CYTOCHROME-PRODUCING ANAEROBIC VIBRIO, VIBRIO

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1 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 (Schnellen, 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 alkalinity 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 fumarateor malate-containing cultures in contrast to the bicarbonate-containing cultures. Morphological examination revealed that a previously undetected, highly motile vibrio had grown profusely 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 acceptors for the oxidation of formate and permitted growth under anaerobic conditions. The isolation and study of pure cultures of the vibrio substantiated this hypothesis and also demonstrated that this obligate anaerobe could only grow in the presence of a limited number of electron donors (H2, HCOOH) and electron acceptors (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 isolation of the vibrio and a general description of its characteristics. MATERIALS AND METHODS 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, %; 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 enrichment medium was made in an identical medium with 0.1% fumarate substituted for the bicarbonate. Since autoclaving produced considerable alkalinity in the medium, presumably due to decomposition of tap water carbonates, the medium was neutralized with acid after autoclaving 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 cultivated in this medium. All incubations were at 37 C. RESULTS Primary enrichment medium. The enrichment culture medium for methanogenic bacteria from Isolation of pure culture. The source of the which the organism was eventually isolated was organism was bovine rumen fluid which was similar to that described by Barker (1936) used as the inoculum in the primary enrichment 911

2 912 WOLIN, WOLIN, AND JACOBS [VOL. 81 medium for the purpose of obtaining organisms which produce methane from formate. Methanogenic organisms were obtained in the enrichment which were morphologically similar to M. formicicum (Schnellen, 1947). The methanogenic culture was transferred, using a 1% inoculum, at least 100 times (in the primary enrichment medium), and the presence of vibrios was not apparent from microscopic examination. The use of fumarate or malate as substitutes for bicarbonate in the medium brought about a large increase in the numbers of a highly motile vibrio. The vibrio was isolated from the secondary enrichment medium by plating on a solid medium which was prepared by adding 2% agar to the fumarate-containing medium. The medium was solidified on the side of a flat-sided bottle after flushing with N2, capped, and inoculated with dilutions of the secondary enrichment culture. The isolated colonies obtained were composed of highly motile vibrios. Subcultures grew in the secondary enrichment medium but no methane was produced as determined by gas chromatographic analysis. No substantial growth or methane production was obtained by subculturing in the primary enrichment medium. Nutrition and general physiology. A few experiments led to the development of the culture medium described under Materials and Methods. It was established that both formate and fumarate were required for growth of the organism. A search was made for compounds which could substitute for formate in the presence of fumarate. H2 is the only compound we have found which will substitute for formate. The only compounds TABLE 1 Pairs of compounds requiredfor growth of the vibrio First of Pair (Electron Acceptor) Growth with Second of Pair (Electron Donor) H2 HCO,H None Fumarate Malate Nitrate None Ten milliliters of medium inoculated with 1 drop of a 24-hr culture. Growth measured as optical density of 48-hr cultures at 660 m, in a spectrophotometer. Tubes were incubated in air except where H2 was used as an electron donor. we have found which will substitute for fumarate in the presence of formate are malate and nitrate. Growth with combinations of formate or hydrogen and fumarate, malate, or nitrate is shown in Table 1. A small amount of slow growth is obtained with formate alone which is not eliminated by using a washed inoculum. This small extent of growth is not due to a limiting substance in the yeast extract because increasing the yeast extract concentration does not significantly increase the yield. The growth on formate alone is probably a reflection of a limited ability to use O2 as an electron acceptor (discussed in greater detail below). Except for the limited growth on formate, the data are consistent with the interpretation that an electron donor compound, formate or H2, and an electron acceptor compound, fumarate, malate, or nitrate, are required for growth of the vibrio. Substances which will not support growth in the presence of fumarate and in the absence of formate are C2-C5 straight chain fatty acids, lactate, glucose, C1-C4 straight chain primary alcohols, glycerol, mannitol, methionine, glycine, and serine. Substances which will not support good growth in the presence of formate and in the absence of fumarate are bicarbonate, sulfate, crotonate, pyruvate, succinate, lactate, glucose, methanol, acetate, and methionine. All substances were tested at a concentration of 0.3% in the medium. An amino acid mixture, consisting of 0.5% acid hydrolyzed casein (Nutritional Biochemicals Corporation) and 0.01% each of L-tryptophan and L-cystine, does not support growth in the presence of either formate or fumarate. The vibrio is not photosynthetic since no growth is obtained in a medium containing formate and bicarbonate with illumination by visible light. Carbohydrates are not fermented by the organism nor do they support growth. Possible acid production and growth on carbohydrates were tested with 0.01 % of formate and fumarate in 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 presence of 0.01 % of formate and fumarate. Choline was tested to determine whether a relationship existed between the vibrio and Vibrio cholinicus (Hayward and Stadtman, 1959).

3 19611 CYTOCHROME-PRODUCING ANAEROBIC VIBRIO 913 Yeast extract was included in the medium because it was found to stimulate growth of the vibrio. An amino acid mixture can replace the yeast extract. A mixture of L-glutamate, L-aspartate, L-alanine, and L-cysteine supports as much growth as is obtained with yeast extract alone. No vitamins are required by the vibrio. Growth is initiated at ph 6.5 to ph 8.0 (but not at 6.0) and at 25 C (but not at 15 C). Catalase is absent. A test for urease was made by adding urea to the amino acid medium mentioned above which supported growth in the absence of (NH4)2S04. Examination of samples of the cultures grown with and without urea showed no increase in the ammonia concentration in the urea-containing culture, indicating the absence of urease. Ammonia was determined as described 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 the organism is grown on H2 or formate and nitrate. Little or no growth is obtained on blood agar in a H2 atmosphere. Morphological characteristics. The organism is a small, curved rod approximately 0.6 by 3 Iu occurring singly, in pairs, and in spiral chains. A photograph of a preparation stained by the method of Leifson (1930) is shown in Fig. 1. The organism is actively motile. It has a single, polar flagellum. Deep agar colonies in the secondary enrichment culture medium are 0.5 to 2 mm in diameter and appear yellow with dark centers. Surface colonies on ordinary transfer medium incubated under nitrogen are about 2 mm in diameter, are highly translucent, and have the appearance of droplets of water. The organism stains poorly in the Gram stain and is gramnegative. It can be stained with basic dyes such as crystal violet or methylene blue. No spores have ever been observed, but the heat sensitivity of the vibrio has not been tested. Relationship to oxygen. The relationship of the organism to oxygen was considered from two standpoints. Consideration was given to the possibility of the utilization of oxygen as an electron acceptor in place of fumarate, malate, or nitrate. Another consideration was the sensitivity of growth on formate and fumarate to the presence of oxygen. Both of these points were tested by streaking the organism on agar slants.k~~~~~~~~s Fig. 1. Flagella stain of the vibrio (1,600X magnification). and incubating with various concentrations of N2 and 02. With formate and fumarate in the culture medium, growth was always obtained in an atomosphere of N2, but never in 95% N2 and 5% 02 or in air. With 98% N2 and 2% 02, growth was sporadic and never exceeded the growth obtained in N2. When fumarate was omitted from the medium, a trace amount of growth was obtained in N2 and even less in 98% N2 and 2% 02, but growth was absent in 5% 02 and in air. It seemed possible that the trace amount of growth obtained in N2 could be due to contamination of this gas with traces of oxygen. This possibility was confirmed by comparing the growth of the organism with hydrogen as the gas phase in the presence and absence of 5% palladium asbestos. The results in Table 2 demonstrate that the organism has a limited ability to use 02 as an electron acceptor. Penassay broth (Difco) was used as a basal medium in this experiment because it is clearer after autoclaving than the usual culture medium and thereby allows easier detection of small amounts of growth. Whereas limited growth is obtained in a H2 or N2 atmosphere when an electron donor is present, no more than a trace of growth is obtained in H2 when palladized asbestos is introduced into the desiccator to remove traces of 02. Formate or

4 914 WOLIN, WOLIN, AND JACOBS [VOL. 81 TABLE 2 Utilization of oxygen by the vibrio Additions to Basal Medium Growth in Various Atmospheres N2-CO2 H2-COs H2-Cat t None Formate, 0.3% Fumarate, 0.3% Formate, 0.3% and fumarate, 0.3% Basal medium consisted of Pennassay broth (Difco) plus 0.05% thioglycolate. Twenty milliliters of medium in a 125-ml flask were inoculated with 0.2 ml of a 24-hr culture. The gas atmospheres contained 20%o C02. Palladium was present as 5% palladium asbestos in a petri dish in the bottom of the desiccator. * Growth was measured as optical density of 60-hr cultures at 660 nvs in a spectrophotometer with uninoculated medium used as a blank. Light path = 1 cm. hydrogen are necessary for 02 utilization because no growth is obtained in N2 with fumarate present and formate absent. Growth in 02 is probably limited by the previously described 02 sensitivity of the vibrio. In the terminology suggested by McBee, Lamanna, and Weeks (1955), the vibrio would be called an oxybiontic obligate anaerobe. Isolation from rumen fluid. Since the organism was originally isolated from an enrichment culture which was transferred many times after the original culture was prepared, it was of interest to determine whether a more direct isolation procedure would detect the organism in bovine rumen fluid. A surface plating method (Snyder, 1947) gave satisfactory results with the pure culture when the petri dishes were incubated under N2 or H2 with 5% palladium asbestos. Ordinary pour plates were unsatisfactory because of the spreading of colonies trapped between the agar and the glass. Dilution blanks were composed of 0.05% sodium thioglycolate (Difco) in 0.05 M K2HPO4, ph 7.0. The usual culture medium was used with 1.5% agar added. Platings of bovine rumen fluid resulted in low colony counts and no vibrios were detected which had the characteristics of the vibrio we have described. Enrichment cultures prepared by inoculating rumen fluid in various dilutions in the liquid culture medium also failed to yield the organism upon subsequent plating. The vibrio could be successfully isolated, however, by preparing enrichment cultures in a medium practically identical to the primary enrichment medium from which the original culture was isolated. The vibrio grew only slightly in this medium and was recovered by a transfer of the enrichment to the usual culture medium and plating of this secondary transfer after the outgrowth of the vibrio. When this procedure was followed with dilutions of rumen fluid, the vibrio could be recovered from 10-5 ml of rumen fluid. Because of the complicated procedure employed, this value cannot be used to obtain an accurate estimate of the number of these vibrios in the rumen. Reduction of fumarate. The reduction of fumarate by H2 was tested with resting cells of the vibrio. With limiting concentrations of fumarate, 1 mole of H2 was taken up per mole 0J 0 U) -J 0w 0 H2 UPTAKE WITH DIFFERENT LEVELS OF FUMARATE l t -K-L-_ 6 X. *6!M 2pM MINUTES Fig. 2. Stoichiometry of fumarate reduction. H2 uptake measured at 37 C in a conventional Warburg apparatus. Each vessel contained 100 pumoles K%HPO4, ph 7.2, and 0.15 ml of cell suspension (cells from 250 ml of medium were washed and resuspended in 24 ml of 0.01% 8-mereaptoethanol). Fumarate was added in the amounts indicated above the corresponding final H2 uptake values.

5 19611 CYTOCHROME-PRODUCING ANAEROBIC VIBRIO 915 of fumarate added (Fig. 2). This ratio indicates that fumarate is reduced to succinate. In a separate experiment resting cells were incubated with 26.75,moles of fumarate and H2 in a desiccator at 37 C for 3 hr. After precipitating the cells with HCl and centrifuging, the supernatant solution was examined for fumarate by measuring the absorbancy at 240 mjl as described by Racker (1950). All of the fumarate had disappeared. The acidified solution was placed on a celite column and the acids present were eluted with ether as described by Swim and Utter (1957). The ether eluate was evaporated to dryness. The dry material was dissolved in water and assayed for succinate with a pig heart succinoxidase preparation (Umbreit et al., 1951); 24.2,umoles of succinate were recovered from the original reaction mixture which represented a 90.5% conversion of fumarate to succinate. The reduction of malate by H2 can be demonstrated with resting cells, but has not been studied in detail. It appears likely that malate is converted to fumarate prior to reduction to succinate. Reduction of nitrate. Although nitrite accumulates in the medium when the vibrio is grown with H2 or formate and nitrate, the stoichiometry of the reduction of nitrate by H2 obtained with resting cells indicates that the nitrate is reduced beyond the nitrite stage (Table 3). The stoichiometry suggests almost a complete reduction of nitrate to NH3. Tests of the Warburg flask contents, after H2 uptake had ceased, showed that no nitrite had accumulated with any of the levels of nitrate used. Perhaps the accumulation of nitrite in the culture medium is due to an inhibi- TABLE 3 H2 uptake with limiting amounts of NaNO3 NaNO3 Added Total H2 Uptake Theoretical H2 Uptake for Reduction to NH3 Amoles pmoles Amoles Protocol for the experiment was the same as in Fig. 2 except that NaNO3 was substituted for fumarate and the cell suspension was concentrated from 1 liter of the culture medium containing 0.3% NaNO3 instead of fumarate. The final volume of cell suspension was 20 ml of which 0.2 ml was present in the Warburg vessels. z cr m0 U, SPECTRA OF SONIC EXTRACT i OXIDIZED (Hile) 3/I \ REDUCED(HH \0 m _ I> WAVELENGTH- m.u Fig. 3. Spectra of cell-free extract. A suspension containing 31 mg (dry weight) per ml of washed cells grown on formate and fumarate was disrupted by sonic oscillation and centrifuged to remove large debris. After flushing hydrogen or helium through the extracts in modified anaerobic cuvettes, spectra were recorded on a Cary recording spectrophotometer with water in the reference cuvette. Light path = 1 cm. tion of the further reduction of nitrite by some environmental factor. Increasing formate in the medium to greater than 4 times the nitrate concentration (on a molarity basis) does not eliminate nitrite production. Lack of hydrogenlyase. It has been observed that no gas is collected in an inverted vial during growth when formate is used as an electron donor in the presence of fumarate or nitrate even when the formate level is increased beyond the amounts which would be required to produce a stoichiometric yield of succinate or NH3. The lack of hydrogen production suggests that the organism does not contain a complete hydrogenlyase sytem although it contains formic dehydrogenase and hydrogenase. This has been confirmed with resting cell experiments. Under conditions where CO2 evolution from formate and fumarate or formate and methylene blue can be demonstrated, no gas evolution from formate alone can be detected. Hydrogenase can also be demonstrated at the same time with benzyl viologen, methylene blue, or fumarate as electron acceptors. 12).

6 916 WOLIN, WOLIN, AND JACOBS (VOL. 81 The results are similar to those obtained by Gest and Peck (1955) with certain anaerogenic strains of Escherichia coli. Presence of cytochromes. The noticeably pink color of packed cells indicated that the vibrio contains a pigment. The absorption spectrum of sonic extracts of cells grown on fumarate or nitrate with formate shows the presence of cytochromes. When fumarate is supplied in the growth medium, the spectrum (Fig. 3) shows a peak at approximately 410 m,u. Upon reduction of the cytochromes with hydrosulfite or H2, peaks appear at approximately 419, 522, and 553 m,u indicating the presence of a cytochrome of the c type. The shoulders on these peaks indicate the presence of a cytochrome of the b type with peaks that have been estimated as occurring at 560 and 528 m,a in the a and,b regions of the spectrum. Extracts of nitrate-grown cells show only the spectrum characteristic of the c type of cytochrome. DISCUSSION The organism described is similar to Desulfovibrio desulfuricans and Vibrio cholinicus with respect to morphology, the requirement for anaerobiosis, and the presence of cytochromes. A cytochrome c is present in both D. desulfuricans (Postgate, 1956) and V. cholinicus (Hayward and Stadtman, 1960). The inability to reduce sulfate to hydrogen sulfide and ferment choline certainly distinguishes this organism from these previously described species. The strict requirement of the vibrio for H2 or formate and fumarate, malate, or nitrate for growth is another distinguishing characteristic. The authors consider the organism to be a new species of the genus Vibrio. The name Vibrio succinogenes, sp. n. is proposed. The other two species of anaerobic vibrios described in Bergey's manual of determinative bacteriology (Breed, Murray, and Smith, 1957), Vibrio niger and Vibrio sputorum are distinct from V. succinogenes. V. niger ferments glucose and V. sputorum grows well on blood media. It should be pointed out, however, that the physiology of V. sputorum and the microaerophilic species Vibrio coli, Vibrio jejuni, and Vibrio fetus is poorly understood. These species do not ferment sugars, and their means of obtaining energy for growth is unknown or only partially known. The unusual relationship to oxygen exhibited by the vibrio warrants further discussion. The organism can use oxygen as an electron acceptor, but only at low partial pressures of oxygen. When oxygen is present at concentrations above 2% in the atmosphere, it inhibits growth even when fumarate is supplied as an electron acceptor. Thus the vibrio is an obligate anaerobe because it is unable to grow in the presence of air (in the absence of reducing agents). Since anaerobic growth with fumarate and nitrate as electron acceptors is always better than growth with oxygen, it seems best to emphasize the anaerobic characteristics of the vibrio. It should be emphasized that growth with fumarate or nitrate as electron acceptors proceeds in the complete absence of oxygen. The organism apparently obtains energy for growth from the coupled oxidation-reduction reactions which are obligatory for growth of the organism. Energy would presumably be obtained from an anaerobic oxidative phosphorylation process. Other bacteria which apparently obtain energy for growth by a similar process are methane-forming bacteria, such as M. formicicum and MIethanococcus vanniellii, D. desulfuricans grown with H2 and sulfate (Butlin, Adams, and Thomas, 1949), and aerobes, such as Micrococcus denitrificans grown with H2 and nitrate. It seems reasonable to expect that the cytochromes of the vibrio will be found to play a role in the oxidationreduction reactions required for growth. A subsequent report will describe some of the chemical and enzymatic properties of the cytochromes. The ecology of the vibrio is obscure at present. It can be isolated from bovine rumen fluid, but whether it is an important inhabitant of the rumen either numerically or functionally remains to be elucidated. Improvement of the tedious isolation procedure would seem to be a necessary prerequisite for studies of the natural distribution of the organism. ACKN OWLEDGMENT This investigation was supported in part by a research grant (E2363) from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. SUMMARY A new obligately anaerobic cytochromeproducing vibrio has been isolated from bovine rumen fluid. The vibrio is restricted to a limited

7 19611 CYTOCHROME-PRODUCING ANAEROBIC VIBRIO 917 number of oxidation-reduction reactions for the production of energy for growth. These oxidationreduction reactions involve H2 and formate as the only known satisfactory electron donors and fumarate, malate, or nitrate as the electron acceptors. Oxygen can serve as an electron acceptor which supports limited growth only when it is present at concentrations of 2% or less in the atmosphere. Higher oxygen concentrations are toxic to the organism. Carbohydrates are not fermented by the vibrio. Studies with washed cell suspensions have demonstrated that fumarate is reduced to succinate in the presence of H2 and the stoichiometry of nitrate reduction by H2 indicates almost a complete reduction of nitrate to ammonia. Nitrite does accumulate, however, in growing cultures when nitrate is used as an electron acceptor. The vibrio appears to lack a hydrogenlyase system which accounts for the lack of hydrogen production during growth when formate is used as an electron donor. Spectra of crude extracts show the presence of cytochromes b and c when the vibrio is grown with fumarate as an electron acceptor. When nitrate is substituted for fumarate in the growth medium, only the c type of cytochrome is observed in crude extracts. The organism has been placed in the genus Vibrio, and given the name Vibrio succinogenes, sp. n. REFERENCES BARKER, H. A Studies upon the methaneproducing bacteria. Arch. Mikrobiol., 7, BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH 1957 Bergey's manual of determinative bacteriology, 7th ed. The Williams & Wilkins Co., Baltimore. BUTLIN, K. R., M. E. ADAMS, AND M. THOMAS 1949 The isolation and cultivation of sulfatereducing bacteria. J. Gen. Microbiol., 3, GEST, H., AND H. D. PECK, JR A study of the hydrogenlyase reaction with systems derived from normal and anaerogenic coliaerogenes bacteria. J. Bacteriol., 70, HAYWARD, H. R., AND T. C. STADTMAN 1959 Anaerobic degradation of choline. I. Fermentation of choline by an anaerobic cytochromeproducing bacterium, Vibrio cholinicus n. sp. J. Bacteriol., 78, HAYWARD, H. R., AND T. C. STADTMAN 1960 Anaerobic degradation of choline II. Preparations and properties of cell-free extracts of Vibrio cholinicus. J. Biol. Chem., 235, LEIFSON, E A method of staining bacterial flagella and capsules together with a study of the origin of flagella. J. Bacteriol., 20, McBEE, R. H., C. LAMANNA, AND 0. B. WEEKS 1955 Definitions of bacterial oxygen relationships. Bacteriol. Rev., 19, POSTGATE, J. R Cytochrome C3 and desulphoviridin; pigments of the anaerobe Desulphovibrio desulphuricans. J. Gen. Microbiol., 14, RACKER, E Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim. et Biophys. Acta, 4, SCHNELLEN, C. G. T. P Onderzoekingen over de methaangisting. Dissertation, Technical University, Delft. De Maasstad, Rotterdam. SNYDER, T. L The relative errors of bacteriological plate counting methods. J. Bacteriol., 54, SWIM, H. E., AND M. F. UTTER 1957 Isotopic experimentation with intermediates of the tricarboxylic acid cycle. In Methods in enzymology vol. 4, pp Edited by S. P. COLOWICK AND N. D. KAPLAN. Academic Press, Inc., New York. UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER 1951 Manometric techniques and tissue metabolism, 2nd ed. Burgess Publishing Co., Minneapolis.