The fermentation of the ketoacids is discussed in a subsequent paper (Whiteleyand Ordal, 1957).The deamination of serine and threonine

appears to be mediated by dehydrase(s), withpyridoxal phosphate as the coenzyme, in somebacteria (reviewed by Snell, 1953), whereasadenylic acid and glutathione (Wood and Gun-salus, 1949), and biotin and adenylic acid (Lich-stein and Umbreit, 1947) have been reported asstimulatory in deamination by other bacteria.Preparations of M. aerogenes aged according tothe method of Lichstein and Christman (1948)failed to show any stimulation in the deaminationof serine when biotin, adenylic acid, or biotinplus adenylic acid were added. Attempts to dis-sociate cofactors participating in the deaminationof serine by dialysis of extracts were unsuccessful.However, a few extracts which had been aged byprolonged storage in the cold, showed a 50 percent increase in serine deamination when pyri-doxal phosphate was added. Glutathione andadenylic acid had no effect.

Glutamate. Cell suspensions and dried cellpreparations degrade glutamate readily to formthe end products shown in column 3 of table 1but cell free extracts capable of degrading gluta-mate could not be prepared. Glutamine (notshown) is fermented by cell suspensions anddried cell preparations at the same rate as gluta-mate and yields the same end products exceptthat twice the amount of ammonia is formed.It will be noted that approx 1 ,umole of carbondioxide and acetate are formed per Mumole ofglutamate decomposed. This observation, coupledwith the finding that none of the acids of thetricarboxylic acid cycle are fermented, indicatesthat glutamate degradation does not occur viathe tricarboxylic acid,cycle. The amount ofhydrogen produced may vary but generally doesnot exceed 1.0 ,jmoles of glutamate, and lactateis not formed. No free organic acids other thanacetate and butyrate have been found either asend products or as intermediates in fermentationswith or without inhibitors. The CoA and phos-phate derivatives of acetate and butyrate werethe only activated compounds that could bedetected in reaction mixtures in the course offermentation.

Mesaconate, citramalate and pyruvate have

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been shown to be intermediates in the fermenta-tion of glutamate by Clostridium tetanomorphum(Wachsman and Barker, 1955b; Barker et al.,1956; Wachsman, 1956). Mesaconate and citra-malate are not fermented by any preparation ofM. aerogenes and can be quantitatively recoveredfrom reaction mixtures. As stated above, pyru-

vate is readily degraded by M. aerogenes butdifferences have been found in the end productsformed from pyruvate and glutamate. Suspen-sions of cells of M. aerogenes grown in peptonemedium form carbon dioxide, hydrogen, acetate,lactate, and traces of butyrate from pyruvate(Whiteley and Ordal, 1957), whereas carbondioxide, hydrogen, acetate and butyrate are

formed from glutamate (table 1). A comparisonof the rates of fermentation of a mixture ofpyruvate and glutamate (table 4) with the rateof degradation of each compound separately hasshown that the mixture is fermented at a rateexceeding the sum of the individual rates. Differ-ences have also been found in the effects ofmetabolic inhibitors on pyruvate and glutamatefermentations (table 2).The fermentation of glutamate is not depend-

ent on the type of preparation used and is notinfluenced by the addition of ATP or CoA, or byacetate, -y-aminobutyrate, acetoacetate, ,3-OHbutyrate, crotonate, formate, or formamide. Thelast 2 compounds could be considered as possibleend products in histidine fermentation and were

added to determine whether the glutamatefermentation could be altered to resemble that ofhistidine. The other compounds added may bepossible participants or intermediates in butyrateformation and were added to fermentations ofglutamate in both nitrogen and hydrogen atmos-pheres to determine whether the amount ofbutyrate could be increased. Similar experimentswere carried out in which these compounds were

added to fermentations of pyruvate. Neither therate of fermentation nor the quantities of endproducts formed were altered by the addition ofthese compounds although the preparations are

capable of dehydrogenating 1-OH butyrate at a

low rate and are able to activate acetoacetateand ,1-OH butyrate.

Histidine. Investigations of the pathway ofhistidine degradation in tissues and bacteriahave shown that the first step is deamination tourocanate (Tabor, 1954). Since this compoundcan be degraded by. M. aerogenes at a rate com-

parable to that of histidine (table 5), it appeared

TABLE 4Rates of fermentation of glutamate and pyruvate by

dried cell preparation of Micrococcus aerogenes

Substrate ~~~TotalSubstrate Gas/20 Min

AL1. Pyruvate..................... 472. Glutamate ................... 503. Pyruvate and glutamate 130

Reaction mixture as for table 1 with 40,Amolesof each substrate, and 50 mg protein.

TABLE 5Rates of fermentation of histidine, urocanate, and

glutamate by dried cell preparations ofMicrococcus aerogenes

Substrate Gas/10 1NHM!AiL jsmoles

1. Histidine...................... 101 6.22. Urocanate .................... 933. Histidine and urocanate....... 1064. Glutamate.................... 105 4.45. Histidine and glutamate ...... 169 9.1

Reaction mixture as for table 1 with 40,molesof each substrate. Ammonia not determined in 2)and 3) above.

likely that urocanate was an intermediate inhistidine fermentation by M. aerogenes. The fol-lowing experimental results have been obtainedwhich support this possibility: (1) urocanate isformed, as determined by spectrophotometry andpaper chromatography, during the fermentationof histidine, and larger amounts of urocanate canbe made to accumulate by the addition of fluoride,arsenate, and arsenite, or by carrying out thefermentation in an alkaline medium; (2) exceptfor ammonia, the same end products are formedin essentially identical quantities from the fer-mentation of histidine and urocanate; (3) a mix-ture of urocanate and histidine is fermented atthe same rate as either compound separately(table 5); and (4) the fermentation of both com-pounds is affected in the same way by the addi-tion of various inhibitors (table 2).

It has been shown (Tabor, 1954) that in tissuesand other microorganisms urocanate is furtherdegraded via formamido-L-glutamic acid andformyl-L-glutamic acid to glutamate, anunoniaand formate, or in C. tetanomorphum (Wachsman

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and Barker, 1955a) and Aerobacter aerogenes(Magasanik, 1953) to glutamate and formamide.Neither glutamate nor formyl derivatives ofglutamate could be detected during the fermen-tation of histidine by M. aerogenes. However, ifM. aerogenes degrades histidine by either of the 2pathways described above, glutamate would notaccumulate since it is a fermentable substrate. Itwould be anticipated then that the quantities ofend products formed from histidine would beidentical with those formed from glutamateexcept for the additional ammonia and formate(or formamide) and that compounds which in-hibit glutamate fermentation would also inhibithistidine fermentation. However, considerabledifferences are found in the effect of variousinhibitors (table 2) and in the end productsformed from glutamate and histidine (columns3 and 4 of table 1). Lactate is produced fromhistidine but not from glutamate, and theamounts of acetate and butyrate formed arequite dissimilar. Formamide was not found andonly traces of formate are produced from his-tidine. As stated above, the addition of formateor formamide does not influence the fermentationof glutamate, nor does it affect the degradation ofhistidine. Neither formamide nor formate isdegraded by M. aerogenes when tested alone orwith a fermentable substrate.

DISCUSSION

It appears likely that all preparations of M.aerogenes degrade serine and threonine via pyru-vate and a-ketobutyrate, respectively, as inter-mediates. This conclusion is supported by thefinding that these a-keto acids may be isolatedfrom fermentations of the amino acids. Furthersupport comes from a comparison of the degrada-tion of serine and threonine with their respectivea-keto acids with regard to the quantities of endproducts, rates of fermentation and effects ofinhibitors. The formation of pyruvate from serineby aged preparations of M. aerogenes requirespyridoxal phosphate but not glutathione oradenylic acid. Hence the enzyme mediating thisreaction in M. aerogenes resembles the L-serinedehydrase of Neurospora (Yanofsky and Reissig,1953) rather than the serine deaminase ofEscherichia coli (Wood and Gunsalus, 1949).The fermentation of glutamate by M. aerogenes

yields the same products as that by C. tetanomor-phum (Barker, 1937), so that it seemed possiblethat the same mechanism of degradation could be

involved. For C. tetanomorphum this has beenshown (Wachsman and Barker, 1955b; Barkeret al., 1956; Wachsman, 1956) to implicatemesaconate, citramalate, pyruvate, and a-keto-glutarate. M. aerogenes is not able to degrademesaconate, citramalate, or a-ketoglutarate andthese compounds could not be detected in reac-tion mixtures fermenting glutamate. It is possible,however, that activated derivatives of mesacon-ate and citramalate are involved but are formedtransiently and in such small amounts that theyhave not been detected in reaction mixtures.Further, pyruvate is the source of butyrate in thefermentation of glutamate by C. tetanomorphum(Wachsman and Barker, 1955b), whereas M.aerogenes produces only traces of butyrate frompyruvate, although substantial amounts of thisacid are formed from glutamate. The effects ofadding various inhibitors to fermentations ofpyruvate and glutamate and the increased rate offermentation of mixtures of these two compoundsalso suggests that pyruvate may not be a normalintermediate in the decomposition of glutamate.

Butyrate formation in M. aerogenes couldoccur by the pathway found in other bacteria(reviewed by Barker, 1951) since extracts areable to activate acetate, acetoacetate, ,B-OHbutyrate and also are able to dehydrogenate,B-OH butyrate. However, the quantity of buty-rate formed either from pyruvate or from gluta-mate was not increased by the addition of acetate,acetoacetate, ,B-OH butyrate, or crotonate,together with ATP and CoA, under any condi-tions of testing. The amount of butyrate formedfrom glutamate was not altered by the additionof pyruvate.

Experimental results of studies on the degrada-tion of histidine by M. aerogenes indicate that thefirst step is a deamination to urocanate. It doesnot seem possible that urocanate could be thendegraded via urea, acrylate, or imidazole, sincenone of these compounds is decomposed by M.aerogenes, nor is imidazole carboxylic acid. Itappears unlikely, also, that urocanate is degradedvia glutamate or that histidine fermentationcould be considered the equivalent of glutamatefermentation with formate and ammonia asadditional end products. This statement is sup-ported by the following experimental findings:(1) different amounts of acetate, butyrate andlactate are formed from histidine and glutamate,(2) the fermentations of histidine and glutamateare affected differently by inhibitors, (3) rate

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determinations show independent degradationsof the two substrates, and (4) extracts of M.aerogenes have been prepared which are able toferment histidine but not glutamate.

It is possible, however, that a derivative ofglutamate is formed from histidine and is de-graded differently than glutamate suppliedexogenously. Activated amino acids have beenreported (Hoagland et al., 1956; Cantoni, 1952)and it is conceivable that activated glutamate,existing as such or bound to an enzyme, could beproduced from histidine. It is also possible thatthe presence of formate (or the equivalent) andammonia produced from histidine, alters thefermentation of glutamate formed as an inter-mediate. A condensation of formate (or itsequivalent), with a C2 compound produced fromglutamate could result in the formation of pyru-vate, thereby providing a hydrogen acceptor anddiverting "available hydrogen" from butyrateformation. Preliminary experiments with labeledformate indicate that the above condensationreaction can occur in M. aerogenes and similarresults have been obtained with other anaerobicmicrococci (Novelli, 1955). Failure to change thefermentation of glutamate with respect to endproducts by the addition of formate and suitablecofactors may be attributed to the fact that thedegradation of histidine does not result in therelease of free formate from the intermediatecompounds (Tabor and Rabinowitz, 1956).

ACKNOWLEDGMENTS

It is a pleasure to thank Drs. H. C. Douglas,C. B. van Niel, and E. J. Ordal for suggestionsand many helpful discussions in the course ofthis work. I wish to thank Mr. John Murphy fortechnical assistance in part of this work.

SUMMARY

Quantitative data for the fermentation ofserine, threonine, glutamate and histidine bystrain 228 of Micrococcus aerogenes are presented.A "Stickland reaction" does not occur in thisstrain or in any of the related anaerobic micro-cocCi.

Serine is degraded to ammonia, carbon dioxide,hydrogen, and acetate with pyruvate as an inter-mediate product. The deamination of serine isstimulated by pyridoxal phosphate but not bybiotin, glutathione, adenylic acid or combinationsof these cofactors.

Threonine is fermented to ammonia, carbon

dioxide, hydrogen, and propionate, with a-keto-blityrate as an intermediate.

Glutamate is degraded to ammonia, carbondioxide, hydrogen, acetate and butyrate. MIesa-conate, citramalate and a-ketoglutarate are notformed from glutamate or degraded by anypreparation of M. aerogenes.

Histidine is fermented to ammonia, carbondioxide, acetate, butyrate, lactate, and traces offormate, with urocanate as an intermediateproduct. A comparison of end products, as wellas other experimental evidence, indicates that ifglutamate is formed as an intermediate productin histidine fermentation, its subsequent degrada-tion is different from that of exogenously suppliedglutamate.

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