Polyphasic Taxonomy Analysis For Sinobacteraceae And Sinomonas

Two novel bacteria strains designated CW-KD 4^T and CW-108^T were isolated from forest soil collected from Hefei,Anhui Province,China,by using a classic enrichment method. Strains CW-KD 4^T and CW-108^T showed obviously distincted morphological, chemotaxonomic and phylogenetic characteristics from their closest neighbors.Based on the polyphasic taxonomic results,the two isolated strains CW-KD 4^T and CW-108^T were assigned to a new family in the class Gammaproteobacteria and a new genus in the family Micrococcaceae,for which a new family name 'Sinobacteraceae' and a new genus 'Sinomonas' were proposed for them,respectively. CW-KD 4^T cells were Gram-negative,strictly aerobic,non-motile and long-rods(0.3-0.4×2.4-2.6μm).Colonies were circular,convex and pale yellow-coloured on LB or TYB agar and were only about 0.5 to 0.8 mm in diameter after 4-5 days cultivation at 30℃.Colonies were ropy and not easy picked up with inoculation loops.It does not require Na⁺ or other special nutrition for growth.Growth occurs at 10-35℃(optimum,25-30℃) and pH 5-8 (optimum,6.0-7.0).Growth detected at 0-2%NaCl and it showed better growth without NaCl.Starch was hydrolyzed,but casein,cellulose,chitin,DNA,gelatin and Tweens 80 were not hydrolyzed.Acid was produced from cellobiose,L-rhamnose,D-ribose,sucrose. Oxidase and weak urease activities were present;arginine decarboxylase,arginine digydrolase catalase,DNase,β-galactosidase,lysine decarboxylase,lipase ornithine decarboxylase,phenylalanine decarboxylase and activities were absent.Nitrate and nitrite are not reduced.H₂S(TSI reaction) is not produced.Methyl red and Voges-Proskauertests were negative.KCN(0.0075%) was not tolerated.The predominant isoprenoid quinone of strain CW-KD 4^T was Q-8 with minor components including Q-9,MK-7 and MK-6;the major fatty acids were C_16:0,C_18:1ω7c and summed feature3.The G+C content of the DNA was about 65.1mol%.Based on 16S rRNA gene sequence analysis,the novel strain represented a deep-rooting lineage within the class Gammaproteobacteria and was clustered with the genera Hydrocarboniphaga and Nevskia and some other unidentified bacteria.Strain CW-KD 4^T showed 16S rRNA gene sequence similarities even lower than 90%to its type genera Hydrocarboniphaga and Nevskia,respectively.On the basis of the distinctive phenotypic and genotypic characteristics,strain CW-KD 4^T should be represented a new family with single genus and a novel species in the class Gammaproteobacteria,for which the name Sinobacter flavus gen.nov.,sp.nov.was proposed to the newly described family 'Sinobacteraceae'. CW-108^T cells were Gram-negative,strictly aerobic,non-motile and bent rods(0.5-0.9×1.7-4.5μm).The strain grew optimally at 30-37℃and pH 6.0-8.0.Colonies are circular, convex and yellow-coloured after 2 days cultivation at 30℃on TYB or PYES agar.Growth occurs at 15-42℃(optimum 30-37℃) and pH 5.0-9.0(optimum pH 7.0-8.0).Growth occurs with 0-3%NaCl,but does not occur with 4%NaCl.Tween 80 was hydrolyzed but casein,starch,and tyrosine were not hydrolyzed.Urease,β-galactosidase and lipase activities were present;arginine dihydrolase,α-methyl-D-glueosidase,ornithine decarboxylase and lysine decarboxylase activities were absent.Nitrate was reduced and nitrite was not.H₂S(Triple Sugar Iron test) was not produced.Voges-Poskauer tests were positive.Indole was not produced.Aeseulin,D-cellobiose,erythritol,D-fructose, D-glucose,D-lactose,glycerol,inositol,2-keto-glueonate,D-maltose,D-mannose,mannitol, D-melezitose,β-methyl-D-xyloside,D-ribose,sorbitol,D-sucrose and D-turanose were utilized.Chemotaxonomically,the amino acids Lys,Ala,Glu and Gly were detected in a molar ratio of 1:3.5:1:0.2 in strain CW-108^T peptidoglyean;these results suggest that the new isolate showed A3αas peptidoglycan type.The cell-wall sugars contained galaetose, mannose and ribose.Polar lipids were DPG,PG,PI and PME.The major fatty acids were I-C_15:0,AI-C_15:0 and AI-C₁₇₀.The predominant respiratory quinone was MK-9(H₂) with minor components including MK-8(H₂).The G+C content of the genomic DNA was about 71.1 mol%.Phylogenetic analysis showed that swain CW-108^T formed a deep separated-lineage in family Micrococcaceae combine with listed species Arthrobacter atrocyaneus(strain DSM 20127^T).Sequence similarities of strains CW-108^T and DSM 20127^T were lower than 95.2%to other validly described species in family Micrococcaceae; but the two closest strains shared 16S rDNA sequence similarity up to 99.4%.DNA-DNA hybridization experiment showed the level of relatedness of 52%between the two neighbors.Based on the polyphasie taxonomic results,strain CW-108^T and strain DSM 20127^T should be assigned into two different species of a novel genus within the family Micrococcaceae for which Sinomonas tiara gen.nov.,sp.nov.is proposed for the newly isolated strain,and Sinomonas atrocyaneus comb.nov.is proposed for strain DSM 20127^T.