Lysobacter chinensis sp. nov., a cellulose-degrading strain isolated from cow dung compost

A novel bacterial strain, TLK-CK17T, was isolated from cow dung compost sample. The strain was Gram-staining negative, non-gliding rods, aerobic, and displayed growth at 15–40 °C (optimally, 35 °C), with 0–5.0% (w/v) NaCl (optimally, 0.5) and at pH 6.5–8.5 (optimally, 7.0–7.5). The assembled genome of strain TLK-CK17T has a total length of 4.3 Mb with a G + C content of 68.2%. According to the genome analysis, strain TLK-CK17T encodes quite a few glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in compost. On the basis 16S rRNA gene sequence analysis, strain TLK-CK17T showed the highest sequence similarity (98.9%) with L. penaei GDMCC 1.1817 T, followed by L. maris KCTC 42381 T (98.3%). Cells contained iso-C16:0, iso-C15:0, and summed feature 9 (comprising C17:1ω9c and/or 10-methyl C16:0), as its major cellular fatty acids (> 10.0%) and ubiquinone-8 as the exclusively respiratory quinone. Diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol prevailed among phospholipids. Based on the phenotypic, genomic and phylogenetic data, strain TLK-CK17T represents a novel species of the genus Lysobacter, for which the name Lysobacter chinensis sp. nov. is proposed, and the type strain is TLK-CK17T (= CCTCC AB2021257T = KCTC 92122 T).


Introduction
The genus Lysobacter was proposed by , and it belongs to the family Lysobacteraceae, phylum 'Pseudomonadota'. As of December 2021, the genus includes 66 validly published species with correct name; the type species is Lysobacter enzymogenes . The main characteristics of the genus are Gram-staining negative, rod-shaped with pink to yellow colonies, and contain high DNA G + C content (61.7-70.1%) (Christensen 2005;Li et al. 2018). Strains of Lysobacter, thought to play vital roles in the environment for their high enzyme production capacity, are ubiquitous in various ecosystems. At present, they have been isolated from various habitats, including different types of soil such as cave soil (Chen et al. 2016), forest soil (Margesin et al. 2018), cultivated soil (Siddiqi and Im 2016), manganese factory soil (Li et al. 2018), arid area soil (Lee et al. 2017a) and abandoned gold mine (Liu et al. 2011). Here, we report a polyphasic taxonomical description of a novel, cellulose-degrading bacterium strain, designated TLK-CK17 T . The phenotypic, chemotaxonomic and genotypic properties indicate that strain TLK-CK17 T represents a novel species within the genus Lysobacter, for which the name Lysobacter chinensis sp. nov. is proposed.

Isolation and cultivation
In our investigation of the diversity of cultured bacteria in cow dung compost, Xinjiang Uygur Autonomous Region, China (43°81′N, 87°57′E), strain TLK-CK17 T was isolated. The compost sample was suspended in sterile water and serially diluted to 10 -1 , 10 -2 and 10 -3 , then 100 µL from each dilution was spread onto 1/3 nutrient agar (NA) plates. The plates were incubated at 30 °C and checked for growth after 2-3 days. After incubation, a single colony was purified by sub-culturing under the same conditions. For further investigation, the isolate was sub-cultured on NA plates at 35 °C and preserved at − 80 °C as glycerol suspension (20.0%, w/v). Meanwhile, closely related strains L. penaei GDMCC 1.1817 T , and L. maris KCTC 42381 T were obtained from the Guangdong Microbial Culture Collection Center (GDMCC) and the Korean Collection for Type Culture (KCTC), respectively.

DNA extraction and genome sequencing
Genomic DNA of strain TLK-CK17 T was extracted and purified using a bacterial genomic DNA kit (Takara), following the manufacturer's recommendations. The taxonomic position of strain TLK-CK17 T was first determined by 16S rRNA gene sequence using the forward primer 27F and the reverse primer 1492R as described previously (Liu et al. 2014). Whole-genome sequencing was performed on the Illumina HiSeq PE150 platform. A-tailed, ligated to paired-end adaptors and PCR amplified with a 350 bp insert was used for the library reconstruction. The raw reads were assembled using SOAPdenovo version 2.04 software (Li et al. 2008(Li et al. , 2010. The genes of strain TLK-CK17 T were identified by NCBI Prokaryotic Genome Annotation Pipeline server online and the Pfam database (Angiuoli et al. 2008;http:// pfam. xfam. org/), and the genes involved in metabolic pathways were analyzed in detail using the information present in RAST (Rapid Annotation using Subsystem Technology; https:// rast. nmpdr. org). The G + C content of the chromosomal DNA was calculated using genome sequencing. The digital DNA-DNA hybridization (dDDH) values were calculated using the Genome-to-Genome Distance Calculator (GGDC 2.0) (Meier-Kolthoff et al. 2013). The average nucleotide identity (ANI) values were calculated using the algorithm of Goris et al. (2007) by using the EzGenome web service (https:// www. ezbio cloud. net/).

Processing of sequence data and phylogenetic analysis
The complete 16S rRNA sequence of strain TLK-CK17 T was uploaded to the EzGenome web service and the NCBI GenBank to indentify the strain based on the sequences available. Multiple alignments with corresponding sequences of the closely related strains were aligned using CLUSTAL_X (Thompson et al. 1997). Phylogenetic analysis was conducted by neighbour-joining (NJ) (Saitou et al. 1987), maximumparsimony (MP) (Fitch et al. 1971) and maximumlikelihood (ML) (Felsenstein et al. 1981) methods in MEGA 7.0 program (Kumar et al. 2016) using the Kimura two-parameter model (Kimura et al. 1980), and the gaps were treated using a partial deletion method. Bootstrap analysis with 1000 replications was conducted, aimed at estimating the topology of the phylogenetic tree (Felsenstein et al. 1985). Moreover, the phylogenetic relationship based on nucleotide sequences was analysed via UBCG (Na et al. 2018), and the phylogenetic trees were constructed by using FastTree (Price et al. 2010) with GTR + CAT parameters and IQTree (Trifinopoulos et al. 2016) with GTR + F + I + G4 model and 1000 bootstrap replicates on the basis of 25 genomes.

Phenotypic and biochemical characteristics
Gram-staining and morphological features were tested with cells grown on NA plates at 35 °C for 24 h. Gram-staining was performed using a Gram stain kit (bioMérieux) according to the manufacturer's instructions. Cell morphology and size were examined with light microscopy (E600; Nikon) and transmission electron microscopy (JEM-1200; JEOL). Motility was determined using the hangingdrop method and gliding motility was determined as described by Bowman 2000. Reduction of nitrate was performed as described by Cowan and Steel 1974. Catalase, oxidase and lipase (Tweens 20 and 80) activities and hydrolysis of alginate, starch and CMcellulose were tested as described previously (Dong and Cai et al. 2001). Anaerobic growth was tested after incubation for 2 weeks at 35 °C on NA with or without 0.1% (w/v) KNO 3 , in an anaerobic chamber filled with a gas mixture (10% H 2 , 10% CO 2 and 80% N 2 ). Growth at different temperatures (0, 4, 10, 15, 20, 28, 30, 33, 37, 40, 45 and 50 °C) and at different concentrations of NaCl (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0%, w/v) were investigated on nutrient broth (NB) for up to 10 days. Growth at pH 5.5-9.5 (at intervals of 0.5 pH unit) was determined by measuring the optical density (wavelength 600 nm) of cultures in NB with the pH adjusted prior to sterilization by adding the appropriate buffers, including MES (for pH 5.5 and 6.0), PIPES (for pH 6.5 and 7.0), HEPES (for pH 7.5 and 8.0), Tricine (for pH 8.5) and CAPSO (for pH 9.0 and 9.5). Antibiotic sensitivity was assessed as described by the Clinical and Laboratory Standards Institute (CLSI et al. 2012), and inoculated plates were incubated at 35 °C for up to 48 h. Other physiological and biochemical characteristics of strain TLK-CK17 T and closely related strains were determined using the API 20E, API ZYM and API 50CHB identification systems (bioMérieux) and the Biolog GEN III identification system, according to the manufacturers' instructions.

Chemotaxonomic analysis
Cell biomass of strains TLK-CK17 T , L. penaei GDMCC 1.1817 T , and L. maris KCTC 42381 T were obtained from cultures grown in NB and on a rotary shaker (150 r.p.m) at 35 °C. After 24-h growth cells were centrifuged, washed three times in sterile distilled water and freeze-dried. The cellular fatty acid methyl esters were prepared and identified according to the Microbial Identification System (Sherlock version 4.5; database: TSBA40; MIDI; Sasser et al. 1990). The isoprenoid quinone strain TLK-CK17 T and two closely related strains were extracted from freeze-dried cell material using the two-stage method described by Tindall et al. (2007) and subsequently analysed by High performance liquid chromatographic (HPLC; Kroppenstedt et al. 1982). Polar lipids were extracted using a chloroform/methanol system and analysed by using two-dimensional thinlayer chromatography, as described previously (Minnikin et al. 1984). For two dimensional Thin Layer Chromatography (TLC) of polar lipid analysis, TLC on silica gel 60 F254 plates (Merck) were developed with chloroform/methanol/water (65:25:4, by vol.) for the first dimension and chloroform/acetic acid/ methanol/water (80:15:12:4, by vol.) for the second dimension. The total lipid profiles were stained with 10% molybdatopho-sphoric acid and the functional groups were determined using spray reagents specific for each one. Complete details are provided by Fang et al. (2017).

Phylogenetic analysis
According to the comparisons with the complete 16S rRNA gene sequence (1545 bp) in the EzTaxon database, the highest level of sequence similarity occurred with L. penaei GDMCC 1.1817 T (98.9%) and L. maris KCTC 42381 T (98.3%). The phylogenetic position of the novel isolate, determined using various tree-making algorithms, confirmed that strain TLK-CK17 T was a member of the genus Lysobacter, forming a coherent cluster with the two abovementioned members of this genus in the NJ, ML and MP trees with low bootstrap values, respectively (Fig. 1). Additional phylogenetic analyses based on a more comprehensive data set of validly published name strains genomes was presented in Fig. 2. On the basis of 16S rRNA gene sequence phylogenetic and phylogenomic analysis, two strains L. penaei GDMCC 1.1817 T and L. maris KCTC 42381 T were chosen as reference strains in this study.
Phenotypic and biochemical characterisation NA and NB 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 35 ℃, colonies were observed to be 1.0-1.5 mm in diameter, circular, smooth and apricot. Strain TLK-CK17 T was found to be Gram-staining negative and catalase negative bacterium. Meanwhile, it showed a positive reaction for oxidase (weakly) and nitrate reduction, nitrate can be reduced to nitrite. Cells are aerobic, 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-CK17 T are non-gliding rods, the mean cell size is 0.3-0.5 µm in width and 1.5-2.0 µm in length (Supplementary Fig. 2). The strain was positive for the hydrolysis of Tweens 20, 80, casein and CM-cellulose, but negative for alginate and starch. Antibiotic susceptibility test indicated that the strain was sensitive to chloramphenicol (30 μg), ceftriaxone (30 μg), 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). Strain TLK-CK17 T contained iso-C 16:0 (24.3%), iso-C 15:0 (23.8%), and summed feature 9 (comprising C 17:1 ω9c and / or 10-methyl C 16:0 , 15.4%) as predominant fatty acids (> 10.0%) in common with closely related strains. However, the ratios of the different components are different. The complete fatty acid composition was shown in Table 1. Ubiquinone-8 (Q-8) was the exclusively respiratory quinone. The polar lipid profile of strain TLK-CK17 T consists in phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG) as dominant elements, and one unknown polar lipid (L), which was similar to two closely related strains ( Supplementary Fig. 2). However, strain TLK-CK17 T and closely related strains were distinguishable from each other based on differences in other polar lipids. Strain TLK-CK17 T differed from L. penaei GDMCC 1.1817 T by the absence of one unidentified phospholipid (PL). Strain TLK-CK17 T differed from L. maris KCTC 42381 T by the existence or absence of two unknown polar lipids (L1 and L2).

Whole genome sequence analysis
The genome size and G + C content of strain TLK-CK17 T are 4,300,099 bp and 68.2%, respectively. Despite the close relationship, large difference in the genome size was found, the genome of strain TLK-CK17 T was larger than the genomes of L. penaei GDMCC 1.1817 T (3.2 Mb), and L. maris KCTC 42381 T (4.1 Mb), respectively. By contrast, strain TLK-CK17 T and them have a similar DNA G + C  (Table S1). The ANI values of strains TLK-CK17 T with L. penaei GDMCC 1.1817 T and L. maris KCTC 42381 T 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. 1988;Thompson et al. 2013). These results indicated that strain TLK-CK17 T represents a novel species of the genus Lysobacter. The related genome data strain TLK-CK17 T and two closely related type strains are listed in Tables S1 and S2.
The genome contributes to an important understanding of the genetic evolution of bacteria, disease prevention and treatment, and the development of antibiotics; thus, the genome of strain TLK-CK17 T was analysed to decipher the genetic code involved in the environmental suitability. 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 glycosyltransferases (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 suggests that they play important roles in the breakdown and modification of carbohydrates in cow dung composting. Compared with L. penaei GDMCC 1.1817 T and L. maris KCTC 42381 T , result showed that GH, GT and CBM family numbers were more in strain TLK-CK17 T , while the L. penaei GDMCC 1.1817 T contained more CE and AA family members. We considered that it might be closely related to their isolated environment, strains L. penaei GDMCC 1.1817 T and L. maris KCTC 42381 T 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-CK17 T 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 Table 1 Cellular fatty acid compositions of strain TLK-CK17 T and phylogenetically related species of the genus Lysobacter Strains: 1, TLK-CK17 T ; 2, L. penaei GDMCC 1.1817 T ; 3, L. maris KCTC 42381 T . Data were obtained in the present study unless indicated. Values are percentages of the total fatty acids, and only fatty acids comprising > 0.5% are shown.
The fatty acids in bold are the major cellular fatty acid (> 10.0%). Results are scored as follows: TR, Trace (< 0.5%); -, not detected * Summed features are groups of two or three fatty acids that could not be separated by gas/liquid chromatography with the Microbial Identification System (MIDI). Summed feature 1 contains iso-C 15:1 H and/or C 13:0 3-OH; summed feature 3 contains C 16:1 ω6c and/or C 16:1 ω7c; summed feature 8 contains C 18:1 ω7c and/or C 18:1 ω6c; summed feature 9 contains iso-C 17:1 ω9c and/or 10-methyl C 16:0 of strain TLK-CK17 T 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-CK17 T with other bacteria whether or not stimulate the production of antimicrobial compounds, so as to quickly remove pathogenic microorganisms in livestock manure.

Conclusion
The results of the phylogenetic analysis, phenotypic analysis and chemotaxonomic studies presented above support the view that strain TLK-CK17 T should be assigned to the genus Lysobacter. Based on the phenotypic analysis a comparison was made between the characteristics of strain TLK-CK17 T and closely related strains and a number of differences were observed, as shown in Table 2. Overall, considering its 16S rRNA gene sequence similarity (< 98.9%) to members of closely related taxa, its unique branching position in phylogenetic trees and the differences that the isolate exhibited described above, the isolate cannot be assigned to any recognized species. Therefore, strain TLK-CK17 T represents a novel species of 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-CK17 T is OK143236. Description of Lysobacter chinensis sp. nov.
Author contributions All authors contributed to the study conception and design. YYL wrote the manuscript and analysed the cultivation data. LYZ performed the genomic and phylogenetic analysis. YXP and PBL isolated the strain and performed the initial cultivation and strain deposition. YXX, JPD and ZQS contributed to text preparation and revised the manuscript. LF performed the electron microscopic analysis and prepared the SEM pictures. XWW and ZFW took the samples. All authors read and approved the final manuscript.
Funding This work was supported by the Xinjiang Academy of Agricultural Sciences Young Science and Technology Backbone Innovation Ability Training Project (xjnkq-2022019), Regional Collaborative Innovation Special Project of Xinjiang Uygur Autonomous Region (Nos. 2021E02022) and Forestry Development Subsidy Fund Project of Xinjiang Uygur Autonomous Region (Nos. XJLYKJ-2021-15).

Data availability
The genome and 16S rRNA gene sequence are available from GenBank under the accession numbers provided in the manuscript.

Conflict of interest
The authors declare that there is no conflict of interest.
Ethics approval This article does not contain any studies with animals performed by any of the authors.