Phylogeneticcharacteristics
An almost-complete 16S rRNA gene sequence (1501 bp) was obtained from strain YIM S02567T. Results from the EzBioCloud server indicated that the closest hit to the validly published species was B. sinopodophylli TEYR-7T (with 95.38 % similarity), while the type species of the genus B. flavus VG341T (94.69 % similarity) and P. tarda WR061T (94.67 % similarity). In the phylogenetic tree based on the maximum-likelihood algorithm (Fig. 1), strain YIM S02567T formed a tight phyletic group with P. tarda WR061T with a bootstrap value of 76 % and belonged to a cluster of species including P. tarda WR061T, B. sinopodophylli TEYR-7T, and B. flavus VG341T. The overall topology of the neighbour-joining and maximum parsimony tree (Fig. S3 and Fig. S4, available in the online Supplementary Material) was essentially the same as that of the maximum-likelihood tree. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM S02567T belonged to the family Propionibacteriaceae, therefore B. sinopodophylli TEYR-7T and P. tarda WR061T were used as references for the subsequent related tests.
Genomecharacteristics and analysis
The genome assembly size of strain YIM S02567T had a total size of 3,796,070 bp with N50 length of 405,848 bp. The genomic DNA G + C content was 71.20 mol%. A total of 3,583 protein-encoding genes, three rRNA genes and 45 tRNAs genes were predicted. The number of genes assigned to COGs was 2928 and the number of genes assigned to KEGG was 1423. Other genome characteristics of strain YIM S02567T and other members in the family Propionibacteriaceae were summarized in Table S1. The ANIb values between YIM S02567T and the closely related type strain were 76.2 % (B. sinopodophylli TEYR-7T), 73.3 % (B. flavus VG341T) and 72.2 % (P. tarda WR061T), respectively. This indicates that strain YIM S02567T is closer to the genus Brevilactibacter. A phylogenomics tree closely related type species of the family Propionibacteriaceae based on the supermatrix method was shown in Fig. 2. Strain YIM S02567T formed a coherent and monophyletic clade closed to the genus Brevilactibacter, which supported that strain YIM S02567T represents a novel species of the genus Brevilactibacter.
According to genome prediction for the secondary metabolite synthesis analysis, it showed that the genome of strain YIM S02567T had gene clusters associated with lanthipeptide and terpene. Lanthipeptide contains the noncanonical amino acid lanthionine and antibacterial activities and anti-HIV activity were exhibited (Ferir et al. 2013). This must be an exciting substance. The result suggested that strain YIM S02567T may be beneficial for the research of lanthipeptide biosynthesis gene clusters that contributes to the development of anti-HIV.
Morphological, physiological, and biochemical characterization
Cells of strain YIM S02567T were Gram-staining positive, non-motile, facultatively anaerobic, cocci-shaped, 0.5-0.7 μm in diameter (Fig. S1) and formed orange colonies. Growth of YIM S02567T was observed at 10-45 ℃ (optimum temperature, 20-25 ℃) and at pH 6.0-9.5 (optimum, pH 8.5-9.0). The tolerance to NaCl was up to 4.0 % (w/v) with optimum growth in the presence of 2.0 % (w/v) NaCl. Tests for catalase activity, hydrolysis of starch, hydrolysis of gelatin, and aesculin ferric citrate were positive, however, those for nitrate reduction, oxidase activity, indole production, H2S production, hydrolysis of cellulose were negative. Strain YIM S02567T produced propionic acid, acetic acid and lactic acid from PYG medium supplemented with 10 g·l-1 glucose after one week of cultivation under anaerobic conditions. Other physiological and biochemical characteristics of strain YIM S02567T and a comparison of special results with closely related species were listed in Table 1 and the species description.
Chemotaxonomic characteristics
The cellular fatty acid profile of strain YIM S02567T was characterized by the predominance of anteiso-C15:0 and C16:0. The closely related species contained the same major fatty acid (anteiso-C15:0), but strain YIM S02567T also included the major fatty acid C16:0 (14.91 %). The cellular fatty acid profiles of strain YIM S02567T and its closely related strains are given in Table 2. The predominant menaquinone of strain YIM S02567T was determined to be menaquinone MK-9(H4), and the whole cell-wall diamino acid was analyzed to be meso-diaminopimelic acid, which was all the same as those of the closely related species reported. The whole-cell sugars detected were glucose, mannose, ribose, galactose, arabinose and rhamnose. Polar lipids of strain YIM S02567T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), two unidentified glycolipids (GL1, GL2), and an unidentified lipid (L1) (Fig. S2). Two type species of the genus Brevilactibacter also contained DPG, PG, GL1, and GL2 as similar polar lipids. In addition, they had different polar lipids (Fig. S2).
Genealogical taxonomy conclusion
Strain YIM S02567T shared < 97% similarities of 16S rRNA gene sequence and showed cocci-shaped distinguished from related valid described species in a cluster of the phylogenetic tree. The phylogenomics tree indicated that strain YIM S02567T formed a coherent with the genus Brevilactibacter. Furthermore, strain YIM S02567T showed differences from closely related type species on nitrate reduction, enzyme activities, and major cellular fatty acids, assimilation of several carbon resources, respiratory quinone, polar lipids and type of diamino acid. Based on morphological, physiological, phylogenetic and biochemical features, strain YIM S02567T can be considered as a novel member of the genus Brevilactibacter, for which the name Brevilactibacter soli sp. nov. is proposed.
DESCRIPTION OF BREVILACTIBACTER SOLI SP. NOV.
Brevilactibacter soli (So′li. L. neut. gen. n. soli of soil, the source of the type strain).
Cells are cocci-shaped, facultatively anaerobic, Gram-staining positive, non-motile bacterial strain, 0.5-0.7 μm in diameter. Colonies are orange, smooth, and convex on R2A medium. The temperature range for growth is 10-45 °C (optimum at 20-25 °C). The pH range for growth is 6.0-9.5 (optimum pH 8.5-9.0). NaCl tolerance is up to 4.0 % (w/v) with optimal growth at 2.0 % (w/v) NaCl. Positive for catalase activity, hydrolysis of starch, hydrolysis of gelatin, Tweens 40 and aesculin ferric citrate, however, negative for nitrate reduction, oxidase activity, indole production, H2S production, hydrolysis of cellulose. Propionic acid, acetic acid and lactic acid are produced anaerobically from PYG medium supplemented with 10 g·l-1 glucose. In API ZYM test strips, it is positive for acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, and β-glucosidase, weakly positive for alkaline phosphatase, valine arylamidase, cystine arylamidase and chymotrypsin, and negative for lipase (C14), trypsin, N-acetyl-β- glucosaminidase and α-mannosidase, and β-fucosidase activities. In the API 20NE test strips, positive for D-glucose fermentation, hydrolysis of esculin, gelatin, and hydrolysis of 4-nitrophenyl β-D-galactopyranoside, negative for nitrate reduction, indole production, arginine dihydrolase, and urea. Weakly positive for assimilation of D-glucose, D-mannose and positive for assimilation of L-arabinose, D-maltose and malic acid, negative for assimilation of adipic acid, capric acid, phenylacetic acid, D-mannitol, N-acetylglucosamine, potassium gluconate, and trisodium citrate. In API 50CH test strips, acid is produced from L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, D-sorbitol, methyl α-D-glucopyranoside, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, starch, glycogen, gentiobiose, D-turanose, and produced weakly from glycerol, D-arabinose, D-ribose, L-xylose, D-galactose, L-sorbose, N-acetyl-D-glucosamine, D-melezitose, xylitol, D-lyxose, D-tagatose, L-fucose, D-arabitol, L-arabitol, potassium 2-keto gluconate, potassium 5-keto gluconate, but not from erythritol, D-adonitol, methyl β-D-xylopyranoside, dulcitol, inositol, methyl α-D-mannopyranoside, inulin, D-fucose, D-potassium gluconate. In the Biolog Gen III Micro Plate system, the following carbon sources have positive reactions: propionic acid, acetic acid, acetoacetic acid, α-D-lactose, D-maltose, D-raffinose, D-turanose, D-gluconic acid, glucuronamide, but negative reactions for methyl β-D-glucoside, D-salicin, inosine, D-serine, D-arabitol, D-aspartic acid, D-serine, gelatin, glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-pyroglutamic acid, L-serine, D-gluconic acid, mucic acid, quinic acid, D-saccharic acid, P-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, L-lactic acid, citric acid, D-malic acid, L-malic acid, bromo-succinic acid, γ-amino-butryric acid, α-hydroxy-butyric acid, α-keto-butyric acid. The cell-wall diamino acid is meso-diaminopimelic acid and the predominant menaquinone is MK-9(H4). The cellular polar lipids are DPG, PG, GL1, GL2, and L1. The whole-cell sugars contain glucose, mannose, ribose, galactose, arabinose and rhamnose. The major cellular fatty acids contain anteiso-C15:0 and C16:0.
The type strain YIM S02567T (=CCTCC AB 2020128T=CGMCC 1.18504T=KCTC 49478T) was isolated from a soil sample collected from Gejiu City, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, PR China. The genomic DNA G + C content was 71.20 mol%. The GenBank accession numbers for the 16S rRNA gene sequence and genomic sequence are MW032491 and JACYOT000000000, respectively.