INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1982, p. 1-14 Vol. 32, No. 1 0020-7713/82/01OO01-14$02 .OO/O Rhodococcus luteus nom. nov. and Rhodococcus maris nom. nov. 0. A. NESTERENKO, T. M. NOGINA, S. A. KASUMOVA, E. I. KVASNIKOV, AND S. G. BATRAKOV D. K. Zabolotny Institute of Microbiology and Virology of the Academy of Sciences of the Ukrainian SSR, 252143, Kiev, USSR Two groups of bacteria isolated from natural substrates were assigned to the genus Rhodococcus Zopf 1891, emend. Goodfellow and Alderson 1977. We propose the name Rhodococcus luteus nom. nov. for the first group, which corresponds to the description of the organism previously known as “Mycobacte- rium luteurn” Sohngen 1913. The type strain of R. luteus is IMV 385 (= AUCNM A-594). The name proposed for the second group of strains, which corresponds to the description of the organism previously known as “Flavobacterium maris” Harrison 1929, is Rhodococcus maris nom. nov. The type strain of R. rnaris is IMV 195 (= AUCNM A-593). The properties of the two species are described, and the characters useful for the identification of the species are given. From soils of the Soviet Union and from skins phy depend on differences in the molecular- and the intestinal tracts of carp (Cyprinus car- weight ranges of the free mycolic acids: Nocar- pio), two groups of bacteria, distinguishable by dia, 48 to 58 carbons; Rhodococcus, 34 to 50 their morphological and colonial characters, carbons; and Corynebacterium, 22 to 38 carbons were isolated on mineral salt agar enriched with (2,28,29). Thus, the Rfvalue of LCN-A may be n-alkanes. These isolates were assigned to the useful in distinguishing among nocardiae, rhodo- genus Rhodococcus Zopf 1891, as emended by cocci, and true corynebacteria. Goodfellow and Alderson (11) to include strains Analyses of other types of lipids may also be previously identified either as members of the valuable in differentiating rhodococci from relat- genus “Gordona” (42) or of the “rhodochrous” ed bacteria. Menaquinone analyses, for exam- complex (7). (Names in quotation marks were ple, indicate that representatives of some animal not included on the Approved Lists of Bacterial corynebacteria, rhodococci (including the type Names [37] and have not been validly published species, Rhodococcus rhodochrous; see 1l), and since 1 January 1980; therefore, they have no Brevibacterium linens all have menaquinones standing in bacterial nomenclature.) with eight isoprene units and one hydrogenated Among the various characters used in describ- double bond [MK-8(Hz)] as the prevalent type; ing these organisms, chemical markers are of arthrobacters, glutamic acid-producing sapro- special value in distinguishing Rhodococcus phytic corynebacteria, and mycobacteria have from related genera (13, 28). Nocardiae, myco- MK-9(H2) as the main menaquinone component; bacteria, rhodococci, and true corynebacteria in contrast, representatives of Nocardia have contain meso-diaminopimelic acid (DAP), arabi- MK-8(H4) as the main menaquinone component nose, and galactose in their whole-cell hydroly- (28). The non-hydroxylated fatty acids of nocar- sates (cell wall chemotype IV) (27), whereas diae, rhodococci, and mycobacteria contain high brevibacteria have only meso-DAP, and arthro- proportions of straight-chain and unsaturated bacters contain no meso-DAP or arabinose (13, acids and of 10-methyloctadecanoic (tuberculo- 23, 32, 35, 44). The above-mentioned organisms stearic) acid, whereas most true corynebacteria can be distinguished from one another by thin- do not contain tuberculostearic acid; the simple layer chromatography of ethanol-ether extracts fatty acids of arthrobacters and B. linens are of their cells. The true corynebacteria, nocar- composed mainly of iso- and anteiso-acids (12, diae, and rhodococci contain a characteristic 28). lipid component (LCN-A), composed of free Analyses of deoxyribonucleic acid base com- mycolic acid (14,23,30), whereas mycobacteria, positions revealed that the guanine-plus-cyto- brevibacteria, and arthrobacters do not (23, 30, sine contents of the deoxyribonucleic acids of 32). The LCN-A of Nocardia has a higher Rf My cobacterium, Nocardia, R hodococcus, Arth- value than that of many rhodococci; the lowest robacter, and B. linens are in the range of 60 to mobility of LCN-A was observed with the Cory- 70 mol%, whereas those of true corynebacteria nebacterium strains (23, 30, 32). Differences in range from 48 to 59 mol% (13, 28). mobility of LCN-A on thin-layer chromatogra- In the study reported here, the characters and 1 2 NESTERENKO ET AL. INT. J. SYST. BACTERIOL. the taxonomic positions of the isolated strains TABLE 1. Isolated strains used in this study were determined. Serial no. Laboratory no.a Site of isolation MATERIALS AND METHODS Rhodococcus luteus (group I bacteria) 1 IMV 8- Soilb Bacterial strains. A list of the strains studied and 2 IMV 21 Soil their sources are given in Tables 1 and 2. The strains 3 IMV 24 Soil were isolated on mineral salt agar (KN03, 1 g; MgS04, 4 IMV 27 Soil 0.1 g; Na2HP04, 0.6 g; KH2P04, 0.14 g; NaC1, 1 g; 5 IMV 68 Soil mixture of n-alkanes (CI2to CZ2),20 g; tap water, 500 6 IMV 103 Soil ml; distilled water, 500 ml) by the method of Yamada 7 IMV 111 Soil et al. (45). The temperature of incubation was 28°C. 8 IMV 115 Soil Morphological and cultural tests. The cell form and 9 IMV 120 Soil size and the reaction to the Gram stain were deter- 10 IMV 158 Soil mined on smears from 16- to 24-h-old, 72-h-old, and 11 IMV 163 Soil 12-day-old cultures, on nutrient agar (NA) (4), glycerol 12 IMV 177 Soil agar (GA) (17), and wort agar (WA) (4) slants. Motility 13 IMV 202 Soil and acid fastness were tested in cultures grown on NA 14 IMV 206 Soil for 18 h. The mode of cell division was studied by 15 IMV 242 Soil time-lapse microscopy. Microcultures of the bacteria 16 IMV 269 Soil were made on NA by the method of Komagata et al. 17 IMV 270 Skin of carp (24). The macroscopic appearance of the growth was 18 IMV 323 Soil examined on NA plates and on NA, GA, and WA 19 IMV 333 Soil slants 2 weeks after inoculation. 20 IMV 372 Soil Physiological tests. Hugh and Leifson’s (21) test was 21 IMV 374 Intestinal tract of carp used to determine the fermentation or oxidation of 22 IMV 385 Soil glucose. The production of acid from different carbo- 23 IMV 401 Soil hydrates, the utilization of organic acids, the produc- 24 IMV 406 Soil tion of urease, the reduction of nitrate to nitrite, and 25 IMV 416 Intestinal tract of carp the decomposition of casein, guanine, hypoxanthine, 26 IMV 417 Soil starch, tyrosine, and xanthine were determined by the 27 IMV 419 Soil methods of Gordon and Smith (18,19) and Gordon and 28 IMV 427 Soil Mihm (16, 17). Phosphatase production was deter- 29 IMV 445 Soil mined by the method of Giane-Williams and Skerman 30 IMV 455 Soil (10). The hydrolysis of Tweens was investigated by the 31 IMV 462 Soil method of Sierra (36), and the utilization of acetamide 32 IMV 604 Soil and benzamide as sole carbon and nitrogen sources was studied by the method of Tsukamura (42). The Rhodococcus maris (group I1 bacteria) production of p-nitrophenoloxidase was determined 33 IMV 195 Soil by the method of Bonicke and Juhasz (6). The ability 34 IMV 217 Soil to utilize n-alkanes was studied on the mineral salt 35 IMV 277 Soil agar mentioned above and by the method described by 36 IMV 283 Intestinal tract of carp Kvasnikov et al. (26). Descriptions of the other tests 37 IMV 324 Soil utilized have been previously published (4, 38). 38 IMV 330 Skin of carp Chemotaxonomic tests. The monosaccharides and the form of DAP in whole-cell hydrolysates were a IMV, Institute of Microbiology and Virology, investigated as previously reported (31). Lipid LCN-A Kiev, USSR. was detected by the method of Mordarska et al. (30). Soils, as a rule, were impregnated with oil. For preparation of the chromatographic plates, we used the 1- to 2-h fraction (or 10 to 30 pm) of silica gel “L” 5/40 pm (Lachema, n.p. Bmo, Czechoslovakia). erythropolis NCIB 8863 occupies an intermediate posi- The chromatograms were developed in the system n- tion (0.56). hexanediethyl ether-glacial acetic acid (50:50:2, vol/ The deoxyribonucleic acid base composition was vol) (23). The spots of LCN-A detected in the bacteria determined by the method described by Sukapare et under investigation were always compared with those al. (41). of three reference strains: Rhodococcus sp. (“ ‘M.’ The simple fatty acids were detected by the method rhodochrous”) strain NCTC 576, R. erythropolis (N. described by Andreev and Galchenko (3). Gas-liquid calcarea) NCIB 8863, and “C. divaricaturn” (B.divar- chromatography was performed with a Hewlett-Pack- icaturn) NCIB 9379. Free mycolic acids of Rhodococ- ard 5380A apparatus. cus sp. strain NCTC 576 contains from 38 to 47 carbon Menaquinones were analyzed by the method of atoms (22), and those from R. erythropolis NCIB 8863 Batrakov et al. (5). contain 34 to 46 carbons (29). “C.divaricaturn” NCIB Identification of strains. The 66 isolates were divided 9379 contains the mycolic acid analogs characteristic into two groups on the basis of their morphological, of C. diphtheriae: 26 to 38 carbons (23, 28). The lipid cultural, and physiological characteristics. The prop- LCN-A of Rhodococcus sp. strain NCTC 576 has a erties of the groups were compared with those of the high Rf value (0.59), and that of “C. divaricaturn” type strains (Table 2) (11, 34) of most of the currently NCIB 9379 has a low value (0.54); the LCN-A of R.