Cupriavidus Necator Gen

INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1987, p. 323-326 Vol. 37, No. 4 0020-77 13/87/04323-04$02.OO/O Copyright 0 1987, International Union of Microbiological Societies

Cupriavidus necator gen. nov., sp. nov.: a Nonobligate Bacterial Predator of Bacteria in Soil?

N. S. MAKKAR AND L. E. CASIPA, JR." Microbiology Program, The Pennsylvania State University, University Park, Pennsylvania I6802

A nonobligate, bacterial predator of bacteria in soil is described. It was previously designated as strain N-1. It is a gram-negative, aerobic, mesophilic, short rod that multiplies by binary fission. It is motile and has peritrichous flagella. It has simple nutritive requirements when not acting as a predator. Glucose is not utilized as a carbon source. Instead, it uses fructose or any of several amino acids. As a predator, it attacks both gram-positive and gram-negative bacteria, including certain other nonobligate bacterial predators of bacteria in soil. It is highly resistant to copper, and its growth initiation is strongly stimulated by copper. Copper has no effect on the cellular multiplication that occurs after growth has initiated. A new genus, Cupriavidus, is proposed for this bacterium. The type species is Cupriavidus necator sp. nov., and strain N-1 (ATCC 43291) is the type strain of this species.

Strain N-1 is a gram-negative, rod-shaped bacterium oc- copper. Based on this information, and on the predation curring naturally in soil. It is a nonobligate bacterial predator characteristics of N-1, we propose that it be designated as a of various other gram-positive and gram-negative bacteria in member of a new species and genus. soil (1, 10, 12). Nonpredacious mutants have not been encountered. It was isolated from soil by the technique of MATERIALS AND METHODS Sillman and Casida (10). This technique also showed that strain N-1 attaches to prey cells. N-1 does not require prey Strain N-1 was maintained on 0.1-strength heart infusion cells for its growth. If the level of available nutrients in soil (HI) (Difco Laboratories, Detroit, Mich.) agar. Unless oth- decreases, however, it will attack other bacteria. This attack erwise stated, the incubation temperature for all trials was can be demonstrated by use of the indirect bacteriophage 27°C. Media were solidified by adding 1.5% agar (Difco). A analysis technique performed directly on soil (1, 4, 12). For synthetic medium was devised that contained 0.1% KH2P04, example, in soil, N-1 was shown to attack Agromyces 0.02% Na2S04, 0.02% NaCl, 0.1% NH4CI, and a suitable ramosus, Arthrohacter globiformis, Azotohacter vinelandii, carbon source (pH 7.0). MgS04 . 7H20at 0.1% was added if Bacillus subtilis, Bacillus thuringiensis, Ens$er adhaerens, the medium was to be used for growth of Alcaligenes Escherichia coli, Micrococcus luteus, Staphylococcus au- faecalis. The blood agar used to examine hemolysis was reus, and two Streptomyces species. There may be other Trypticase soy agar (blood agar base; BBL Microbiology prey among those species that were not tested. Note that A. Systems, Cockeysville, Md.) containing 5% defibrinated ramosus, E. adhaerens, and the two Streptomyces species sheep blood. Oxidative versus fermentative metabolism was are themselves nonobligate predators of other bacteria. tested by the method of Hugh and Leifson (6) using our Examples of bacteria not attacked by N-1 in soil (12) are synthetic medium containing 1.0% fructose as carbon Agrobacterium tumefaciens, Nocardia salmonicolor, Sal- source. The Hugh and Leifson medium was not used be- monella typhosa, and the gram-negative soil bacterial pred- cause of interference by the alkaline reaction that resulted ator isolates B, 5, L-2, and M-8. from peptone degradation. Growth under anaerobic condi- A. ramosus and strain N-1 are both powerful predators of tions was determined in a GasPak anaerobic jar (BBL other bacteria and can attack each other. This has been Microbiology Systems). The medium was nutrient agar or described as an attack-counter attack phenomenon (1). the synthetic medium containing 0.1% L-glutamic acid. An- Based on a chemical signal (a magnesium-related growth aerobic growth was also evaluated by using tubes of thiogly- initiation factor) elaborated by strain N-1, A. rumosus pro- colate medium (Difco). Burk's broth (normally used for duces a mycelium that approaches, makes contact with, and Azotobacter spp.) was modified to contain 0.064% K2HP04, then lyses a portion of the N-1 cells. The surviving N-1 cells, 0.016% KH2P04, 0.02% MgS04 * 7H20, 0.005% however, then lyse the A. ramosus mycelium, but not the CaS04 . 2H20, 0.02% NaCI, 0.00025% NaMo04 . 2H20, rod-form cells that manage to fragment from the mycelium 0.00018% ferric citrate, and 1.0% fructose (pH 7.2). before and during the N-1 attack. This lysis of the A. The catalase test was performed by adding 1 drop of 3% ramosus mycelium is caused by a second growth initiation H202to the surface of a colony. For the oxidase test, growth factor (GIF) produced by N-1 (2). This GIF chelates copper from the surface of an agar medium was smeared on a piece and is very toxic to A. ramosus. of filter paper that had been moistened with a freshly In the present paper, we describe the morphological, prepared 1.O% solution of tetramethyl-p-phenylenediamine biochemical, respiratory, and nutritional characteristics of dihydrochloride (Sigma Chemical Co., St. Louis, Mo.). strain N-1. We also describe the interaction of N-1 with Development of a violet or purple color within 10 s was considered a positive test. Nitrate reduction was determined fQr a stationary culture grown 48 h in nutrient broth contain- * Corresponding author. ing 0.1% NaN03; nitrite was detected by the method de- t Paper no. 7625 in the journal series of the Pennsylvania Agri- scribed in the Manual of Microbiological Methods (11).The culture Experiment Station. litmus milk reaction was evaluated by using the medium

323 324 MAKKAR AND CASIDA INT. J. SYST.BACTERIOL. described in the Manual of Microbiological Methods (11). Indole production was tested by using Kovac reagent on a culture grown in nutrient broth. Filter paper strips impreg- nated with lead acetate were used to detect H2S production for cultures grown in nutrient broth for up to 7 days. Starch hydrolysis was determined with iodine solution for cultures grown 5 days on nutrient agar supplemented with 1.0% soluble starch (Difco). Christensen urea agar (3) was modified by replacing glucose with 1% fructose to test for urea hydrolysis. Hydrolysis of gelatin was tested on 12% (wthol) gelatin in distilled water. The method of Gordon et al. (5) was modified by adding agar to solidify the broth for esculin degradation. Degradation of tyrosine was examined on nutrient agar supplemented with 0.5% tyrosine. The medium used for hippurate hydrolysis contained 0.5% NaCI, 0.02% MgS04 . 7H20, 0.1% NH4H2P04,0.1% K2P04,0.3% sodium hippurate, and 0.001% phenol red (pH 7.0). Hydrolysis of the hippurate was indicated by growth and a pink color due to alkali production. A medium containing 0.5% peptone, 0.3% FIG. 1. Flagellation of strain N-1. Bar, 1.0 km. yeast extract, 1.0% tributyrin, and 1.5% agar indicated tributyrin degradation by clear zones around the growth. The medium described by Sierra (9) was supplemented with source, N-1 grew aerobically. Only scant growth was ob- 1.0% Tween 80 or Tween 20 to evaluate hydrolysis of these served anaerobically (in anaerobe jars). On thioglycolate compounds. Hydrolysis was indicated by the presence of a medium, N-1 grew in the oxidized region. As a control for zone of opacity due to precipitation of calcium soaps around this observation, A. faecalis grew in the oxidized zone, the growth. Deoxyribonuclease (DNase) activity was deter- whereas Escherichia coli, a known facultative anaerobe, mined on DNase test agar (Difco); the unhydrolyzed deoxy- grew throughout the medium. The metabolism of N-1 was ribonucleic acid (DNA) was precipitated with 1 N HCI. oxidative, based on the Hugh-Leifson test performed in Nutrient agar with various concentrations of NaCl was synthetic medium containing fructose. N-1 grew and pro- employed to test for NaCl tolerance. Growth of N-1 at duced acid (pH 5.3) without gas in the top portion of the various pH values and temperatures was tested on nutrient medium in the aerobic tubes. No growth or acid was agar. Carbon utilization tests were done on synthetic me- observed in the anaerobic tubes. dium agar to which a filter-sterilized solution of a carbon Biochemical characteristics. Strain N-1 was catalase posi- source was added to give a final concentration of 0.1%. tive and oxidase positive. It did not show hemolysis on blood Simmons citrate agar (Difco) was used for citrate utilization. agar. Esculin, gelatin, starch, and urea were not hydrolyzed. Motility was determined by phase-contrast microscopy. DNase activity was negative. Hippurate, tributyrin, Tween Flagellar arrangement was observed by electron microscopy 80, and Tween 20 were hydrolyzed. Tyrosine was degraded, (Phillips EM-300) of 14- to 18-h cultures negatively stained Indole and H2S were not produced. The litmus milk reaction with 0.25% phosphotungstic acid (pH 6.5 with KOH). was basic with reduction of the litmus. Acetate, L-aspartate, citrate, fructose, fumarate, gluconate, L-glutamate, glutar- RESULTS ate, P-hydroxybutyrate, lactate, L-leucine, oxalacetate, and succinate were utilized as the carbon source for growth. Morphology. Strain N-1 cells were gram-negative, short Utilization of sucrose was equivocal. Acetate, L-glutamate, rods measuring 0.7 to 0.9 pm by 0.9 to 1.3 pm. The L-leucine, and fructose were used after a short delay (13 to measurements were based on electron micrographs of neg- 25 h); the delays for L-alanine and L-valine were, respec- atively stained cells. N-1 was motile by 2 to 10 peritrichous tively, 61 and 92 h. Carbon sources that were not utilized flagella (Fig. 1). Division was by binary fission. In older were arabinose, adonitol, benzoate, glucose, glycerol, lac- cultures, or when the cells were placed in contact with soil in tose, L-lysine, mannitol, mannose, melibiose, L-methionine, soil column slides (l), the rods decreased somewhat in size rhamnose, and xylose. and became rounded. They elongated only when they started Nutrition, pH, and temperature. Strain N-1 grew well on to multiply. In soil, these rounded forms appeared to be fructose or acetate broth media with NH4C1 as the only dormant (1). N-1 was desiccation resistant based on drying nitrogen source. Excellept growth was obtained by using of the cells in the absence of soil on cover slips over Drierite N-1 synthetic medium broth with L-glutamic acid as the desiccant. carbon and nitrogen source. The optimum temperature for After 2 days of incubation at 27"C, colonies on nutrient growth was 27°C; growth was good at 37°C. Growth at 15°C agar were off-white, glistening, mucoid, smooth, and convex occurred after a delay; there was no growth at 55°C. Growth with an entire edge. They were 2 to 4 mm in diameter. After occurred over a pH range of 5.5 to 9.2. The optimum pH was many transfers in the laboratory, N-1 produced small num- 7.0 to 8.0. N-1 did not grow on media containing 3% NaCl. bers of a nonmucoid variant whose colonies were slightly Growth was poor at 2% NaCI, but good at 1%NaCI. Growth smaller. They also were more flat and did not glisten. A did not occur in Burk's nitrogen-free medium with glucose or bacteriophage that lysed the mucoid form of N-1 did not lyse sucrose as the carbon source. Limited growth did occur, the nonmucoid variant. N-1 and the variant, however, were however, with fructose as the carbon source, but further similar in all other respects. transfers from this to fresh, similar medium did not yield Oxygen requirement. On nutrient agar, or on synthetic growth. medium agar containing L-glutamic acid as the carbon G+C content. The guanine-plus-cytosine (G +C) content VOL. 37, 1987 CUPRIAVIDUS NECATOR GEN. NOV., SP. NOV. 325

affecting its growth rate. Based on the above, we propose that a new genus, Cupriavidus, be established for strain N-1. The characteristics of the genus are as follows. Description of Cupriavidus gen. nov. Cupriavidus (L. n. cuprum, copper; L. adj. avidus, eager for, loving; M. L. neut. n. Cupriavidus, lover of copper). Coccoid rods, 0.7 to 0.9 km by 0.9 to 1.3 km. Gram negative. Motile by 2 to 10 peritrichous flagella. Chemoheterotrophic. Organic nitrogen source not required. Glucose not utilized. Oxidative metabolism with fructose. Oxidase positive. Catalase positive. Nitrate reduced. Gelatin, starch, and urea not hydrolyzed. Indole and H2S not produced. Utilizes several amino acids, but not ~-1ysineor L-methionine, as the only source of carbon and nitrogen for growth. Optimum temperature is 27°C. Optimum pH is 7.0 to 8.0. NaCl at 3% inhibits growth. Resistant to copper. Growth initiation is stimulated by copper. Colonies on nutrient agar after 2 days at 27°C are off- white, glistening, mucoid, smooth, and convex with an entire edge; 2 to 4 mm diameter. Isolated from soil. Nonobligate predator causing lysis in soil of various gram- positive and gram-negative bacteria. Can lyse certain other nonobligate bacterial predators. Growth does not require presence of prey species. - I --- Description of Cupriavidus necator sp. nov. Cupriavidus necator (L.n. necator, slayer). Description as for the genus. of I 210 I 4To I $0 I do Id0 I IhO I I40 TIME (HI Type species of the genus Cupriavidus. The type strain of C. necator is strain N-1, a culture of which has been deposited FIG. 2. Growth of strain N-1 in synthetic medium containing 0.1% ~-alanineand various amounts of CuC12 . 2H20. Magnesium with the American Type Culture Collection under the num- was not added to the medium. Copper levels: 0 (W), 400 pM (O),600 ber ATCC 43291. The description of the type strain is the JIM (O), and 800 pM (0). same as that given above for the genus and species. During laboratory cultivation, C. necator may produce small numbers of a nonmucoid variant. The variant resem- of the DNA of N-1 was 57 2 1 mol% (as determined by the bles the mucoid form except for a drier, flatter appearance thermal melting point). and smaller colonies. However, a bacteriophage, isolated Copper and magnesium requirements. N-1 grew in the from soil, that lyses the mucoid form does not lyse the synthetic medium without addition of magnesium (Fig. 2). nonmucoid variant. Also, addition of magnesium did not change the growth rate or time delay before initiation of growth. Taxonomically, 140r N-1 bears some resemblance to Alcaligenes species (see below). A. faecalis, however, did not grow in the above medium unless magnesium was added (Fig. 3). Copper was toxic to the growth of A. faecalis (Fig. 3). In contrast, N-1 was resistant to copper (8), and its growth initiation (Fig. 2) responded directly and in a graded fashion to the amount of copper that was available. The cellular multiplication that followed growth initiation was unaffected by the copper. Presence in soil. Strain N-1 was originally isolated from a soil in the vicinity of University Park, Pa., using the technique of Sillman and Casida (10). Although isolation of N-1 was not attempted, a lytic bacteriophage for N-1 has now been recovered from a garden soil from Oakland, Calif. This predicts the presence of N-1 in this soil. Proposed taxonomy. As noted above, the cells of strain N-1 are aerobic rods. Therefore, N-1 should be included in Section 4 of Bergey 's Manual of Systematic Bacteriology, Vol. 1 (7), which includes gram-negative, aerobic rods and cocci. However, strain N-1 differs significantly from all of the genera in Section 4. It has several characteristics in common with the genus Alcaligenes, including the lack of utilization of glucose (for most strains of Alcaligenes) and TIME (HI the DNA G+C content. Nevertheless, it can be differenti- FIG. 3. Growth of A. faeculis in synthetic medium containing ated from Alcaligenes spp. on the basis of the predatory 0.1% L-alanine and various amounts of CuCl2 . 2H20. MgS04 . activity and the fructose utilization of N-1. These organisms 7H20 at 0.1% was added to the medium. Copper levels: 0 (01,200 also differ based on the resistance of N-1 to copper and the pM (O), and 400 JIM (A). Growth did not occur at 600 and 800 pM stimulation by copper of the growth initiation of N-1 without copper. 326 MAKKAR AND CASIDA INT. J. SYST.BACTERIOL.

ACKNOWLEDGMENTS Pang. 1974. Nocurdia coeliaca, Nocardia autotrophica, and the Nocardin strain. Int. J. Syst. Bacteriol. 24:54-63. This work was supported by grant DAAG29-85-K-0084 from the 6. Hugh, R., and E. Leifson. 1953. The taxonomic significance of U.S. Army Research Office. fermentative versus oxidative metabolism of carbohydrates by We thank W. D. Taylor for determination of the DNA G+C various gram-negative bacteria. J. Bacteriol. 66:24-26. contents. 7. Krieg, N. R., and J. G. Holt (ed.). 1984. Bergey’s manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore. LITERATURE CITED 8. Makkar, N. S., and L. E. Casida, Jr. 1987. Technique for 1. Byrd, J. J., L. R. Zeph, and L. E. Casida, Jr. 1985. Bacterial estimating low numbers of a bacterial species strain(s) in soil. control of Agromyces ramosus in soil. Can. J. Microbiol. Appl. Environ. Microbiol. 53:887-888. 31: 1157-1163. 9. Sierra, G. 1957. A simple method for the detection of lipolytic 2. Casida, L. E., Jr. 1987. Relation to copper of N-1, a nonobligate activity of microorganisms and some observations on the influ- bacterial predator. Appl. Environ. Microbiol. 53:1515-1518. ence of the contact between cells and fatty substances. Antonie 3. Christensen, W. B. 1946. Urea decomposition as a means of van Leeuwenhoek J. Microbiol. Serol. 23:15-22. differentiating Proteus and paracolon cultures from each other 10. Sillrnan, C. E., and L. E. Casida, Jr. 1986. Isolation of non- and from Salmonella and Shigella types. J. Bacteriol. 52: obligate bacterial predators of bacteria from soil. Can. J. Micro- 461466. biol. 32:760-762. 4. Germida, J. J., and L. E. Casida, Jr. 1983. Ensifer adhaerens 11. Society of American Bacteriologists. 1957. Manual of microbio- predatory activity against other bacteria in soil, as monitored by logical methods. McGraw-Hill, New York. indirect phage analysis. Appl. Environ. Microbiol. 451380- 12. Zeph, L. R., and L. E. Casida, Jr. 1986. Gram-negative versus 1388. gram-positive (actinomycete) bacterial predators of bacteria in 5. Gordon, R. E., D. A. Barnett, J. E. Handerhan, and C. Hor-Nay soil. Appl. Environ. Microbiol. 522319-823.