INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1988, p. 124-127 Vol. 38, No. 1 0020-7713/88/010124-04$02 .OO/O Copyright 0 1988, International Union of Microbiological Societies

Three New Methylobacterium Species: M. rhodesianum sp. nov. M. zatmanii sp. nov., and M. fujisawaense sp. nov. P. N. GREEN,l* I. J. BOUSFIELD,l AND D. HOOD2 National Collections of Industrial and Marine Bacteria Ltd., Torry Research Station, Aberdeen AB9 8DG, Scotland, and Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England2

Three new Methylobacterium species (M. zatmanii, M. rhodesianum, and M. fujisawaense) are proposed on the basis of deoxyribonucleic acid-deoxyribonucleic acid homology. Strains are assigned to these and other species of Methylobacterium on the basis of both the homology data and previously published phenotypic data.

Recent deoxyribonucleic acid (DNA)-DNA homology NCIB 12249T (Pseudomonas strain 1 [16]), which was iso- studies on the genus Methylobacterium have shown that lated from a fermentor operating with formaldehyde as a sole there are at least five distinct homology groups (lA, lB, lC, carbon source, as the type strain. As a result of recent work, 2B, and 2D) (9) within the genus that should be assigned including DNA-DNA homology studies (9), we also propose separate species status. Two of these groups correspond to that the following strains be considered as strains of M. the species M. radiotolerans and M. extorquens, but the rhodesianum: Pseudomonas sp. strains NCIB 10598 and others cannot be equated with any currently described 10611, strain M159-1 (13), Rhodes-Roberts strain CS51, and species of Methylobacterium. The type strains of M. orga- NCIB 12440 “Corynebacterium rubrum.” (It should be r,iophilum, M. rhodinurn, and M. mesophilicum, the only noted that the National Collection of Industrial Bacteria other species recognized at present, were shown to be strain of “C. rubrum” is different from the strain deposited distinct from each other and from the five homology groups. in the DSM as DSM 1857). Other strains assigned to this Thus, we consider the three anonymous homology groups to species solely on the basis of phenotypic data are listed in represent as yet undescribed species of Methylobacterium. Table 1. The present paper presents formal proposals for and descrip- Methylobacterium zatmanii sp. nov. (zat.man’i.i M. L. m. tions of these new species. noun zatmanii, named after the British biochemist L. J. Strains assigned to the eight species of Methylobacterium Zatman for his work on the bacterial metabolism of one- on the basis of both homology and phenotypic data are listed carbon compounds.) Mode of respiration, colony and cell in Table 1. Table 2 gives the phenotypic features that morphology, flagellar arrangement, Gram reaction, and ox- differentiate among these species and among other probable idase and catalase as previously described (6). centers of variation within the genus. Grows at 20 and 37°C but not at 10°C. Growth factors are Descriptions of the three new species are given below not required. Casein, cellulose, gelatin, lecithin, Tweens, (methodologies as previously described [S]). and DNA are not degraded. Urease is produced, but p- Methylobacterium rhodesianum sp. nov. (rhbd’es.i.an.um galactosidase, indol, and H2S are not. The methyl red and Id. L. neut. adj. rhodesianum named after the British Voges-Proskauer tests are negative. Nitrate is not reduced, taxonomist Muriel Rhodes-Roberts for her contributions to Susceptible to kanamycin, tetracyclines, novobiocin, pseudomonad taxonomy). Mode of respiration, colony and neomycin, gentamycin, albamycin T, streptomycin, cepha- cell morphology, flagellar arrangement, Gram reaction, and lothin, framycetin, and cephaloridine but resistant to nali- oxidase and catalase as previously described (6). dixic acid, oleandomycin, spiramycin, polymyxin B, colistin Grows at 15 and 30°C but not at 10°C. Most strains will sulfate, penicillin G, and bacitracin. also grow at 37°C. Growth factors are not required. Casein, The following compounds serve as sole carbon sources: cellulose, gelatin, lecithin, and DNA are not degraded. fructose, glycerol, malonate, succinate, fumarate, 2-oxoglu- Urease is produced, but P-galactosidase, indol, H,S, L- tarate, lactate, malate; acetate, pyruvate, ethanol, propylene ornithine and L-1 ysine decarboxylases, and L-arginine dihy- glycol, methanol, monomethylamine, ethylamine, and for- tlrolase are not. The methyl red and Voges-Proskauer tests mate. Some strains utilize tartrate and trimethylamine. Does are negative. Some strains reduce nitrate to nitrite. not utilize monosaccharides other than fructose, citrate, Sensitive to kanamycin, tetracyclines, gentamicin, al- sebacate, aspartate, glutamate, or betaine. hamycin T, streptomycin, and framycetin but resistant to The guanine-plus-cytosine content of the DNA ranges tx-ythromycin, nalidixic acid, oleandomycin, spiramycin, from 69.4 to 70.3 mol% (9); for the type strain, this value is polymyxin B, colistin sulfate, penicillin G, and bacitracin. 70.3 mol%. The following compounds serve as sole carbon sources: We propose NCIB 12243T (Pseudomonas strain 135 [16]), fructose, glycerol, malonate, succinate, fumarate, 2-oxoglu- which was isolated from a fermentor operating with formal- tarate, lactate, malate, acetate, pyruvate, ethanol, propylene dehyde as a sole carbon source, as the type strain. As a glycol, methanol , monomethy lamine, ethylamine, formate, result of recent work, including DNA-DNA homology stud- and betaine. Does not utilize monosaccharides other than ies (9), we also propose that the following strains be consid- fructose, citrate, sebacate, tartrate, or trimethylamine. ered as strains of M. zatmanii: Pseudornonas spp. strains The guanine-plus-cytosine content of the DNA ranges NCIB 10604,10609, and 10610. Other strains assigned to this from 69.8 to 71.2 moles percent (mol%) (9). We propose species solely on the basis of phenotypic data are listed in Table 1. Methylobacterium fujisawaense sp. nov. (fu.ji.sa’wa.en.si? * Corresponding author N. L. neut. adj. fujisawaense coming from the Fujisawa

124 VOL.38, 1988 NOTES 125

TABLE 1. Strains assigned to Methylobacterium species Species Strain or collection no. Reference M. organophilum XX (NCIB 1127gT)" Patt et al. (14) M. extorquens NCIB 9399Tu Janota-Bassalik and Pedyk (11) Pseudomonas AM1 (NCIB 9133)" Peel and Quayle (15) Pseudomonas M27 (NCIB 9686) Anthony and Zatman (1) "Protominobacter ruber" (NCIB 2879)" Den Dooren de Jong (3) 453, 456, 465, 530, 539," 691, 724, 789, N-3, N-4, N-5, Kouno and Ozaki (13) N-10, N-12" 82A,b NCIB 10409 190B,' AM1 var. 471 "Pseudomonas methylica" strain 2 Kirikova (12) B020,d B026 111,' 270 19;f28, 50 M. rhodinum NCIB 942lTU Heumann (8) M. radiotolerans 0-1 (NCIB 10815T)" Ito and Iizuka (10) Pseudomonas spp. (NCIB 9142" and 9143) Hayward (7) 434," 532, 0-20 Kouno and Ozaki (13) M. mesophilicum A47 (NCIB 11561T)" Austin and Goodfellow (2) M. rhodesianum sp. nov. Pseudomonas strain 1 (NCIB 12249T)" Rock et al. (16) Pseudomonas spp. (NCIB 10597'-10602, 10611") "Corynebacterium rubrum"" Graf and Bauer (4) CS51"*b 82, 83, 711, M159-1," Kouno and Ozaki (13) B023,d B040,d 381B' "Protaminobacter ruber" Sat0 et al. (17) "Protaminobacter ruber subsp. machidanus" (ATCC 21614) M. zatmanii sp. nov. Pseudomonas strain 135 (NCIB 12243=)" Rock et al. (16) Pseudomonas spp. (NCIB 10603,' 10604, 10606-10608, 10609-10610," 10612) 109,' 212, 216, 339, 390, 393, 463, 466, 471 590, N-11 Kouno and Ozaki (13) M.fujisawaense sp. nov. 0-31 (NCIB 12417T)u Kouno and Ozaki (13) 044,' 115, 269" 35, 37, 45," 58, 420, 437, 470, 488 617," 670, 679, 696, Kouno and Ozaki (13) 767, 781, 787, 788, 823, 0-6, 0-7, 0-46 BWd "Mycoplana rubra" (NCIB 11272)h D123" 8p9, 26, 31, 36, 37, 39

" Strains assigned on the basis of DNA-DNA homology. Other strains are assigned on the basis of phenotypic characteristics only. M. E. Rhodes-Roberts, University College of Wales, Aberystwyth, United Kingdom. T. S. Chandra, Indian Institute of Science, Bangalore, India. T. G. Mitchell, British-American Tobacco Co., Southampton, United Kingdom. ICI Ltd., Billingham, Cleveland, United Kingdom. H. Stolp, University of Bayreuth, Bayreuth, Federal Republic of Germany. B. Austin, Heriot-Watt University, Edinburgh, United Kingdom. Boots Co. Ltd., Nottingham, United Kingdom. 126 NOTES INT. J. SYST.BACTERIOL.

TABLE 2. Features that differentiate among species and some unassigned Methylobacterium" strains Substrates utilized as sole C sourceb

*a,

U5 Species or Strain

a, 0 - s * U * 0 U e B 2 5 s cl c4 Y c-r w 1A M. zatmanii - - - -+- - -+-v++v-+ 1B M. extorquens - - - - -v- -++v++--v 1C M. rhodesianum - - - -+v- -++-++- -+ 1E NCIB 9141 and - - - -+- - -+++++- -+ 9145 (9) 1F N-6 (9) - - - -+- - -++-+++-+ 1G M. rhodinum w-+ - -+++-++-++- -+ 1H M. orphanophilum + - - -+- - -+- -+++v+ 1J 602 and 317 (9) - - - -v++++-- +- - -+ ------2A 790 (9) - - - - -+++- - 2B M. radiotoleruns + + + + - + + + + + - v - -+ 2D M.fujisawaense + + + + V + + + + - V V - - -+ - -+- - - - 2E M. mesophilicum + + + + - + + V - 2F N-2 (9) ++++++++------+ 2G R14 (9) - - - -+++++- -+- - -+ - - - 2H D12 (9) - - - -+++-+- -+------25 B46 (9) - - - -+++- y+- 7 and 35d +- -++v- -++-++- -+

a Owing to the slow growth of some strains on certain substrates, carbon utilization tests were read after 14 days of incubation at 30°C (5). Doubtful results were checked by twice subculturing in liquid medium. V, Variable result; W, weak growth. Most strains which utilize sebacate can also utilize pimelate, suberate, azelate, and adipate. Isolated by H. Stolp, University of Bayreuth, Bayreuth, Federal Republic of Germany. region of Japan). Mode of respiration, colony and cell mor- Other strains assigned to this species solely on the basis of phology, flagellar arrangement, Gram reaction, and oxidase phenotypic data are listed in Table 1. atnd catalase as for the type species. Grows at 15 and 37°C. Some strains grow at 10°C. Growth factors are not required. Gelatin, starch, cellulose, lecithin, LITERATURE CITED DNA, casein, and Tweens are usually not degraded. Urease is produced, but indol, H,S, P-galactosidase, L-ornithine and 1. Anthony, C., and L. J. Zatman. 1964. The microbial oxidation of methanol. I. Isolation and properties of Pseudomonas M27. r.-lysine decarboxylases, and L-arginine dihydrolase are not. Biochem. J. 92:609-614. Methyl red and Voges-Proskauer tests are negative. Nitrate 2. Austin, B., and M. Goodfellow. 1979. Pseudomonas mesophi- is not reduced by most strains. lica: a new species of pink bacteria isolated from leaf surfaces. Sensitive to kanamycin, tetracyclines, gentamicin, al- Int. J. Syst. Bacteriol. 29:373-378, hamycin T, streptomycin, and framycetin but resistant to 3. Den Dooren de Jong, L. E. 1927. Uber protaminophage Bakte- rialidixic acid, oleandomycin, polymyxin B, colistin sulfate, rien. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. penicillin G, and bacitracin. 2 71:193-232. The following compounds can serve as sole carbon 4. Graf, W., and L. Bauer. 1973. Red bacterial growth (Coryne- sources: D-glucose, L-arabinose, D-xylose, D-fucose, D-ga- bacterium rubrum n. spec.) in tap water systems. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1Orig. Reihe A lactose, glycerol, malonate, succinate, fumarate, L-aspar- 236:513-530. tate, L-glutamate, sebacate, 2-oxoglutarate, lactate, malate, 5. Green, P. N., and I. J. Bousfield. 1982. A taxonomic study of acetate, pyruvate, citrate, citraconate, ethanol, methanol, some gram-negative facultatively methylotrophic bacteria. J. and propylene glycol. Some strains can utilize fructose. Gen. Microbiol. 12tk623-638. Monomethylamine, trimethylamine, and betaine are not 6. Green, P. N., and I. J. Bousfield. 1983. Emendation of Methy- utilized. Grows on peptone-rich nutrient agar (Oxoid CM55). fobacterium Patt, Cole, and Hanson 1976; Methylobacterium The guanine-plus-cytosine content of the DNA ranges rhodinum (Heumann 1962) comb. nov. corrig. ; Methylobacte- from 70.8 to 71.8 mol% (9); for the type strain, this value is rium radiotoleruns (It0 and Iizuka 1971) comb. nov. corrig.; and 71.8 mol%. Methylobacterium mesophilicum (Austin and Goodfellow 1979) comb. nov. Int. J. Syst. Bacteriol. 335375477. (= We propose NCIB 12417T strain 0-31 [13]) as the type 7. Hayward, A. C. 1960. Relationship between Protaminobacter s,train. As a result of recent work, including DNA-DNA ruber and some red pigmented pseudomonads. J. Appl. Bacte- homology studies (9), we also propose that strains 45, 269, rial. 23:ii. and 617 (13) be considered as strains of M. fujisawaense. 8. Heumann, W. 1962. Die Methodik der Kreuzung sternbildener VOL.38, 1988 NOTES 127

Bakterien. Biol. Zentralbl. 81:341-354. methanol-utilizing bacteria, p. 11-21. In Proceedings of the 9. Hood, D., C. S. DOW,and P. N. Green. 1987. DNA:DNA International Symposium on Microbial Growth on C, Com- hybridization studies on the pink pigmented facultative methy- pounds. Society of Fermentation Technology, Osaka, Japan. lotrophs. J. Gen. Microbiol. 133:709-720. 14. Patt, T. E., G. C. Cole, and R. S. Hanson. 1976. Methylobacte- 10. Ito, H., and H. Iizuka. 1971. Taxonomic studies on a radiore- rium, a new genus of facultatively methylotrophic bacteria. Int. sistant Pseudornonas. XII. Studies of the micro-organisms of J. Syst. Bacteriol. 26:226229. cereal grain. Agric. Biol. Chem. 3515661571. 15. Peel, D., and R. Quayle. 1961. Microbial growth on C, com- 11. Janota-Bassalik, L., and D. Pedyk. 1961. Ability of Fiavobacte- pounds. I. Isolation and characterization of Pseudomonas AMI. rium extorquens Bassalik to utilise various sources of carbon Biochem. J. 81:465469. with particular reference to glucose. Acta Microbiol. Pol. 16. Rock, J. S., I. Goldberg, A. Ben-Bassat, and R. I. Mateles. 1976. 10~225-238. Isolation and characterization of two methanol utilizing bacte- 12. Kirikova, N. N. 1970. Properties of two strains of Pseudornonas ria. Agric. Biol. Chem. 40:2129-2135. utilizing one-carbon compounds. Microbiology 39: 12-16. (Eng- 17. Sato, K., S. Ueda, and S. Shimizu. 1977. Form of vitamin B,, and lish translation of Mikrobiologiya.) its role in a methanol-utilizing bacterium, Protarninobacter 13. Kouno, K., and A. Ozaki. 1975. Distribution and identification of ruber. Appl. Environ. Microbiol. 33515-521.