Wolinella Recta, Wolinella Curva, Bactevoides Ureolyticus, and Bactevoides Gvacilis Are Microaerophiles, Not Anaerobes Y.-H

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 218-222 Vol. 41, No. 2 0020-7713/91/020218-05$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Wolinella recta, Wolinella curva, Bactevoides ureolyticus, and Bactevoides gvacilis Are Microaerophiles, Not Anaerobes Y.-H. HAN,l R. M. SMIBERT,2 AND N. R. KRIEG1* Microbiology and Immunology Section, Department of Biology, and Department of Anaerobic Microbiology,2 Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Although the nonfermentative, asaccharolytic, putative anaerobes Wulinella curva, Wolinella recta, Bacterui- des ureolyticus, and Bacteroides gracilis are phylogenetically related to the true campylobacters, the type strains of these species exhibited 0,-dependent microaerophilic growth in brucella broth and on brucella agar. The optimum 0, levels for growth of these strains ranged from 4 to 14% in brucella broth and from 2 to 8% on brucella agar, when H, was provided as the electron donor. No growth occurred under 21% O,, and scant or no growth occurred under anaerobic conditions unless fumarate or nitrate was provided as a terminal electron acceptor. Aspartate, asparagine, and malate also served as apparent electron acceptors. The organisms were catalase negative and, except for B. gracilis, oxidase positive. Catalase added to brucella broth enhanced growth. 0, uptake by all species was inhibited by cyanide and 2-heptyl-4-hydroxyquinolineN-oxide. We concluded that these organisms are not anaerobes but instead are microaerophiles, like their campylobacter relatives.

Among the proteobacteria, a major taxonomic problem (21), it is oxidase positive, a characteristic usually associated has been finding phenotypic characteristics that correlate with organisms that can respire with 02.This raises the with the various phylogenetic groups delineated by rRNA question of whether W. succinogenes is in fact an anaerobe. analyses. This problem is exemplified by studies of campylo- Although W. succinogenes grows anaerobically by using bacters and campylobacter-like organisms. On the basis of fumarate as a terminal electron acceptor (5,6, 10-13), Wolin the results of rRNA sequence analyses (15), the nonfermen- et al. (24) and Jacobs and Wolin (5, 6) have shown that it is tative, asaccharolytic , putative anaerobes Wolinella recta, also capable of using 0, as a terminal electron acceptor Wolinella curva, Bacteroides ureolyticus, and Bacteroides under microaerobic conditions (approximately 2% 0,). Be- gracilis belong to the “true campylobacters,” a group that cause anaerobes do not use 0, as a terminal electron includes all Campylobacter species except Campylobacter acceptor for respiration, W. succinogenes must be consid- cryaerophila , Campylobacter nitrofigilis, Campylobacter ci- ered a H,-requiring microaerophile. naedi, and Campylobacter fennelliae, as well as Helicobac- Similarly, W. recta, W. curva, B. ureolyticus, and B. ter pylori (formerly called Campylobacter pylori) (14, 17, gracilis are presently considered to be anaerobes (4, 19-21), 23). The addition of the two Wolinella and two Bacteroides but their phylogenetic placement with the campylobacters, species to the true campylobacters makes it difficult to as well as the fact that three of the species (W. recta, W. describe the group in phenotypic terms. These four species curva, and B. ureolyticus) are oxidase positive, suggests that do resemble campylobacters in the following respects: (i) they may actually be microaerophiles. This would make like campylobacters, W. recta and W. curva are motile by their inclusion with the true campylobacters much more means of polar flagella; (ii) W. recta, W. curva, and B. satisfying in phenotypic terms. Some previous reports sug- ureolyticus are oxidase positive; (iii) W. curva has a vibrioid gest that this may be the case. Tanner et al. (19,20) reported shape; and (iv) like some campylobacters (Campylobacter that some strains of W.recta, W. curva, and B. gracilis could concisus, Carnpylobacter mucosalis, C. cinaedi, and C. grow under microaerobic conditions (4%0,). Jackson and fennelliae), W. recta, W. curva, B. gracilis, and B. ureolyti- Goodman (4) showed that B. ureolyticus could grow under cus all require H, or formate as an electron donor. However, microaerobic conditions and that it has cytochrome b and there are some important differences: (i) W. recta, B. ureo- cytochrome c. However, it has not been shown previously lyticus, and B. gracilis are straight rods; (ii) B. ureolyticus that these organisms are capable of 0,-dependent growth. In and B. gracilis are nonmotile; (iii) B. gracilis is oxidase this report, we provide evidence in support of this conten- negative; and (iv) W. recta, W. curva, B. gracilis, and B. tion. ureolyticus have all been considered to be anaerobes. The latter difference seems to be a particularly fundamental difference. MATERIALS AND METHODS The problem of including anaerobes with microaerophiles in a phylogenetic group existed previously in another group, Bacterial strains. The strains used in this study were W. viz., rRNA group I1 of Thompson et al. (23). This group curva ATCC 35224T (T = type strain), W. recta ATCC contains the putative anaerobe Wolinella succinogenes and 3323gT, B. ureolyticus NCTC 10941T, and B. gracilis ATCC the microaerophiles H. pylori, C. cinaedi, and C. fennelliae 33236T. Stock cultures were grown under an atmosphere (14,17, 23). Although W. succinogenes was described as an containing 6% O,, 3% CO,, 23% N,, and 68% H, at 37°C in anaerobe of Bergey ’s Manual of Systematic Bacteriology semisolid brucella medium (brucella broth [Difco] supplemented with 0.15% agar) and transferred weekly. Inocula and general culture conditions. The top 1 cm was removed from a 2-day-old culture grown in semisolid bru- * Corresponding author. cella medium and mixed to yield a homogeneous suspension.

218 VOL.41, 1991 MICROAEROPHILIC BACTERIA 219

One drop (0.05 ml) of this suspension was inoculated into TABLE 1. Growth responses of Wolinellu and Bucteroides each 5-ml portion of test broth. For inoculation of agar species in brucella broth in the presence of different 0, media, one loopful of the suspension was streaked over the concentrations when H, was used as the electron donor" entire surface of a test slant or plate. Turbidity (optical density at 660 nm) For testing growth responses in liquid media, organisms 0, concn (%Ih were cultured in 5-ml volumes of media contained in 50-ml W. curvu W.rectu B. ureolyticus B. gracilis cotton-stoppered serum bottles. For testing growth re- 0 0.07 -+ 0.01' 0.07 -+ 0.01 0.05 -+ 0.01 0.05 L 0.01 sponses on solid media, organisms were cultured on 10-ml 2 0.37 t 0.04 0.21 5 0.01 0.57 2 0.13 0.18 t 0.02 agar slants or in petri plates containing 20 ml of medium. 4 0.44 -+ 0.01 0.30 * 0.01 0.72 -+ 0.07 0.18 t 0.02 Cultures were incubated statically at 37°C in sealed ves- 6 0.46 -+ 0.02 0.31 & O.Md 0.81 t 0.02 0.21 2 0.03 sels (Oxoid anaerobic jar system) equipped with a vent to 8 0.49 -+ 0.04 0.27 t 0.02 0.83 t 0.08 0.20 t 0.03 allow filling with various gas mixtures. Gas atmospheres 10 0.58 & 0.04 0.18 t 0.02 0.86 2 0.02 0.20 * 0.05 were obtained manometrically by evacuating the Oxoid jars 12 0.58 & 0.01 0.02 t 0.01 0.88 A 0.02 0.10 * 0.04 and refilling them with various combinations of CO,, H,, N,, 14 0.56 -+ 0.03 0.01 t 0.01 0.85 t 0.05 0.03 * 0.02 16 0.43 -+ 0.07 0.01 t 0.00 0.56 2 0.01 0.01 5 0.00 and 0,. When anaerobic conditions were required, 0, was 18 0.02 t 0.01 0.01 & 0.00 0.04 2 0.00 0.01 * 0.00 omitted and an activated palladium catalyst was used to 21 0.00 t 0.00 0.00 2 0.00 0.01 2 0.00 0.00 * 0.00 remove residual 0,. Measurement of growth. Growth responses in liquid media "Cultures were grown in 5-ml portions of brucella broth contained in cotton-stoppered SO-ml serum bottles and incubated at 37°C for 48 h. were estimated turbidimetrically at 660 nm with a Bausch & Jars containing the bottles were initially evacuated to various extents to Lomb Spectronic 2000 spectrophotometer by using 1-cm give the desired residual levels of oxygen manometrically. Carbon dioxide cuvettes. Growth responses on solid media were estimated (final concentration, 3%) was added, and the jars were restored to 1 atm by washing the cells from the agar surface with 5 ml of (101.29 kPa) with H,. When 0% 0, was desired (anaerobic conditions), a palladium catalyst was used to remove the residual 0,. When 21% 0, was physiological saline (0.85% NaCl) and measuring the turbid- desired, the jars were evacuated and pure 0, gas was added to give this level ity of the resulting suspension. of oxygen before addition of CO, and H,; this allowed a sufficient level of H2 Oxidase and catalase tests. The oxidase test was performed to be provided for growth of all four species, including W. recta, which by using 1% tetramethyl-p-phenylenediamine dissolved in requires at least 30% H,. Values are the averages L standard deviations for three or four replicate dimethyl sulfoxide (22). Growth was removed with a plati- cultures. num wire loop from cultures grown on brucella agar slants Values in boldface type represent maximum growth responses. for 48 to 72 h. When the growth was smeared onto filter paper moistened with the test reagent, the development of a purple color within 10 s was considered positive. For the was also inhibitory, the percentage of inhibition of oxygen catalase test, 1 ml of a freshly prepared 3% H,O, solution uptake by rotenone was determined as follows: [l - (oxygen was added to a 24- to 48-h-old culture grown in semisolid uptake rate in the presence of rotenone plus DMFA)/(oxygen brucella medium. The subsequent generation of bubbles was uptake rate in the presence of DMFA alone)] X 100. considered a positive reaction (18). Oxygen uptake rates. Cells were grown in a biphasic culture system (50 ml of brucella broth overlaid onto 200 ml RESULTS of brucella agar [agar concentration, 2.5%]) at 37°C for 48 h Oxygen-dependent growth. W. curva, W. recta, B. ureo- in an atmosphere containing 6% O,, 3% CO,, 23% N,, and lyticus, and B. gracilis showed the typical characteristics of 68% H,. Cells were harvested by centrifugation and sus- microaerophilic growth in unsupplemented brucella broth pended in 40 mM Tris-HC1 buffer (pH 7.0). Oxygen con- (Table 1) and on unsupplemented brucella agar slants when sumption was measured with an oxygen electrode (model 53 H, was used as the electron donor (Table 2). They exhibited oxygen monitor; Yellow Springs Instrument Co.) inserted only very slight growth under anaerobic conditions and no into a water-jacketed Clark cell type chamber (Gilson Med- growth under 21% 0,. In unsupplemented brucella broth, ical Electronics) maintained at 37°C with a circulating water the optimum levels of 0, for growth were 10 to 14% for W. bath. The system was calibrated for dissolved 0, concentra- curva, 4 to 8% for W. recta, 10 to 14% for B. ureolyticus, and tions by using the method of Robinson and Cooper (16). 4 to 8% for B. gracilis. On brucella agar slants the optimum Oxygen uptake rates were linear with time and were ex- 0, levels for growth were lower than the optimum 0, levels pressed in nanomoles of oxygen consumed per minute per for growth in brucella broth (6 to 8% for W. curva, 2% for W. milligram of protein. Protein concentrations were deter- recta, 6 to 8% for B. ureolyticus, and 2% for B. gracilis on mined by the method of Bradford (1). brucella agar slants) (Table 2). Similar optimum 0, levels To measure the effects of cyanide, rotenone, and 2-heptyl- were found for growth on brucella agar plates (data not 4-hydroxyquinoline N-oxide (HOQNO) on oxygen uptake, shown). In these experiments, the 11% H, that was provided each inhibitor was added initially to a cell suspension in as an electron source was usually sufficient to support the absence of an electron donor. After steady-state oxygen growth; however, W. recta required at least 30% H, for consumption was maintained for 2 min, sodium formate growth (Table 3). (final concentration, 1 mM) was added to the cell suspen- The addition of catalase to brucella broth resulted in sion. Formate was also added to control cell suspensions stimulation of growth of all species under microaerobic which lacked the inhibitor. In the case of KCN and conditions (Table 4). HOQNO, the percentage of inhibition of oxygen uptake was When brucella broth was supplemented with 50 mM determined as follows: [l - (oxygen uptake rate in the formate as an electron donor in the absence of H,, all four presence of inhibitor/oxygen uptake rate in the absence of species grew under microaerobic conditions (4% 0,, 3% inhibitor)] x 100. The KCN and HOQNO stock solutions CO,, 93% N,). Turbidity values and optimum 0, levels at 48 were prepared in 40 mM Tris-HC1 (pH 7.0) and 0.01 N KOH, h were 0.23 and 12% for W. curva, 0.31 and 14% for W. respectively, while the stock solution of rotenone was pre- recta, 0.45 and 18% for B. ureolyticus, and 0.10 and 2% for pared in N,N-dimethylformamide (DMFA). Because DMFA B. gracilis. No growth (turbidity, 10.02) occurred in unsup- 220 HAN ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Microaerophilic growth of Wolinella and Bacteroides TABLE 4. Effect of catalase on the growth of Wolinella species on brucella agar slants in the presence of various 0, and Bacteroides speciesa concentrations when H, was used as the electron donora Turbidity (optical density at 660 nm) Culture Turbidity (optical density at 660 nm) ;:::- 0, concn age (h) addedb W. curva W. recta B. ureolyticus B. gracilis (%Ib W. curva W. recta B. ureolyticus B. gracilis 0 No 0.01 t 0.00" 0.01 t 0.00 0.01 f 0.00 0.00 k 0.00 0 0.06 * 0.01' 0.05 f 0.02 0.05 -f- 0.02 0.06 t 0.01 6 Yes 0.17 t 0.01 0.16 t 0.01 0.21 t 0.01 0.17 2 0.02 2 0.83 f 0.03 0.38 2 0.12d 1.10 t 0.04 0.87 f 0.09 No 0.04 t 0.00 0.04 f 0.01 0.04 t 0.00 0.01 t 0.00 4 1.65 f 0.06 0.27 t 0.10 1.70 + 0.06 0.05 t 0.04 12 Yes 0.26 f 0.02 0.27 t 0.01 0.26 k 0.01 0.18 t 0.04 6 1.84 f 0.10 0.07 t 0.03 1.71 2 0.07 0.05 f 0.02 No 0.13 * 0.00 0.11 t 0.00 0.16 +- 0.00 0.04 t 0.00 8 1.84 f 0.03 0.06 t 0.01 1.66 f. 0.16 0.02 t 0.00 18 Yes 0.31 f 0.03 0.32 * 0.01 0.33 2 0.03 0.19 t 0.00 10 0.38 f 0.06 0.04 t 0.00 1.13 t 0.02 0.02 f 0.00 No 0.18 t 0.04 0.14 t 0.02 0.24 t 0.00 0.09 +_ 0.01 12 0.15 t 0.05 0.02 t 0.00 0.47 k 0.21 0.02 2 0.01 24 Yes 0.33 t 0.05 0.34 * 0.03 0.42 t 0.04 0.20 2 0.02 14 0.07 t 0.01 ND' 0.28 ? 0.02 ND No 0.27 L 0.04 0.22 t 0.04 0.39 t 0.05 0.12 2 0.01 16 0.05 t 0.01 ND 0.17 ? 0.02 ND 48 Yes 0.52 f 0.08 0.49 t 0.05 0.94 2 0.04 0.27 2 0.04 18 0.02 t 0.00 ND 0.04 k 0.00 ND No 0.42 t 0.04 0.29 t 0.02 0.73 t 0.08 0.18 +- 0.01 21 0.01 f 0.00 0.01 t 0.00 0.01 f 0.00 0.01 If: 0.00 72 Yes 0.73 f 0.03 0.56 t 0.03 0.97 t 0.06 0.33 t 0.02 No 0.67 2 0.04 0.44 2 0.06 0.93 k 0.11 0.24 t 0.02 a Cultures were grown on brucella agar slants with loosened screw caps and t f k incubated at 37°C for 48 h. 96 Yes 0.67 * 0.02 0.57 0.03 1.03 0.02 0.33 0.03 See Table 1, footnote b. No 0.56 t 0.01 0.40 f 0.04 0.93 * 0.09 0.26 k 0.02 The values are the turbidities of cell suspensions that were obtained by Cells were grown in 5-ml volumes of brucella broth in 5-ml cotton- washing growth from slants with 5 ml of saline. The values are the averages 5 standard deviations for three or four replicate cultures. stoppered serum bottles. The jar containing the stoppered bottles contained 78% H,. Values in boldface type represent maximum growth responses. an atmosphere of 4% 0,, 3% CO,, 15% N,, and ND, Not determined. Filter-sterilized catalase was added to give a final concentration of 2,000 U/ml. After 4 days, the enzyme was still active, as indicated by bubbling when a 3% H20, solution was added. See Table 1, footnote c. plemented brucella broth. Acetate, L-lactate, pyruvate, a-ketoglutarate, and L-malate (all at a final concentration of 50 mM, pH 7.0) failed to support growth of any species. conditions. Nitrite was readily detected in cultures incu- Succinate (50 mM) supported slight growth of B. ureolyticus bated in an atmosphere containing 6% O,, 3% CO,, 23% N,, (turbidity, 0.05) but not the other species. and 68% H, for 7 days in semisolid brucella medium con- Under anaerobic conditions (3% CO,, 97% H2) and in the taining 100 mM KNO,. However, the specific activity of the absence of any added terminal acceptor, the four species nitrate reductase of W. recta has not been determined.) exhibited only scant growth in brucella broth (turbidity Sodium nitrite (5 mM) supported slight growth of all species values at 48 h ranged from 0.04 to 0.09). Supplementation of except W. recta (turbidity values ranged from 0.09 to 0.12). the medium with fumarate, L-aspartate, L-asparagine, or Higher concentrations of sodium nitrite (10 to 50 mM) L-malate (final concentration, 50 mM; pH 7.0) resulted in resulted in decreased growth responses or complete inhibi- good growth of the four species (turbidity values at 48 h tion of growth. ranged from 0.21 to 0.83). Potassium nitrate (50 mM) sup- Oxygen uptake. Cell suspensions of all of the species ported growth of W. curvu, B. gracilis, and B. ureolyticus exhibited 0, uptake when either H, (0.11 mM), formate (I (turbidity values ranged from 0.16 to 1.02) but not of W. mM), or succinate (10 mM) was provided as the electron recta (turbidity, 0.04). (The failure of the latter organism to donor (Table 5). The rates of 0, uptake were highest with grow with nitrate is interesting because this organism ap- formate. Unlike the other species, W. recta exhibited very pears to have a nitrate reductase, at least under microaerobic low 0, uptake rates when H, was used as the electron donor. Oxygen uptake by all four species was inhibited by KCN, an inhibitor of the terminal oxidase of 0,-utilizing respira- A TABLE 3. Growth responses of Wolinella and Bacteroides tory chains (Table 6). KCN concentration of 1 mM species in the presence of various H, concentrations in a 4% 0, atmospherea TABLE 5. Rates of oxygen uptake by Wolinella and Bacteroides H, concn Turbidity (optical density at 660 nm) species with various electron donorsa (%Ib W. curva W. recta B. ureolyticus B. gracilis Electron Concn Rate of oxygen uptake 0 0.03 t 0.01" 0.02 t 0.00 0.03 2 0.00 0.02 t 0.00 donor (mM) W. curva W. recta B. ureolyticus B. gracilis 2.5 0.33 t 0.04 0.02 t 0.00 0.32 t 0.06 0.04 t 0.02 5 0.42 2 0.02 0.02 t 0.00 0.61 +- 0.20 0.12 t 0.01 Formate 1 432 t 8' 639 2 76 360 t 1 206 t 6 10 0.44 t 0.03 0.02 f 0.00 0.69 k 0.15 0.21 2 0.02 H2 0.11 390 f 46 14 t 1 87 2 4 151 t 6 15 0.41 t 0.03 0.02 t 0.01 0.77 t 0.15 0.24 k 0.02 Lactate 1 Of0 020 020 oto 20 0.42 t 0.04 0.02 f 0.01 0.71 k 0.21 0.24 k 0.03 Malate 1 Of0 oto 050 oto 30 0.41 t 0.04 0.23 t 0.01 0.67 k 0.08 0.22 t 0.01 Succinate 1 Of0 OtO 020 oto 40 0.46 * 0.01 0.22 -f- 0.01 1.17 t 0.04 0.19 t 0.00 10 821 4t0 2021 16t4 50 0.47 t 0.00 0.23 f 0.01 1.17 t 0.06 0.20 t 0.00 NADH 1 Of0 oto o+o 020 20 166 t 11 167 t 42 138 2 24 215 t 23 See Table 1, footnote a. ~~~ ~~ ___ ~ Initially, jars containing the bottles were partially evacuated to give 4% " The cells were grown on brucella medium in a biphasic system at 37°C for residual oxygen. Carbon dioxide (final concentration, 3%) and various con- 48 h under an atmosphere containing 6% O,, 3% CO,, 23% NZ,and 68% H,. centrations of H, were added, and the jars were restored to 1 atm (101.29 kPa) ' Values are expressed as nanomoles of 0, taken up per minute per with N,. milligram of protein. The values are the averages ? standard deviations for L' See Table 1, footnote c. three replicates. VOL.41, 1991 MICROAEROPHILIC BACTERIA 221

TABLE 6. Inhibition of oxygen uptake by KCN, rotenone, and W. curva, W. recta, B. ureolyticus, and B. gracilis are 0.75, HOQNO when 1 mM potassium formate was used 1.66, 2.06, and 2.04, respectively. Proton translocation is as the electron donor" inhibited by the protonophore carbonylcyanide rn-chloro- % Inhibition of oxygen uptake ratec phenylhydrazone . Inhibitor6 Under anaerobic conditions, the four species were capable (mM) W. curva W. recta B. ureolytious B. gracilis of using fumarate and (with the exception of W.recta) nitrate KCN 1 75 13 45 53 as terminal electron acceptors. Although aspartate and as- 2 78 19 52 69 paragine seemed to be electron acceptors, it is possible that 5 88 51 69 89 fumarate was the actual acceptor; the organisms may con- 10 97 92 89 95 20 100 100 95 100 vert asparagine to aspartate by means of an asparaginase and 40 NT~ NT 100 NT aspartate to fumarate by means of an aspartase. Indeed, W. Rotenone 20 15 4 7 9 succinogenes has been shown to be capable of supplying HOQNO 1 99 86 81 97 itself with fumarate in this manner (2,7). Malate also seemed to serve as an electron acceptor, but again the organisms a Cells were grown as described in Table 5, footnote a. To determine the level of inhibition of oxygen uptake, the inhibitor was added to the cell might have supplied themselves with fumarate by means of a suspension and then sodium formate (final concentration, 1 mM) was injected. fumarase. Enzymatic activities such as these might explain Stock solutions of KCN, rotenone, and HOQNO were prepared by dissolving the inhibitors in 40 mM Tris-HC1 buffer (pH 7.0), DMFA, and 0.01 why some species exhibited a slight growth response under N KOH, respectively. anaerobic conditions in the absence of any added electron The percentage of inhibition by KCN or HOQNO was determined as acceptor. This slight growth might have been due to the low follows: [l - (oxygen uptake rate in the presence of inhibitodoxygen uptake levels of aspartate and asparagine which are undoubtedly rate in the absence of inhibitor)] x 100. The percentage of inhibition by rotenone was determined as follows: [l - (oxygen uptake rate in the presence present in complex media such as brucella medium. of rotenone plus DMFA)/(oxygen uptake rate in the presence of DMFA The ability of exogenous catalase to enhance the growth of alone)] x 100. all four species might have been due to the destruction of NT, Not tested. H202 produced in the presence of 0,. Preliminary experiments performed in our laboratory have indicated that cell suspensions of all four species do form some H,O, in the resulted in various levels of inhibition (range, 13 to 75%) depending on the species, and a concentration of 10 mM course of their uptake of O,, as indicated by a reduction in resulted in 89 to 95% inhibition. Oxygen uptake was also the rate of 0, uptake in the presence of added catalase. inhibited by HOQNO, a compound used in respirometric Hydrogen peroxide might damage microaerophilic bacteria studies to inhibit electron transport between cytochrome b by acting on target proteins associated with the outer mem- and cytochrome c. Rotenone, which is used in respirometric brane, redox-active components of the cytoplasmic mem- studies to inhibit electron transport by flavoproteins between brane, or enzymes in the periplasmic space (9). NAD and coenzyme Q and/or cytochrome b, caused only Although W. curva, W. recta, B. ureolyticus, and B. slight inhibition of 0, uptake. gracilis exhibited 0, uptake with formate, H,, and succinate as electron donors, W. recta exhibited only a very low 0, DISCUSSION uptake rate with H,. This corresponds to the fact that W. recta required relatively high hydrogen partial pressure for As defined by Krieg and Hoffman (9), microaerophilic growth (>30% H,) (Table 3). organisms exhibit 0,-dependent growth under microaerobic As indicated by Krieg (8), fundamental questions about conditions, do not grow under anaerobic conditions unless phylogeny versus practicality have arisen in bacterial taxon- suitable alternative electron acceptors are provided, fail to omy because many groups that have been formed on the grow or grow only poorly at the level of 0, present in air basis of results of rRNA analyses have not been easily (21% O,), and are able to utilize 0, as a terminal electron definable in terms of phenotypic similarities. The resulting acceptor. The four species which we studied conform to difficulties could lead to the unattractive prospect of having these criteria. They grew when they were provided with low two separate classification systems, a practical one based on levels of O,, exhibited no growth or only slight growth on phenotypic characteristics and a more esoteric one based on brucella medium under anaerobic conditions in the absence phylogeny. Yet at least some of these difficulties may be due of electron acceptors such as fumarate, and failed to grow to the fact that organisms which have been found to be under 21% 0,. Their ability to use 0, as a terminal electron related to one another by rRNA analysis may not have been acceptor was shown by the fact that their uptake of 0, was characterized phenotypically by comparable methods or inhibited by KCN, by HOQNO, and to a slight extent, by tests. Our discovery that W.recta, W.curva, B. ureolyticus, rotenone. and B. gracilis are microaerophiles instead of anaerobes There is additional evidence (3a) of the microaerophilic nature of these organisms. (i) Dithionite-reduced minus supports the inclusion of these organisms with the true air-oxidized difference spectra indicate that the four species campylobacters. However, there are some differences in morphology, motility, and oxidase reaction among members possess cytochrome b560, cytochrome ~551-553, and CO- binding cytochrome c in membrane fractions and soluble of rRNA group I that continue to make it difficult to define cytochrome ~552and CO-binding cytochrome c in soluble this group in phenotypic terms. It is likely that, for practical fractions; in whole-cell suspensions, these cytochromes be- reasons, the group may have to be divided into two or more come reduced when either H, or formate is provided as the genera to reflect practical considerations. Such a division electron donor. (ii) With either H, or formate as the electron has already been done in the case of H. pylori, which, donor, proton effluxfrom anaerobic cells has been shown to despite its relatedness to W. succinogenes as established by occur upon addition of a pulse of oxygen; for instance, with rRNA analyses (14, 15, 23), was separated from it at the formate as the electron donor, the H+/Oratios obtained with genus level largely on the basis of phenotypic differences (3). 222 HAN ET AL. INT. J. SYST.BACTERIOL.

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