CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO … · CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO...

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CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO SUCCINOGENES, SP. N. M. J. WOLIN, E. A. WOLIN, AND N. J. JACOBS Department of Dairy Science, University of Illinois, Urbana, Illinois Received for publication November 28, 1960 An enrichment culture of a methane-producing organism morphologically and physiologically similar to Mlethanobacterium formicicum (Schnel- len, 1947) has been maintained in our laboratory. The culture originated from an inoculum of bovine rumen fluid and was serially transferred in an anaerobic medium containing formate, sulfide, and salts. An attempt to obtain growth of the methanogenic organism when dicarboxylic acids were substituted for bicarbonate in the medium resulted in increased turbidity and al- kalinity when fumarate and malate were added but not when succinate was added. Analysis by gas chromatography, however, showed that little methane had been formed in the fumarate- or malate-containing cultures in contrast to the bicarbonate-containing cultures. Morpho- logical examination revealed that a previously undetected, highly motile vibrio had grown pro- fusely in the media containing fumarate or malate. The absence of growth of the vibrio in the succinate-containing medium suggested the possibility that fumarate and malate stimulated growth of the vibrio by acting as electron ac- ceptors for the oxidation of formate and per- mitted growth under anaerobic conditions. The isolation and study of pure cultures of the vibrio substantiated this hypothesis and also demon- strated that this obligate anaerobe could only grow in the presence of a limited number of electron donors (H2, HCOOH) and electron ac- ceptors (fumarate, malate, and nitrate). The presence of cytochromes in the organism adds to its complement of unusual characteristics. The present report is concerned with the isola- tion of the vibrio and a general description of its characteristics. MATERIALS AND METHODS Primary enrichment medium. The enrichment culture medium for methanogenic bacteria from which the organism was eventually isolated was similar to that described by Barker (1936) except for the addition of a few vitamins. It consisted of NH4Cl, 0.1%; KH2PO4, 0.04%; MgCl2 6H20, 0.02%; sodium formate, 0.5%; FeSO4, 0.001%; phenol red, 0.0003%; NaHCO3, 0.078%; Na2S-9H20, 0.02%; vitamin B12, 0.02 ,g/ml; and p-aminobenzoic acid, 0.30 ,ug/ml. The medium was prepared using tap water and placed in serum bottles. Bicarbonate and sulfide were sterilized by filtration and added aseptically. Prior to inoculation, the medium was aseptically flushed with a mixture of 95% N2 and 5% C02, until the pH was 7.2 to 7.4, and capped with a serum bottle cap. Inoculations were made with a hypodermic syringe. Secondary enrichment medium. Enrichment for the anaerobic vibrio from the primary en- richment medium was made in an identical medium with 0.1% fumarate substituted for the bicarbonate. Since autoclaving produced con- siderable alkalinity in the medium, presumably due to decomposition of tap water carbonates, the medium was neutralized with acid after auto- claving and autoclaved again. Sterile sulfide prepared in 0.01 M K2iPO4, pH 7.2, was then added. The gas phase was N2. Culture medium. A simple culture medium was developed after isolation of the organism which consists of (NH4)2S04, 0.1 %; K2HPO4, 0.5%; fumaric acid, 0.3%; sodium formate, 0.3%; yeast extract (Difco), 0.1%; MgC12 6H20, 0.02%; and FeSO4, 0.001%. The pH is 7.0 to 7.2. Sterile, autoclaved sodium thioglycolate (Difco) is added aseptically before inoculation to a final concentration of 0.05% and provides sufficient anaerobiosis for routine transfers. Unless stated otherwise, the organism was cul- tivated in this medium. All incubations were at 37 C. RESULTS Isolation of pure culture. The source of the organism was bovine rumen fluid which was used as the inoculum in the primary enrichment 911 on June 27, 2019 by guest http://jb.asm.org/ Downloaded from

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Page 1: CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO … · CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO SUCCINOGENES, SP. N. M. J. WOLIN, E. A. WOLIN, AND N. J. JACOBS Department ofDairy

CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIOSUCCINOGENES, SP. N.

M. J. WOLIN, E. A. WOLIN, AND N. J. JACOBS

Department of Dairy Science, University of Illinois, Urbana, Illinois

Received for publication November 28, 1960

An enrichment culture of a methane-producingorganism morphologically and physiologicallysimilar to Mlethanobacterium formicicum (Schnel-len, 1947) has been maintained in our laboratory.The culture originated from an inoculum ofbovine rumen fluid and was serially transferredin an anaerobic medium containing formate,sulfide, and salts. An attempt to obtain growthof the methanogenic organism when dicarboxylicacids were substituted for bicarbonate in themedium resulted in increased turbidity and al-kalinity when fumarate and malate were addedbut not when succinate was added. Analysis bygas chromatography, however, showed thatlittle methane had been formed in the fumarate-or malate-containing cultures in contrast tothe bicarbonate-containing cultures. Morpho-logical examination revealed that a previouslyundetected, highly motile vibrio had grown pro-fusely in the media containing fumarate ormalate. The absence of growth of the vibrio inthe succinate-containing medium suggested thepossibility that fumarate and malate stimulatedgrowth of the vibrio by acting as electron ac-ceptors for the oxidation of formate and per-mitted growth under anaerobic conditions. Theisolation and study of pure cultures of the vibriosubstantiated this hypothesis and also demon-strated that this obligate anaerobe could onlygrow in the presence of a limited number ofelectron donors (H2, HCOOH) and electron ac-ceptors (fumarate, malate, and nitrate). Thepresence of cytochromes in the organism addsto its complement of unusual characteristics.The present report is concerned with the isola-tion of the vibrio and a general description of itscharacteristics.

MATERIALS AND METHODS

Primary enrichment medium. The enrichmentculture medium for methanogenic bacteria fromwhich the organism was eventually isolated wassimilar to that described by Barker (1936)

except for the addition of a few vitamins. Itconsisted of NH4Cl, 0.1%; KH2PO4, 0.04%;MgCl2 6H20, 0.02%; sodium formate, 0.5%;FeSO4, 0.001%; phenol red, 0.0003%; NaHCO3,0.078%; Na2S-9H20, 0.02%; vitamin B12, 0.02,g/ml; and p-aminobenzoic acid, 0.30 ,ug/ml.The medium was prepared using tap water andplaced in serum bottles. Bicarbonate and sulfidewere sterilized by filtration and added aseptically.Prior to inoculation, the medium was asepticallyflushed with a mixture of 95% N2 and 5% C02,until the pH was 7.2 to 7.4, and capped with aserum bottle cap. Inoculations were made witha hypodermic syringe.

Secondary enrichment medium. Enrichmentfor the anaerobic vibrio from the primary en-richment medium was made in an identicalmedium with 0.1% fumarate substituted for thebicarbonate. Since autoclaving produced con-siderable alkalinity in the medium, presumablydue to decomposition of tap water carbonates,the medium was neutralized with acid after auto-claving and autoclaved again. Sterile sulfideprepared in 0.01 M K2iPO4, pH 7.2, was thenadded. The gas phase was N2.

Culture medium. A simple culture mediumwas developed after isolation of the organismwhich consists of (NH4)2S04, 0.1 %; K2HPO4,0.5%; fumaric acid, 0.3%; sodium formate,0.3%; yeast extract (Difco), 0.1%; MgC12 6H20,0.02%; and FeSO4, 0.001%. The pH is 7.0 to7.2. Sterile, autoclaved sodium thioglycolate(Difco) is added aseptically before inoculationto a final concentration of 0.05% and providessufficient anaerobiosis for routine transfers.Unless stated otherwise, the organism was cul-tivated in this medium. All incubations were at37 C.

RESULTS

Isolation of pure culture. The source of theorganism was bovine rumen fluid which wasused as the inoculum in the primary enrichment

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medium for the purpose of obtaining organismswhich produce methane from formate. Meth-anogenic organisms were obtained in the enrich-ment which were morphologically similar toM. formicicum (Schnellen, 1947). The meth-anogenic culture was transferred, using a 1%inoculum, at least 100 times (in the primaryenrichment medium), and the presence of vibrioswas not apparent from microscopic examination.The use of fumarate or malate as substitutes forbicarbonate in the medium brought about a largeincrease in the numbers of a highly motile vibrio.The vibrio was isolated from the secondary en-richment medium by plating on a solid mediumwhich was prepared by adding 2% agar to thefumarate-containing medium. The medium wassolidified on the side of a flat-sided bottle afterflushing with N2, capped, and inoculated withdilutions of the secondary enrichment culture.The isolated colonies obtained were composedof highly motile vibrios. Subcultures grew in thesecondary enrichment medium but no methanewas produced as determined by gas chromato-graphic analysis. No substantial growth ormethane production was obtained by subcul-turing in the primary enrichment medium.

Nutrition and general physiology. A few experi-ments led to the development of the culturemedium described under Materials and Methods.It was established that both formate and fu-marate were required for growth of the organism.A search was made for compounds which couldsubstitute for formate in the presence of fumarate.H2 is the only compound we have found whichwill substitute for formate. The only compounds

TABLE 1Pairs of compounds requiredfor growth of the vibrio

Growth with Second of PairFirst of Pair (Electron Donor)

(Electron Acceptor)H2 HCO,H None

Fumarate 0.184 0.315 0.023Malate 0.178 0.240 0.000Nitrate 0.205 0.230 0.018None 0.000 0.093

Ten milliliters of medium inoculated with 1drop of a 24-hr culture. Growth measured as op-tical density of 48-hr cultures at 660 m, in a spec-trophotometer. Tubes were incubated in air ex-cept where H2 was used as an electron donor.

we have found which will substitute for fumaratein the presence of formate are malate and nitrate.Growth with combinations of formate orhydrogen and fumarate, malate, or nitrate isshown in Table 1. A small amount of slow growthis obtained with formate alone which is noteliminated by using a washed inoculum. Thissmall extent of growth is not due to a limitingsubstance in the yeast extract because increasingthe yeast extract concentration does not signifi-cantly increase the yield. The growth on formatealone is probably a reflection of a limited abilityto use O2 as an electron acceptor (discussed ingreater detail below).

Except for the limited growth on formate, thedata are consistent with the interpretation thatan electron donor compound, formate or H2, andan electron acceptor compound, fumarate,malate, or nitrate, are required for growth of thevibrio. Substances which will not support growthin the presence of fumarate and in the absenceof formate are C2-C5 straight chain fatty acids,lactate, glucose, C1-C4 straight chain primaryalcohols, glycerol, mannitol, methionine, glycine,and serine. Substances which will not supportgood growth in the presence of formate and in theabsence of fumarate are bicarbonate, sulfate,crotonate, pyruvate, succinate, lactate, glucose,methanol, acetate, and methionine. All substanceswere tested at a concentration of 0.3% in themedium. An amino acid mixture, consistingof 0.5% acid hydrolyzed casein (NutritionalBiochemicals Corporation) and 0.01% each ofL-tryptophan and L-cystine, does not supportgrowth in the presence of either formate orfumarate. The vibrio is not photosynthetic sinceno growth is obtained in a medium containingformate and bicarbonate with illumination byvisible light.

Carbohydrates are not fermented by theorganism nor do they support growth. Possibleacid production and growth on carbohydrateswere tested with 0.01% of formate and fumaratein the medium and the following carbohydrates:glucose, maltose, sucrose, inulin, L-arabinose,raffinose, sorbitol, trehalose, lactose, cellobiose,salicin, dextrin, galactose, xylose, and fructose.Choline did not support growth in the presenceof 0.01% of formate and fumarate. Choline wastested to determine whether a relationshipexisted between the vibrio and Vibrio cholinicus(Hayward and Stadtman, 1959).

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Yeast extract was included in the mediumbecause it was found to stimulate growth of thevibrio. An amino acid mixture can replace theyeast extract. A mixture of L-glutamate,L-aspartate, L-alanine, and L-cysteine supportsas much growth as is obtained with yeast extractalone. No vitamins are required by the vibrio.Growth is initiated at pH 6.5 to pH 8.0 (but notat 6.0) and at 25 C (but not at 15 C).

Catalase is absent. A test for urease was madeby adding urea to the amino acid medium men-tioned above which supported growth in theabsence of (NH4)2S04. Examination of samplesof the cultures grown with and without ureashowed no increase in the ammonia concentrationin the urea-containing culture, indicating theabsence of urease. Ammonia was determined asdescribed by Umbreit, Burris, and Stauffer(1951).Hydrogen sulfide is produced from both

cysteine and thioglycolate but not from sulfate.Nitrite accumulates in the medium when theorganism is grown on H2 or formate and nitrate.Little or no growth is obtained on blood agar ina H2 atmosphere.

Morphological characteristics. The organism isa small, curved rod approximately 0.6 by 3 Iuoccurring singly, in pairs, and in spiral chains. Aphotograph of a preparation stained by themethod of Leifson (1930) is shown in Fig. 1. Theorganism is actively motile. It has a single, polarflagellum. Deep agar colonies in the secondaryenrichment culture medium are 0.5 to 2 mm indiameter and appear yellow with dark centers.Surface colonies on ordinary transfer mediumincubated under nitrogen are about 2 mm indiameter, are highly translucent, and have theappearance of droplets of water. The organismstains poorly in the Gram stain and is gram-negative. It can be stained with basic dyes suchas crystal violet or methylene blue. No sporeshave ever been observed, but the heat sensitivityof the vibrio has not been tested.

Relationship to oxygen. The relationship of theorganism to oxygen was considered from twostandpoints. Consideration was given to thepossibility of the utilization of oxygen as anelectron acceptor in place of fumarate, malate,or nitrate. Another consideration was the sensi-tivity of growth on formate and fumarate to thepresence of oxygen. Both of these points weretested by streaking the organism on agar slants

.k~~~~~~~~SFig. 1. Flagella stain of the vibrio (1,600X

magnification).

and incubating with various concentrations ofN2 and 02. With formate and fumarate in theculture medium, growth was always obtainedin an atomosphere of N2, but never in 95% N2and 5% 02 or in air. With 98% N2 and 2% 02,growth was sporadic and never exceeded thegrowth obtained in N2. When fumarate wasomitted from the medium, a trace amount ofgrowth was obtained in N2 and even less in 98%N2 and 2% 02, but growth was absent in 5% 02and in air. It seemed possible that the traceamount of growth obtained in N2 could be dueto contamination of this gas with traces of oxygen.This possibility was confirmed by comparing thegrowth of the organism with hydrogen as the gasphase in the presence and absence of 5% pal-ladium asbestos. The results in Table 2 demon-strate that the organism has a limited ability touse 02 as an electron acceptor. Penassay broth(Difco) was used as a basal medium in thisexperiment because it is clearer after autoclavingthan the usual culture medium and thereby allowseasier detection of small amounts of growth.Whereas limited growth is obtained in a H2 orN2 atmosphere when an electron donor is present,no more than a trace of growth is obtained in H2when palladized asbestos is introduced into thedesiccator to remove traces of 02. Formate or

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TABLE 2Utilization of oxygen by the vibrio

Growth in Various Atmospheres

Additions to Basal MediumN2-CO2 H2-COs H2-Cat t

None .................. 0.004 0.032 0.012Formate, 0.3%......... 0.060 0.031 0.005Fumarate, 0.3% ........ 0.004 0.095 0.069Formate, 0.3% and fu-marate, 0.3%......... 0.097 0.085 0.082

Basal medium consisted of Pennassay broth(Difco) plus 0.05% thioglycolate. Twenty milli-liters of medium in a 125-ml flask were inoculatedwith 0.2 ml of a 24-hr culture. The gas atmospherescontained 20%o C02. Palladium was present as5% palladium asbestos in a petri dish in the bot-tom of the desiccator.

* Growth was measured as optical density of60-hr cultures at 660 nvs in a spectrophotometerwith uninoculated medium used as a blank. Lightpath = 1 cm.

hydrogen are necessary for 02 utilization becauseno growth is obtained in N2 with fumaratepresent and formate absent. Growth in 02 iSprobably limited by the previously described02 sensitivity of the vibrio. In the terminologysuggested by McBee, Lamanna, and Weeks(1955), the vibrio would be called an oxybionticobligate anaerobe.

Isolation from rumen fluid. Since the organismwas originally isolated from an enrichment culturewhich was transferred many times after theoriginal culture was prepared, it was of interestto determine whether a more direct isolationprocedure would detect the organism in bovinerumen fluid. A surface plating method (Snyder,1947) gave satisfactory results with the pureculture when the petri dishes were incubatedunder N2 or H2 with 5% palladium asbestos.Ordinary pour plates were unsatisfactory becauseof the spreading of colonies trapped between theagar and the glass. Dilution blanks were com-posed of 0.05% sodium thioglycolate (Difco) in0.05 M K2HPO4, pH 7.0. The usual culture mediumwas used with 1.5% agar added.

Platings of bovine rumen fluid resulted in lowcolony counts and no vibrios were detected whichhad the characteristics of the vibrio we havedescribed. Enrichment cultures prepared by in-oculating rumen fluid in various dilutions in the

liquid culture medium also failed to yield theorganism upon subsequent plating. The vibriocould be successfully isolated, however, by pre-paring enrichment cultures in a medium practi-cally identical to the primary enrichment mediumfrom which the original culture was isolated. Thevibrio grew only slightly in this medium and was

recovered by a transfer of the enrichment to theusual culture medium and plating of this second-ary transfer after the outgrowth of the vibrio.When this procedure was followed with dilutionsof rumen fluid, the vibrio could be recoveredfrom 10-5 ml of rumen fluid. Because of thecomplicated procedure employed, this value can-

not be used to obtain an accurate estimate of thenumber of these vibrios in the rumen.

Reduction of fumarate. The reduction offumarate by H2 was tested with resting cells ofthe vibrio. With limiting concentrations offumarate, 1 mole of H2 was taken up per mole

0J

0

U)

w-J

0

0

0 10 30 40MINUTES

Fig. 2. Stoichiometry of fumarate reduction.H2 uptake measured at 37 C in a conventionalWarburg apparatus. Each vessel contained 100pumoles K%HPO4, pH 7.2, and 0.15 ml of cell sus-

pension (cells from 250 ml of medium were washedand resuspended in 24 ml of 0.01% 8-mereapto-ethanol). Fumarate was added in the amountsindicated above the corresponding final H2 uptakevalues.

H2 UPTAKE WITH DIFFERENT

10 |LEVELS OF FUMARATE

-t -K-L-_6___X. *6 !M

6

2pM

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of fumarate added (Fig. 2). This ratio indicatesthat fumarate is reduced to succinate. In a sepa-rate experiment resting cells were incubated with26.75 ,moles of fumarate and H2 in a desiccatorat 37 C for 3 hr. After precipitating the cells withHCl and centrifuging, the supernatant solutionwas examined for fumarate by measuring theabsorbancy at 240 mjl as described by Racker(1950). All of the fumarate had disappeared. Theacidified solution was placed on a celite columnand the acids present were eluted with ether asdescribed by Swim and Utter (1957). The ethereluate was evaporated to dryness. The drymaterial was dissolved in water and assayed forsuccinate with a pig heart succinoxidase prepara-tion (Umbreit et al., 1951); 24.2 ,umoles of suc-cinate were recovered from the original reactionmixture which represented a 90.5% conversionof fumarate to succinate. The reduction of malateby H2 can be demonstrated with resting cells, buthas not been studied in detail. It appears likelythat malate is converted to fumarate prior toreduction to succinate.

Reduction of nitrate. Although nitrite accumu-lates in the medium when the vibrio is grownwith H2 or formate and nitrate, the stoichiometryof the reduction of nitrate by H2 obtained withresting cells indicates that the nitrate is reducedbeyond the nitrite stage (Table 3). The stoichi-ometry suggests almost a complete reduction ofnitrate to NH3. Tests of the Warburg flask con-tents, after H2 uptake had ceased, showed that nonitrite had accumulated with any of the levelsof nitrate used. Perhaps the accumulation ofnitrite in the culture medium is due to an inhibi-

TABLE 3H2 uptake with limiting amounts of NaNO3

NaNO3 Added Total H2 Uptake Theoretical H2 Uptakefor Reduction to NH3

Amoles pmoles Amoles

1 4.15 4.02 6.66 8.04 14.0 16.0

Protocol for the experiment was the same as inFig. 2 except that NaNO3 was substituted forfumarate and the cell suspension was concen-trated from 1 liter of the culture medium con-taining 0.3% NaNO3 instead of fumarate. Thefinal volume of cell suspension was 20 ml of which0.2 ml was present in the Warburg vessels.

1.10

0.90

z

mcr0U,

0.70

0.50

0303

0.10

400 430 460 500 520 550 600

WAVELENGTH- m.u

Fig. 3. Spectra of cell-free extract. A suspensioncontaining 31 mg (dry weight) per ml of washedcells grown on formate and fumarate was disruptedby sonic oscillation and centrifuged to remove

large debris. After flushing hydrogen or heliumthrough the extracts in modified anaerobiccuvettes, spectra were recorded on a Cary re-

cording spectrophotometer with water in thereference cuvette. Light path = 1 cm.

tion of the further reduction of nitrite by some

environmental factor. Increasing formate in themedium to greater than 4 times the nitrate con-

centration (on a molarity basis) does noteliminate nitrite production.Lack of hydrogenlyase. It has been observed

that no gas is collected in an inverted vial duringgrowth when formate is used as an electron donorin the presence of fumarate or nitrate even whenthe formate level is increased beyond the amountswhich would be required to produce a stoichi-ometric yield of succinate or NH3. The lack ofhydrogen production suggests that the organismdoes not contain a complete hydrogenlyasesytem although it contains formic dehydrogenaseand hydrogenase. This has been confirmed withresting cell experiments. Under conditions whereCO2 evolution from formate and fumarate or

formate and methylene blue can be demon-strated, no gas evolution from formate alone can

be detected. Hydrogenase can also be demon-strated at the same time with benzyl viologen,methylene blue, or fumarate as electron acceptors.

SPECTRA OF SONIC EXTRACT

.------ OXIDIZED (Hi3/I \ REDUCED(HH

\0 m420

I>0.300 _

i

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The results are similar to those obtained byGest and Peck (1955) with certain anaerogenicstrains of Escherichia coli.

Presence of cytochromes. The noticeably pinkcolor of packed cells indicated that the vibriocontains a pigment. The absorption spectrum ofsonic extracts of cells grown on fumarate ornitrate with formate shows the presence of cyto-chromes. When fumarate is supplied in the growthmedium, the spectrum (Fig. 3) shows a peak atapproximately 410 m,u. Upon reduction of thecytochromes with hydrosulfite or H2, peaksappear at approximately 419, 522, and 553 m,uindicating the presence of a cytochrome of thec type. The shoulders on these peaks indicatethe presence of a cytochrome of the b type withpeaks that have been estimated as occurring at560 and 528 m,A in the a and ,B regions of thespectrum. Extracts of nitrate-grown cells showonly the spectrum characteristic of the c typeof cytochrome.

DISCUSSION

The organism described is similar toDesulfovibrio desulfuricans and Vibrio cholinicuswith respect to morphology, the requirement foranaerobiosis, and the presence of cytochromes.A cytochrome c is present in both D. desulfuricans(Postgate, 1956) and V. cholinicus (Haywardand Stadtman, 1960). The inability to reducesulfate to hydrogen sulfide and ferment cholinecertainly distinguishes this organism from thesepreviously described species. The strict require-ment of the vibrio for H2 or formate and fu-marate, malate, or nitrate for growth is anotherdistinguishing characteristic. The authors con-sider the organism to be a new species of thegenus Vibrio. The name Vibrio succinogenes,sp. n. is proposed. The other two species ofanaerobic vibrios described in Bergey's manual ofdeterminative bacteriology (Breed, Murray, andSmith, 1957), Vibrio niger and Vibrio sputorumare distinct from V. succinogenes. V. niger fer-ments glucose and V. sputorum grows well onblood media. It should be pointed out, however,that the physiology of V. sputorum and themicroaerophilic species Vibrio coli, Vibrio jejuni,and Vibrio fetus is poorly understood. Thesespecies do not ferment sugars, and their meansof obtaining energy for growth is unknown oronly partially known.The unusual relationship to oxygen exhibited

by the vibrio warrants further discussion. Theorganism can use oxygen as an electron acceptor,but only at low partial pressures of oxygen.When oxygen is present at concentrations above2% in the atmosphere, it inhibits growth evenwhen fumarate is supplied as an electron acceptor.Thus the vibrio is an obligate anaerobe becauseit is unable to grow in the presence of air (in theabsence of reducing agents). Since anaerobicgrowth with fumarate and nitrate as electronacceptors is always better than growth withoxygen, it seems best to emphasize the anaerobiccharacteristics of the vibrio. It should be empha-sized that growth with fumarate or nitrate aselectron acceptors proceeds in the completeabsence of oxygen.The organism apparently obtains energy for

growth from the coupled oxidation-reductionreactions which are obligatory for growth of theorganism. Energy would presumably be obtainedfrom an anaerobic oxidative phosphorylationprocess. Other bacteria which apparently obtainenergy for growth by a similar process aremethane-forming bacteria, such as M. formicicumand MIethanococcus vanniellii, D. desulfuricansgrown with H2 and sulfate (Butlin, Adams, andThomas, 1949), and aerobes, such as Micrococcusdenitrificans grown with H2 and nitrate. It seemsreasonable to expect that the cytochromes of thevibrio will be found to play a role in the oxidation-reduction reactions required for growth. A subse-quent report will describe some of the chemicaland enzymatic properties of the cytochromes.The ecology of the vibrio is obscure at present.

It can be isolated from bovine rumen fluid, butwhether it is an important inhabitant of therumen either numerically or functionally remainsto be elucidated. Improvement of the tediousisolation procedure would seem to be a necessaryprerequisite for studies of the natural distributionof the organism.

ACKNOWLEDGMENT

This investigation was supported in part by aresearch grant (E2363) from the National Insti-tute of Allergy and Infectious Diseases, U. S.Public Health Service.

SUMMARY

A new obligately anaerobic cytochrome-producing vibrio has been isolated from bovinerumen fluid. The vibrio is restricted to a limited

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CYTOCHROME-PRODUCING ANAEROBIC VIBRIO

number of oxidation-reduction reactions for theproduction of energy for growth. These oxidation-reduction reactions involve H2 and formate as theonly known satisfactory electron donors andfumarate, malate, or nitrate as the electronacceptors. Oxygen can serve as an electronacceptor which supports limited growth onlywhen it is present at concentrations of 2% orless in the atmosphere. Higher oxygen concentra-tions are toxic to the organism. Carbohydratesare not fermented by the vibrio.

Studies with washed cell suspensions havedemonstrated that fumarate is reduced to suc-cinate in the presence of H2 and the stoichiometryof nitrate reduction by H2 indicates almost acomplete reduction of nitrate to ammonia. Nitritedoes accumulate, however, in growing cultureswhen nitrate is used as an electron acceptor. Thevibrio appears to lack a hydrogenlyase systemwhich accounts for the lack of hydrogen produc-tion during growth when formate is used as anelectron donor.

Spectra of crude extracts show the presence ofcytochromes b and c when the vibrio is grownwith fumarate as an electron acceptor. Whennitrate is substituted for fumarate in the growthmedium, only the c type of cytochrome is ob-served in crude extracts.The organism has been placed in the genus

Vibrio, and given the name Vibrio succinogenes,sp. n.

REFERENCESBARKER, H. A. 1936 Studies upon the methane-

producing bacteria. Arch. Mikrobiol., 7,420-438.

BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH1957 Bergey's manual of determinative bac-teriology, 7th ed. The Williams & WilkinsCo., Baltimore.

BUTLIN, K. R., M. E. ADAMS, AND M. THOMAS1949 The isolation and cultivation of sulfate-reducing bacteria. J. Gen. Microbiol., 3,46-59.

GEST, H., AND H. D. PECK, JR. 1955 A studyof the hydrogenlyase reaction with systemsderived from normal and anaerogenic coli-aerogenes bacteria. J. Bacteriol., 70, 326-334.

HAYWARD, H. R., AND T. C. STADTMAN 1959Anaerobic degradation of choline. I. Fermen-tation of choline by an anaerobic cytochrome-producing bacterium, Vibrio cholinicus n.sp. J. Bacteriol., 78, 557-561.

HAYWARD, H. R., AND T. C. STADTMAN 1960Anaerobic degradation of choline II. Prepara-tions and properties of cell-free extracts ofVibrio cholinicus. J. Biol. Chem., 235, 538-543.

LEIFSON, E. 1930 A method of staining bacterialflagella and capsules together with a studyof the origin of flagella. J. Bacteriol., 20,203-211.

McBEE, R. H., C. LAMANNA, AND 0. B. WEEKS1955 Definitions of bacterial oxygen relation-ships. Bacteriol. Rev., 19, 45-47.

POSTGATE, J. R. 1956 Cytochrome C3 and desul-phoviridin; pigments of the anaerobeDesulphovibrio desulphuricans. J. Gen. Mi-crobiol., 14, 545-572.

RACKER, E. 1950 Spectrophotometric measure-ments of the enzymatic formation of fumaricand cis-aconitic acids. Biochim. et Biophys.Acta, 4, 211-214.

SCHNELLEN, C. G. T. P. 1947 Onderzoekingenover de methaangisting. Dissertation,Technical University, Delft. De Maasstad,Rotterdam.

SNYDER, T. L. 1947 The relative errors ofbacteriological plate counting methods. J.Bacteriol., 54, 641-653.

SWIM, H. E., AND M. F. UTTER 1957 Isotopicexperimentation with intermediates of thetricarboxylic acid cycle. In Methods inenzymology vol. 4, pp. 584-609. Edited byS. P. COLOWICK AND N. D. KAPLAN. Acade-mic Press, Inc., New York.

UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER 1951 Manometric techniques and tissuemetabolism, 2nd ed. Burgess Publishing Co.,Minneapolis.

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