Phenotypic and biochemical characterisation
NA medium was used for general laboratory cultivation, but the novel strain also grows well on TSA and R2A media. After 24 h growth on NA at 30 C, colonies were observed to be 1.0–1.5 mm in diameter, circular, smooth and apricot. Strain TLK-CK17T was found to be Gram-reaction-negative and catalase negative aerobic bacterium. Meanwhile, it showed a positive reaction for catalase (weakly) and nitrate reduction. Cells are non-motile, grow in 0–5.0% (w/v) NaCl, at a pH range from 6.5 to 8.5 and at temperatures between 15 and 40°C. Optimal growth was observed at 35°C, 0.5% (w/v) NaCl and pH 7.0-7.5. Cells of strain TLK-CK17T are short rods, catalase-negative but oxidase-positive, the mean cell size is 0.3–0.5 µm in width and 1.5–2.0 µm in length (Supplementary Fig. 2). Nitrate can be reduced to nitrite. The strain was positive for the hydrolysis of Tween 20, 80, casein, CM-cellulose, but negative for alginate, starch. Antibiotic susceptibility test indicated that the strain was sensitive to chloramphenicol (30 µg), ceftriaxone (30), Ofloxacin (5 µg) and Ciprofloxacin (5 µg). However, it was resistant to penicillin (10 µg), tetracycline (30 µg), vancomycin (30 µg), ampicillin (10 µg), streptomycin (10 µg), Clindamycin (2 µg), Amoxicillin (25 µg) and Cephalexixn (30 µg)
Whole genome sequence analysis
The genome contributes to an important understanding for the genetic evolution of bacteria, disease prevention and treatment, and the development of antibiotics; thus, the genome of strain TLK-CK17T was analysed to decipher the genetic code involved in the environmental suitability. The genome size and G + C content of TLK-CK17T are 4,300,099 bp and 68.2 mol%, respectively. A length of 3,695,277-bp genes was found based on gene prediction, and the ratio of gene length/genome was 85.9%. Furthermore, 3792 CDSs were contained in the genome, and all were assigned functions. CDSs were further annotated in NR, Swiss-Prot, COGs, KEGGs, GO and Pfam, and their numbers were 3630, 1485, 2762, 3421, 2419 and 2419, respectively. The ANI values of strains TLK-CK17T with L. penaei GDMCC 1.1817T and L. maris KCTC 42381T were 79.9% and 85.6%, respectively, while the GGDC values were 29.6 and 23.8, respectively. For species delineation, ANI values of 95–96% and dDDH values of 70%, respectively, are normally accepted (Wayne et al. 1987; Thompson et al. 2013). These results indicated that strain TLK-CK17T represents a novel species of the genus Lysobacter. The related genome datas strain TLK-CK17T and two closely related type strains are listed in Tables S1 and S2.
The RAST analysis revealed the presence of 302 subsystems, the subsystem coverage was 25% (total 977, non-hypothetical 936, hypothetical 41), and 67 glycoside hydrolase (GHs), 42 glycosyl transferases (GTs), 44 carbohydrate-binding modules (CBMs), 6 Carbohydrate Esterases (CEs), and 4 auxiliary activities (AAs) were identified. Because cow dung is rich in lignocellulose, the coexistence of these genes suggest that they play important roles in the breakdown and modification of carbohydrates in cow dung composting. The assembled genome of strain TLK-CK17T was compared to the genomes of closely related strains L. penaei GDMCC 1.1817T and L. maris KCTC 42381T. Result showed that GH, GT and CBM family numbers were more in strain TLK-CK17T, while the L. penaei GDMCC 1.1817T contained more CE and AA family members (Table S2). We considered that it might be closely related to their isolated environment, L. penaei GDMCC 1.1817T and L. maris KCTC 42381T were isolated from the pacific white shrimp and seawater, respectively.
Lysobacter spp. has been identified as heterotrophic with a wide range of extracellular enzymes and other metabolites against other microorganisms, including fungi and nematodes, so it played an important role in the suppression of pathogenic bacteria (de Bruijn et al. 2015; Xie et al. 2012; Pidot et al. 2014). Our results showed that the strain TLK-CK17T possessed chitinase, protease and glucanase activity, confirming and extending previous research (Zhang et al. 2001; Palumbo et al. 2005). Chitinase, glucanase and protease activities may contribute to antimicrobial activity, since chitin, α- and β-glucans and glycoproteins are the major components of the cell walls of fungi (Figueiredo et al. 2014). Moreover, we analysed that Lysobacter strains showed a high genetic diversity, which could confer an advantage under adverse environmental conditions (Foster et al. 2005). To better understand the potential effect of strain TLK-CK17T to the overall activities of the microbial communities in cow dung compost, our future work will include testing it with other bacterial genera abundant in compost. Interactions of strain TLK-CK17T with other bacteria whether or not stimulate the production of antimicrobial compounds, so as to quickly remove pathogenic microorganisms in livestock manure.
Based on the phylogenetic analyses, strain TLK-CK17T was found to be affiliated with members of the genus Lysobacter in the family Xanthomonadaceae. Strain TLK-CK17T represented a novel species of the genus Lysobacter, as supported by the related genome datas. In addition, phenotypic and biochemical collectively support the fact that strain TLK-CK17T was distinguishable, whilst chemotaxonomic analyses are consistent with their affiliation with the genus Lysobacter. The phenotypic characterisation presented in Table 1 differentiates strain TLK-CK17T as a separate species. The exclusively respiratory quinone was Q-8, as reported for the major respiratory quinone of all members of the genus Lysobacter. The major fatty acids of type strain TLK-CK17T were iso-C16:0, iso-C15:0, and feature 9 (comprising C17:1 ω9c and / or 10-methyl C16:0). Based on the results presented in this study, it is proposed that strain TLK-CK17T represents novel member in the genus Lysobacter, for which the names Lysobacter chinensis sp. nov., is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and whole-genome shotgun project of strain TLK-CK17T is OK143236.
Description of Lysobacter chinensis sp. nov.
Lysobacter chinensis (chi.nen′sis. N.L. masc. adj. chinensis pertaining to China, where the type strain was isolated)
Cells are Gram-stain-negative, aerobic, non-motile, and short rod-shaped with a size of 0.3–0.5×1.5–2.0 µm. Good growth is observed on R2A agar, TSA and NA, but not on MA. Colonies on NA are beige to apricot, smooth, opaque, circular (approximately 1.0-1.5 mm in diameter) with entire edges and convex. Growth occurs on NA at temperatures of 15–40°C (optimum 35°C). The pH range for growth is from pH 6.5 to 8.5 (optimum pH 7.0–7.5). Growth occurs at 0–5.0% NaCl concentrations (optimum 0.5). Catalase-positive and oxidase-negative. Nitrate can be reduced to nitrite. CM-cellulose, starch, casein, Tweens 20 and 80 are hydrolysed, but alginate is not. Positive for alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, acid phosphatase, α-glucosidase, and β-glucosidase, but negative for esterase lipase (C8), lipase (C14), cystine arylamidase, α-chymotrypsin, acid phosphatase, β-glucuronidase, N-acetyl-glucosaminidase and α-mannosidase. Positive for ONPG test, indole production and gelatinase, but negative for H2S production, Voges-Proskauer reaction, Simmons’ citrate utilization. Acid is produced from l-arabinose, d-xylose, d-galactose, d-glucose, d-mannose, amygdalin, ESC, d-cellobiose, d-maltose (weakly), d-lactose, d-melibiose, d-sucrose, d-raffinose, glycogen, d-gentiobiose. Positive for oxidation of d-trehalose, d-cellobiose (weakly), gentiobiose, N-acetyl-β-d-mannosamine (weakly), d-mannose, d-galactose. The major cellular fatty acids are iso-C16:0, iso-C15:0, and feature 9 (comprising C17:1 ω9c and / or 10-methyl C16:0). The major polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The exclusively respiratory quinone is Q-8.
The type strain TLK-CK17T (=CCTCC AB2021257T= KCTC 92122T) was isolated from cow dung compost sample collected from the Xinjiang Uygur Autonomous Region, China (43°81′N, 87°57′E). The genomic DNA G+C content of the type strain was 68.2 mol%.