0020-77 13/78/0028-0032$02.00/0 INTERNA'I'IONAI, JOIIRNA~.OF SYSTI.:MATI(' BA(TTEHIOI,OGY, Jan. 1978, p. 32-36 Vol. 28, No. 1 Copyright 0 1978 International Association of Microbiological Societies Printed in U.S. A. Runella slithyfurmis gen. nov., sp. nov., a Curved, Nonflexible, Pink Bacterium JOHN M. LARKIN AND PATRICIA M. WILLIAMS Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803 Two strains of bacteria regarded as belonging to a new species were isolated from bodies of water near Baton Rouge, La. The cells of these strains were gram-negative, curved rods, the degree of curvature varying among cells in a single culture. A pink pigment was produced on glucose-peptone-yeast extract agar. The strains were nonmotile and nonfermentative, and the guanine-plus- cytosine contents of their deoxyribonucleic acids varied from 49.3 to 49.6 mol%. The species cannot be assigned to any known genus, and therefore a new genus, Runella, is proposed, with R. slzthyformis as the type species. The type strain of this species is strain 4 (= ATCC 29530). At present, it is difficult to place the genus Runella in a family. During examination of the bacteria that in- eosin-methylene blue agar, phenol red-mannitol-salt habit the bodies of fresh water in southern agar, phenyl ethyl alcohol agar, nutrient agar, nutrient Louisiana, we repeatedly encountered bacteria agar plus 5% sucrose, Trypticase soy agar, Trypticase soy agar plus 3% glucose, peptonized milk agar, MS that resembled those of the newly described agar, yeast extract-acetate-tryptoneagar, McConkey genus Flectobucillus (2). The organisms were agar, bismuth sulfide agar, and salmonella-shigella seen both on slides that had been suspended in agar. water and in enrichments made by adding 0.1 g Additional characters.The techniques and meth- of peptonized milk to 100 ml of the water sample. ods used to determine the abilities of the strains to We were successful in isolating two strains of utilize sole carbon sources, to hydrolyze macromole- these organisms, and subsequent characteriza- cules, to produce specific enzymes or end products, tion studies indicated that they are physiologi- and to determine their susceptibilities to antibiotics cally quite different from flectobacilli even have been reported previously (2). though they are similar morphologically. These Two techniques were used to determine the gua- nine-plus-cytosine (G+C) contents of the deoxyribo- a strains represent new species and genus of nucleic acids (DNA) of the strains. One technique, bacteria, descriptions of which are presented. used with strain 6, made use of the Marmur method (6) to purify the DNA, followed by determining the MATEXIALS AND METHODS melting-point profile with a Gilford model 2400 spec- Bacterid strains. Two similar strains were iso- trophotometer equipped with a temperature-con- lated from water from the Baton Rouge, La., area by trolled cuvette compartment (5). In the other tech- repeated streaking of water samples onto MS agar nique, used with strain 4, DNA was partially purified (0.1% each of glucose, peptone, and yeast extract, plus by the method of Meyer and Schliefer (7), and the 1.5% agar) with incubation at room temperature. One mole percent G+C was determined by the absorbance strain, designated as strain 4, was isolated from Uni- ratio technique of Ulitzer (9). Measurements of cell versity Lake, and the other, designated as strain 6, size were obtained with a Filar micrometer. Photomi- was isolated from Elbow Bayou. Cultures of these crographs were obtained with a GiIlet and Seibert strains were maintained on MS agar, with transfers microscope equipped with a Nikon model AFM cam- made at about 2-week intervals. era. Utilization of carbohydrates. The production of acid from carbohydrates by aerobic or anaerobic RESULTS means was determined by the method of Hugh and Leifson (11, in which MS agar was used but with the The results of the analysis of the two strains glucose replaced by 1%of the substrate to be tested are summarized in Table 1. The cells were and the agar concentration lowered to 0.3%. Incuba- curved rods, with the degree of curvature of tion was continued for 8 weeks for cultures giving individual cells within a culture varying from negative results. nearly straight to curved in the shape of a ring. Ability to grow on various media. The abilities of the organisms to grow on various media were inves- Coils of two to three turns were rarely formed, tigated by making a single streak of the organisms and filaments up to 14 pm in length were pro- from a fresh slant onto the surface of the medium duced. This morphological variation prompted and incubating at 25°C for a minimum of 3 weeks. us to determine whether the cells were flexible The media tested were chocolate agar, blood agar, or capable of gliding motility, but observations 32 VOL. 28,1978 RUNELLA SLITHYFORMIS, GEN. NOV., SP. NOV. 33 TABLE1. Characteristics of two strains of Runella slithyfonnis" Character Strain 4 Strain 6 ~______~~ Character Strain 4 Strain 6 Gram reaction ......... - - Indole production ..... - - Pigmentation ......... Pale pink Pale pink Methyl red test ........ - - Fluorescence .......... - - Voges-Proskauer test . - - Formation of coils ..... Rare Rare NO3 reduction ......... - - Motility ............. - - H2S production ........ - - Mol% G+C ........... 49.6 49.3 Hydrolytic activity on: Cell size: Cellulose ........... - - Length (pm) ........ 2.5-4.0 2.0-4.5 Agar ............... - - Diameter (pm) ...... 0.5-0.9 0.6-0.9 Chitin ............. - - Diameter of rings (pm) 2.0-3.0 2.0-2.5 Gelatin ............. - - Filament length (pm) up to 12.0 Up to 14.0 Starch ............. sl+ sl+ Acid produced aerobi- Casein ............. NG NG cally from: Esculin ............. - - Pentoses Tributyrin .......... + + Arabinose ........ Litmus milk ......... No change No change Ribose ........... Utilization of sole carbon Xylose ........... sources: Methyl pentose Acetate ............. - - Rhamnose ........ - + Benzoate ........... - - Hexoses Citrate ............. - - Fructose .......... Formate ............ - - Galactose ......... Glycerol phosphate . - - Glucose .......... Methylamine ....... - - Mannose ......... Propionate .......... - - Sorbose .......... Succinate ........... - - Glucosides Tartrate ............. - - a-Methyl-D-gluco- Malonate ........... - - side ............ Methanol ........... - - Salicin ........... Growth on agar media: Disaccharides Chocolate ........... 1 1 Cellobiose ........ Blood .............. NG NG Lactose ........... - - EMB ............... NG NG Maltose .......... + + NA ................. 3 3 Melibiose ......... NAS ............... NG NG Sucrose ........... + + PRMS ............. NG NG Trehalose ......... NG NG PEA ............... NG NG Trisaccharide TSA ............... NG NG Fbffinose ......... - + TSAS .............. NG NG Polysaccharides TSAG .............. NG NG Dextrin ........... - - PMA ............... 1 1 Inulin ............ + + MS ................ 4 4 Alcohols YEAT .............. 1 1 Glycerol .......... McConkey .......... NG NG Erythritol ........ BS ................. NG NG Dulcitol .......... ss ................. NG NG Mannit01 ......... Antibiotic susceptibility Sorbitol .......... to: Actinomycin D (100 Production of enzymes S S Urease ............. pg/d . , - Lecithinase ......... Ampicillin (10 pg) ... S S Lysine decarboxylase Aureomycin (15 pg) . S S Ornithine decarboxyl- Carbenicillin (50 pg) . S S ase ............. Cephalothin (30pg) . S S Phenylalanine deami- Colistin (10 pg) ...... R R nase ............ Erythromycin (15 pg) S S ONPG ............. Furadantin/macrodantin + + S S Oxidase ............. + + (300 pg) ........ Catalase ............ sl+ sl+ Gentamycin (10 pg) . S S Phosphatase ........ + + Kanamycin (30 pg) . R S Hemolvsin .......... - - 34 LARKIN AN I) WILLIAMS INT. J. SYST. BACTEIIIOL. TABLEl-Continued Character Strain 4 Strain 6 Character Strain 4 Strain 6 ~_.______ Mitomycin C (1 pg/ml) S Sulfamethoxyzole/tri- Neomycin (30 pg) S methopterh (25 S S Penicillin G (10 U) S S Clg) Polymyxin B (300 U) H Tetracycline (30 pg) S S Streptomycin (10 pg) S S Triple sulfa (1 mg) K R " Symbols: +, positive, -, negative; NG, no growth. The growth responses on various media are graded from 1 to 4 for scant to luxurious growth, repectively. Abbreviations: S, susceptible; R, resistant; EMB, eosin- methylene blue; NA, nutrient agar; NAS, nutrient agar plus 5% sucrose; PRMS, phenol red-mannitol-salt agar; PEA, phenyl ethyl alcohol agar; TSA, Trypticase soy agar; TSAG, Trypticase soy agar plus 3% glucose; PMA, peptonized milk agar; YEAT, yeast extract-acetate-tryptoneagar; BS, bismuth sulfide agar; SS, salmo- nella-shigella agar. of tubes. Figure 1 shows the typical morphology of these two strains. DISCUSSION The organisms described above are suffi- ciently different from those of the known genera to merit their recognition as members of a new genus, which we propose to call Runella. In Table 2 are listed some of the characteristics that help to differentiate Runella from other genera that have some characteristics in com- mon with the new genus. Runella resembles Vibrio in DNA G+C con- tent and in being curved, but it differs from Vcbrio in being nonmotile, nonfermentative, in- capable of anaerobic growth, and in producing a pink pigmentation. Runella differs from Cy- tophaga and FZexibacter in DNA G+C content and in being nonmotile; additionally, it differs from Cytophagu in being nonfermentative and unable to hydrolyze cellulose,