Description and genomic characterization of Cohnella amylolytica sp. nov., isolated from Hot Springs in Yunnan province, south-west China

doi:10.21203/rs.3.rs-3147728/v1

Download PDF

Research Article

Description and genomic characterization of Cohnella amylolytica sp. nov., isolated from Hot Springs in Yunnan province, south-west China

https://doi.org/10.21203/rs.3.rs-3147728/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 08 Jan, 2024

Read the published version in Antonie van Leeuwenhoek →

You are reading this latest preprint version

A bacterial strain, Gram staining positive, strictly aerobic, rod-shaped, motile bacterium with flagellum and endospore-forming, designated strain YIM B05605^T, was isolated from soil sampled in Hamazui hot springs, Tengchong City, Yunnan province, China. Optimum growth for the strain occurred at pH 7.0 and 45 °C. The predominant menaquinone of strain YIM B05605^T was MK-7. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), unidentified glycolipid (GL), three unknown aminophospholipids (APLs) and unidentified polarlipid (PL) were part of the polar lipid profle. The major fatty acids were anteiso-C_15:0 and iso-C_16:0. The DNA G+C content of the type strain was 58.76 mol%. Genome-based phylogenetic analysis confirmed that strain YIM B05605^T formed a distinct phylogenetic cluster within the genus Cohnella. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strain YIM B05605^T with the most related species C. fontinalis YT-1101^T were 73.42 % and 15.7 %. Based on phylogenomic and phylogenetic analyses coupled with phenotypic and chemotaxonomic characterizations, strain YIM B05605^T could be classified as a novel species of the genus Cohnella, for which the name Cohnella amylolytica sp. nov., is proposed. The type strain is YIM B05605^T (= CGMCC 1.60052 ^T= KCTC 43462 ^T= NBRC 115921 ^T).

Cohnella

Genomics

Phylogeny

Taxonomy

The genus Cohnella was first proposed by Kämpfer et al.(Kämpfer et al. 2006)，affiliated to the family Paenibacillaceae, phylum Firmicutes. The members of the genus Cohnella are Gram-positive, rod-shaped, spore-forming, aerobic and motile or non-motile bacteria. Most species are thermotolerant, contain MK-7 as the main menaquinone. Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine (García-Fraile et al. 2008). At present, the genus Cohnella contains 34 species with validly published names (http://www.bacterio.net/cohnella.html ) which isolated from various environments, including rhizospheric soil (Jiang et al. 2019, Wang et al. 2015), freshwater green alga (Lee and Jeon 2017), arctic tundra soil(Jiang et al. 2012), buffalo faeces (Khianngam et al. 2012), bark (Meng et al. 2019), haematite ore (Mayilraj et al. 2011), fresh water (Shiratori et al. 2010), wild grass fermentation broth (Won et al. 2022), siberian permafrost (Kudryashova et al. 2018), volcanic pond (Cho et al. 2007) and maize callus cultures (Johnson et al. 2021). During the diversity and novelty study of bacterium in geothermal environment of Tengchong City, Yunnan province, China, the strain designated as YIM B05605^T was isolated from Hamazui hot spring. In the present study, polyphasic taxonomy, genomic and phylogenetic analyses were used to clarify the taxonomic status of strain YIM B05605^T.

Bacterial Isolation

The soil samples were collected from Hamazui hot spring. The isolation medium contained the following components, microcrystalline cellulose 1g, acid hydrolyzed casein 1g, CaCO₃ 0.5g, FeSO₄ 1g, agar 20g, water 1L, pH 6.0, after 14 days of incubation at 45 ℃, supplemented with sodium taurocholate. The strain was maintained on TSA agar at 4 ℃, 20 % (v/v) aqueous glycerol suspension at -80 °C for short-term preservation and 20 % (w/v) skim milk after freeze drying for long-term preservation. Biomass for molecular and chemical research is on TSB (pH 7.0) at 45 ℃ and 200 rpm for 1 week.

16S rRNA gene sequence analysis

To extract the genomic DNA of strain YIM B05605^T, a little cell mass was soaked with 50 μL 5 % (w/v) chelex‐100 solution and boiled 15 min. PCR amplification of 16S rRNA gene sequence was performed using universal primer sets 27F and 1492R (Weisburg et al. 1991). The PCR products were purified using PCR purification kit (Axygen), then cloned into the pEASYT1 vector (TransGen Biotech) following the manufacturer’s standard protocol, and finally sequenced at Sangon Biotech (Shanghai) Co., Ltd. (https://www.sangon.com). The obtained 16S rRNA gene sequence was compared with available 16S rRNA gene sequences of validly published species in EzBioCloud server (https://www.ezbiocloud.net/) to determine the similarities with the corresponding sequences of closely related type species. Multiple sequence alignments were carried out using Clustal W tool (Larkin et al. 2007). The phylogenetic tree was reconstructed by the neighbour-joining ( Saitou and Nei, 1987), maximum-parsimony (Fitch 1987) and maximum-likelihood (Felsenstein 1981) algorithms in MEGA version 7 software (Kumar et al. 2018) with 1000 bootstrap repetitions (Felsenstein 1985). Evolutionary distance matrices of the neighbour-joining tree were calculated using Kimura’s two-parameter model (Kimura 1980).

Genomic analysis and annotation of gene functions

The genomic DNA of strain YIM B05605^T for draft genome sequencing were extracted using the SDS method (Lim et al. 2016). The harvested DNA were tested by the agarose gel electrophoresis and quantified by Qubit® 2.0 Fluorometer (Thermo Scientific). A-tailed ligated paired-end adaptors and PCR amplified with a 350 bp insert were used for the library construction. The draft genomes were sequenced by PacBio and Illumina Hiseq 2000 sequencers at Shanghai Majorbio Bio-pharm Technology Co., Ltd (https://www.majorbio.com). Using SOAPdenovo software version 2.04 to assemble the sequenced reads (Li et al. 2010). The obtained draft genome of strain YIM B05605^T has been submitted to DDBJ/ENA/GenBank under the accession number JAQZSJ000000000. The version described in this paper was version JAQZSJ010000000. The DNA G + C content of strain YIM B05605^T was calculated according to the draft genome.

For the phylogenomic analysis, automated multi-locus species tree (autoMLST) pipeline (https://automlst.ziemertlab.com/) (Alanjary et al. 2019) was used to align series of conserved core genes present in strain YIM B05605^T and closely related species. The genomes used for phylogenomic analysis were based on 16S rRNA gene sequence analysis and automatically selected by software of autoMLST. The final phylogenomic tree based on genomic data from multiple loci was reconstructed using the maximum-likelihood algorithm in software package MEGA version 7, with Thermobacillus xylanilyticus XE^T (AJ005795) as an outgroup. In addition, to construct a phylogenomic tree, the core proteomes of strain YIM B05605^T and other related species (based on 16S rRNA gene sequence analysis) were extracted and aligned in M1CR0B1AL1Z3R web server (https://microbializer.tau.ac.il/) with the minimal percent of identity for proteins to be 80 and the minimal percent of orthologs to be considered as a core gene to be 100 (Avram et al. 2019). The maximum-likelihood phylogenomic tree based on concatenated core proteins was reconstructed using MEGA 7 software with 1000 bootstrap replicates and default parameters. To provide cogent evidence for the classification of strain YIM B05605^T as a novel species within the genus Cohnella, ANI value between the YIM B05605^T with the most closely related species C. fontinalis YT-1101^T and the estimated digital DNA-DNA hybridization (dDDH) (Auch et al. 2010) value were calculated using the genome to genome distance calculator with recommended BLAST + alignment and Formula 2 available at the DSMZ server (https://ggdc.dsmz.de/distcalc2.php) (Meier-Kolthoff et al. 2013).

Functional annotation of the genome in strain YIM B05605^T were performed by Clusters of Orthologous Groups (COG) database (Galperin et al. 2015) and Carbohydrate‐Active EnZymes (CAZy) database (Zhang et al. 2018). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (CSL STYLE ERROR: reference with no printed form.) was carried out to determine the key potential pathways in YIM B05605^T. In order to analyse the bioactive potential of strain YIM B05605^T biosynthesis gene clusters (BGCs) were predicted using Antibiotic and Secondary Metabolite Analysis Shell (antiSMASH) (Blin et al. 2021).

Morphology, physiological and biochemical characteristics

Light microscopy (BX41, Olympus) and transmission electron microscopy (JEM-2100, JEOL) were used to observe the morphology, motility and size of cell, and the cells were grown exponentially, strains grow on TSA medium at 45℃ for 3 days and stored in refrigerator at 4 ℃ for 3 days. The Gram-staining test was performed using Gram-staining kit (Changde BKMAN Biotechnology Co., Ltd). The temperature ranges for growth of strain YIM B05605^T and reference strain were tested by incubation on TSA at 4, 10, 15, 20, 25, 28, 30, 35, 37, 40, 45 ,50, 55 and 60 °C for 1 week. The pH range for growth was measured by incubation on TSA with pH 4-12 (at intervals of 1 pH unit) for 1 week. The NaCl tolerance range for growth was determined by incubation on TSA agar with 0-5 % (w/v) (at intervals of 0.5 % unit) NaCl for 1 week. Catalase activity was detected by bubble production in 5 % (v/v) H₂O₂. Oxidase activity was assessed by using oxidase reagent (bioMérieux). Gelatinase activities, starch hydrolysis, nitrate reduction, urease, milk peptonization and coagulation, and H₂S and melanin production were assessed as described by Gonzalez et al.(Gonzalez et al. 1978). Substrate utilization and chemical sensitivity were determined using Bilog Gen III Microplate. The other enzyme activities and acid production of carbohydrates were tested using the API ZYM, API 20NE and API 50CH systems (bioMérieux) according to the manufacturer’s instructions. Antibiotic susceptibility tests were performed on TSA using disks impregnated with various antimicrobial compounds (Amoozegar et al. 2014).

Chemotaxonomic characterization

For the chemotaxonomic analysis of quinones and polar lipids, strain YIM B05605^T was cultivated in TSB at 45°C for 1 week with 200 rpm. The polar lipids of strain YIM B05605^T was extracted from freeze-dried cells according to the method of Minnikin et al. (Minnikin et al. 1984) and analysed by twodimensional thinlayer chromatography on silica gel 60 plate (10 × 10 cm, Merck) using chloroform-methanol-water (64:27:5, by vol.) as the first dimension and chloroform-methanol-acetic acid-water (80:18:12:5, by vol.) as the second dimension. The following spray reagents were respectively used for determining total lipids, phosphorus‐containing lipids, lipids containing amino groups and glycolipids: 10 % (w/v) ethanolic molybdophosphoric acid, molybdenum blue spray reagent, 0.4 % (w/v) solution of ninhydrin in acetone and α-naphthol (0.3 g α-naphthol: 1.3 ml H₂SO₄: 10 ml 95 % ethanol). Menaquinones were extracted according to Collins et al. (Collins et al. 1977) and separated by HPLC (Tamaoka 1986). Amino acids in cell walls and sugars in whole cell hydrolysates were analyzed according to the procedures described by Hasegawa et al. (Hasegawa et al. 1983; Tang et al. 2009). The Sherlock Microbial Identification System (MIDI) was used to extract, methylate and analyze cellular fatty acids according to the manufacturer’s instructions. For fatty acid analysis, strain YIM B05605^T and reference strain were cultured at 45 ℃ on TSA (Difco) for 4 days. The Microbial Identification software package (Sherlock Version 6.1; MIDI databaseTSBA6) was used to analyze fatty acid methyl esters (Moss and Dees 1975). The separation of fatty acid methyl esters was by using capillary column DB‐5MS (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Agilent Technologies), and analysed by using a Trace 1310 gas chromatograph coupled with a Trace ISQ single quadrupole mass spectrometer equipped with Nist 08 Library software database (Tuo et al. 2019).

Phylogenetic analysis based on 16S rRNA gene sequence

The nearly complete 16S rRNA gene sequence of strain YIM B05605^T contains 1545 nucleotides. Strain YIM B05605^T was closely related to C. candidum 18JY8-7^T with 96.61 % 16S rRNA gene sequence similarity. The neighbor-joining phylogenetic tree showed that strain YIM B05605^T formed a distinct and stable clade within the genus Cohnella with C. fontinalis YT-1101^T as the closest relative, and supported by 100 % bootstrap value (Fig. 1). The similar topological results were also obtained from the maximum-likelihood and maximum-parsimony phylogenetic trees (Figs. S1 and S2). It was obvious that strain YIM B05605^T were phylogenetically affiliated to the genus Cohnella.

Genomic analysis and annotation of gene functions

The dDDH, ANI values between YIM B05605^T and C. fontinalis YT-1101^T were 15.7 %, 73.42 %, respectively. These values were significantly lower than the currently established boundaries for genomic species definition (70 % for dDDH, 95-96 % for ANI) (Wayne 1988; Richter and Rosselló-Móra 2009), which demonstrated that strains YIM B05605^T and C. fontinalis YT-1101^T were not belong to the same species. According to the genome-based phylogenetic tree (Fig. 3), strain YIM B05605^T formed a distinct phylogenetic cluster within the genus Cohnella with a high bootstrap value, confirming the topology determined by 16S rRNA gene sequences. These results proved that strain YIM B05605^T should be assigned to the genus Cohnella and represented novel species.

The genome of strain YIM B05605^T was assembled into 55 contigs with a total length of 5,510,416 bp. The G + C content was 58.76 mol% with N50 value of 243,336 bp. Strain YIM B05605^T was predicted to harbor 4,957 protein-coding genes (CDS), The numbers of tRNA and rRNA were 45 and 4, respectively. The genome of strain C. fontinalis YT-1101^T was assembled into 161 contigs with a total length of 6,658,403 bp. The G + C content was 57.78 mol% with N50 value of 161,101 bp. Strain C. fontinalis YT-1101^T was predicted to harbor 5,690 protein-coding genes (CDS), The numbers of tRNA and rRNA were 56 and 7, respectively. Sequences matching antibiotic resistance genes and virulence factors were retrieved from the genomes of strains YIM B05605^T and C. fontinalis YT-1101^T in accordance with the analysis carried out using CARD, PHI and VFDB database. 314 and 350 antibiotic resistance genes (annotated in CARD database) were respectively predicted in genomes of strains YIM B05605^T and C. fontinalis YT-1101^T. More detailed general features of the genome of strains YIM B05605^T and C. fontinalis YT-1101^T were summarized in Table 1 and the distribution of specific genomic regions was displayed in Fig. 2.

Functional analysis by COG database revealed that strains YIM B05605^T and C. fontinalis YT-1101^T had 4,131 and 5,019 genes, respectively. Both assigned to 21 and 23 categories, respectively. The most abundant category of strain YIM B05605^T was carbohydrate transport and metabolism (G, 16.2 %). In this, gene 4225 encodes a chitinase (table S1). Followed by transcription (K, 12.5 %), signal transduction mechanisms (T, 10.7 %), general function prediction only (R, 9.4 %), amino acid transport and metabolism (E, 7.4 %), cell wall/membrane/envelope biogenesis (M, 6.2 %). The most abundant category of strain C. fontinalis YT-1101^T was function unknown (S, 30.5 %), carbohydrate transport and metabolism (G, 14.3 %). In this, gene 0645, gene 0970 and gene 1012 encode three chitinases (table S2). signal transduction mechanisms (T, 8.7 %), transcription (K, 8.4 %), inorganic ion transport and metabolism (P, 6.4 %), amino acid transport and metabolism (E, 4.9 %). The detailed distributions of genes associated with general COG functional categories in the genome of strains YIM B05605^T and C. fontinalis YT-1101^T were presented in Fig. S3. Functional analysis by CAZy database found that 215 genes in genome of strain YIM B05605^T and 285 genes in strain C. fontinalis YT-1101^T were assigned to CAZy families. The strain YIM B05605^T have 5 functional CAZy classes, the largest class was glycoside hydrolases (GHs, 122 domain), followed by carbohydrate esterases (CEs, 46 domain), glycosyl transferases (GTs, 39 domain), auxiliary activities (AAs, 17 domain) and carbohydrate‐binding modules (CBMs, 1 domain). In addition to the above 5 functional CAZy classes, there is polysaccharide lyases (PLs, 8 domain) in strain C. fontinalis YT-1101^T. The detailed distribution of genes associated with general CAZy functional categories in the genome of strains YIM B05605^T and C. fontinalis YT-1101^T were presented in Fig. S4. Functional analysis by KEGG database revealed that strains YIM B05605^T and C. fontinalis YT-1101^T had 2,606 and 2,763 genes, respectively. In strain YIM B05605^T of carbohydrate metabolism, gene 4010 encodes a chitinase, both gene 3531 and gene 4979 encode starch synthase (table S3). In strain C. fontinalis YT-1101^T of carbohydrate metabolism, gene 0604 encodes a chitinase (table S4).The detailed distribution of genes associated with general KEGG functional categories in the genome of strains YIM B05605^T and C. fontinalis YT-1101^T were presented in Fig. S5.

The prediction of antiSMASH revealed that strain YIM B05605^T contained six biosynthetic gene clusters encoded for secondary metabolites, including redox-cofactor, LAP, terpene, T3PKS, cyclic-lactone-autoinducer and RiPP-like. Among them, lankacidin C , as a microtubule stabilizer by binding to the paclitaxel binding site, by 17-membered polyketide, exhibits considerable antitumor activity (Ayoub et al. 2022). Lankacidin C and lankamycin, a 14-membered macrolide produced in the same strain, can synergistically inhibit protein synthesis by binding to the neighboring sites in the bacterial large ribosomal subunit (Auerbach et al. 2010; Belousoff et al. 2011). Lankacidins exert considerable antitumor activity against various cell lines including L1210 leukemia, B16 melanoma, HeLa, and the breast cancer T47D cells (Ootsu and Matsumoto 1973; Oostu et al. 1975; Ayoub et al. 2016).

Morphology, physiological and biochemical characteristics

Strain YIM B05605^T was Gram-stain positive, strictly aerobic and endospore-forming. Under transmission electron micrographs, the shape of the cells was rod-shaped (approximately 0.37-0.93 × 1.97-3.21 µm) with flagella and the endospores (Fig. 4). Colonies of strain YIM B05605^T was punctiform, circular and white-cream on TSA when incubated at 45 °C for 5 days. Strain YIM B05605^T grew at pH ranging from pH 6.0 to 8.0 with optimum growth at pH 7.0which was slightly different from its experimental control (C. fontinalis YT-1101^T; Table 2). Strain YIM B05605^T grew at temperatures ranging from 37 to 50 °C with optimum growth 45 °C, NaCl tolerance ranging from 0 to 3 % with optimum growth 0.5 %. Like strain C. fontinalis YT-1101^T, strain YIM B05605^T was positive for oxidase and catalase. Negative for nitrate reduction , while C. fontinalis YT-1101^T was positive. Unlike C. fontinalis YT-1101^T, strain YIM B05605^T was positive for hydrolysis of starch and gelatin. Strains YIM B05605^T and C. fontinalis YT-1101^T were negative for hydrolyzed of tweens 20, 40, 60 and 80, H₂S production, cellulose, milk peptonization and coagulatio. In API 20NE and API ZYM strips, strain YIM B05605^T was negative for tryptophan, glucose, D-mannitol, gluconate, esterase lipase C8, leucine arylamidase and α-fucosidase, while C. fontinalis YT-1101^T was positive. Strain YIM B05605^T was positive for acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase, while C. fontinalis YT-1101^T was negative. The data of API 50CH, Biolog GEN III MicroPlate tests and antibiotic susceptibility for strains YIM B05605^T and C. fontinalis YT-1101^T were shown in Table S5, Table S6 and Table S7, respectively.

Chemotaxonomic characterization

The polar lipid of strain YIM B05605^T was comprised of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), unidentified glycolipid (GL), three unknown aminophospholipids (APLs) and unidentified polarlipid (PL) (Fig. S6). MK-7 were the isoprenoid quinone present in strain YIM B05605^T, which was consistent with members of the genus Cohnella. The cell wall peptidoglycan contained glutamic acid, serine, threonine and alanine, the diagnostic diamino acid was meso-diaminopimelic acid. The predominant fatty acid (>10 %) of strain YIM B05605^T were identified as anteiso-C_15:0(41.34 %) and iso-C_16:0(27.60 %) and that of C. fontinalis YT-1101^T were anteiso-C_15:0(44.81 %) and iso-C_16:0(28.37 %). The fatty acid profiles of strains YIM B05605^T and C. fontinalis YT-1101^T showed disparities from the reference strains were presented in Supplementary Table S8.

In this research, a Cohnella strain, namely YIM B05605^T, was isolated from soil collected from Hamazui hot springs, Tengchong City, Yunnan Province, China. On the basis of evidences from the phylogenetic and phylogenomic analyses, physiological and chemotaxonomic features, we conclude that the strain should be assigned to new species of the genus Cohnella, for which the name Cohnella amylolytica sp. nov. is proposed, with YIM B05605^T (= CGMCC 1.60052 ^T= KCTC 43462 ^T= NBRC 115921 ^T) as a type strain.

Description of Cohnella amylolytica sp. nov.

Cells are strictly aerobic, Gram-stain positive, rod-shaped (approximately 0.37-0.93 × 1.97-3.21 µm) with flagella and endospores, colonies on TSA were punctiform, circular and white-cream. Substrate mycelia, aerial mycelia and diffusible pigments were not observed on any of the tested media. Able to tolerate a wide range of temperature (37-50 °C, optimum at 45 °C), NaCl (0-3 %, optimum at 0.5 %) and pH (6-8, optimum at pH 7). Cells are positive for oxidase and catalase, hydrolysis of starch and gelatin. Nitrate reduction, tweens 20, 40, 60 and 80, H₂S production, cellulose, milk peptonization and coagulation are negative. Results from API 50CH tests showed that acids was produced from glycol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl β-D-xylopyranoside, D-galactose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, sorbitol, methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, N-acetylglucosamine, amygdalin, aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-raffinose, starch, D-lyxose, D-arabitol and 5-ketogluconate. Sensitive to chloroamphenicol (30 μg), carbenicillin (100 μg), novobiocin (30 μg), compound sulfamethoxa (25 μg), ampicillin (10 μg), genlamicin (10 μg), kanamycin (30 μg), neomycin (30 μg), penicillin (10 μg), erythromycin (15 μg), tetracycline (30 μg), polymyxin B (100 μg) and streptomycin (10 μg). Positive for esterase C4, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. The cell wall peptidoglycan contained glutamic acid, serine, threonine and alanine, the diagnostic diamino acid was meso-diaminopimelic acid. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, unidentified glycolipid, three unknown aminophospholipids and unidentified polarlipid. The predominant menaquinone is MK-7. The major fatty acids are anteiso-C_15:0and iso-C_16:0. The DNA G+C content of the type strain is 58.76 mol%. The type strain is YIM B05605^T (= CGMCC 1.60052 ^T= KCTC 43462 ^T= NBRC 115921 ^T), isolated from Hamazui hot springs, Tengchong City, Yunnan province, China.

Nucleotide sequence accession numbers

The GenBank accession number for the 16S rRNA gene sequence of strain YIM B05605^T was OQ426529. The DDBJ/ENA/GenBank accession number for the genome sequence of strain B05605^T was JAQZSJ000000000.

Acknowledgements

The authors are grateful to Dr Moriyuki Hamada for kindly providing C. fontinalis YT-1101^T. This study was performed with support of the Science and Technology Planning Project of Yunnan Province (202207AB110016), Major Science and Technology Projects of Yunnan Province (Digitalization, development, and application of biotic resource, 202002AA100007) and the National Natural Science Foundation of China (82160674, 31760003 and 31860017).

Author contributions

Ming-Xian Xiang: Writing-original draft (lead); Cui-Ping Miao: Concept design, revise and reviewing (lead); Juan Wang: bacterial isolation; Yi-Qing Li：Resources (equal); Shu-Kun Tang: Funding acquisition (equal); Resources (equal). Min Yin: Funding acquisition (equal); Resources (equal).

Confict of interest

Authors are required to disclose nonfnancial interests.

Ethical approval

This work did not include humans or animals.

Consent for publication

This work did not include details, images, or videos relating to an individual person.

Alanjary M, Steinke K, Ziemert N (2019) AutoMLST: an automated web server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Res 47:276–282. https://doi.org/10.1093/nar/gkz282
Amoozegar MA, Bagheri M, Makhdoumi-Kakhki A, et al (2014) Oceanobacillus limi sp. nov., a moderately halophilic bacterium from a salt lake. Int J Syst Evol Microbiol 64:1284–1289. https://doi.org/10.1099/ijs.0.057265-0
Auch AF, von Jan M, Klenk H-P, Göker M (2010) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2:117–134. https://doi.org/10.4056/sigs.531120
Auerbach T, Mermershtain I, Davidovich C, et al (2010) The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics. Proc Natl Acad Sci USA 107:1983–1988. https://doi.org/10.1073/pnas.0914100107
Avram O, Rapoport D, Portugez S, Pupko T (2019) M1CR0B1AL1Z3R-a user-friendly web server for the analysis of large-scale microbial genomics data. Nucleic Acids Res 47:W88–W92. https://doi.org/10.1093/nar/gkz423
Ayoub AT, Abou El-Magd RM, Xiao J, et al (2016) Antitumor activity of lankacidin group antibiotics is due to microtubule stabilization via a paclitaxel-like mechanism. J Med Chem 59:9532–9540. https://doi.org/10.1021/acs.jmedchem.6b01264
Ayoub AT, Nishiura N, Teshima A, et al (2022) Bioinspired computational design of lankacidin derivatives for improvement in antitumor activity. Future Medicinal Chemistry 14:1349–1360. https://doi.org/10.4155/fmc-2022-0134
Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Gerdes, S., Glass, E.M., Kubal, M., Meyer, F., Olsen, G.J., Olson, R., Osterman, A.L., Overbeek, R.A., McNeil, L.K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G.D., Reich, C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A., Zagnitko, O. (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9, 75. https://doi.org/10.1186/1471-2164-9-75
Belousoff MJ, Shapira T, Bashan A, et al (2011) Crystal structure of the synergistic antibiotic pair, lankamycin and lankacidin, in complex with the large ribosomal subunit. Proc Natl Acad Sci USA 108:2717–2722. https://doi.org/10.1073/pnas.1019406108
Blin K, Shaw S, Kautsar SA, et al (2021) The antiSMASH database version 3: increased taxonomic coverage and new query features for modular enzymes. Nucleic Acids Res 49:D639–D643. https://doi.org/10.1093/nar/gkaa978
Cho E-A, Lee J-S, Lee KC, et al (2007) Cohnella laeviribosi sp. nov., isolated from a volcanic pond. Int J Syst Evol Microbiol 57:2902–2907. https://doi.org/10.1099/ijs.0.64844-0
Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100:221–230. https://doi.org/10.1099/00221287-100-2-221
Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376. https://doi.org/10.1007/BF01734359
Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4), 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Fitch W (1987) Citation - Classic - Toward Defining the Course of Evolution - Minimum Change for a Specific Tree Topology. Current Contents/Agriculture Biology & Environmental Sciences 14–14.
Galperin MY, Makarova KS, Wolf YI, Koonin EV (2015) Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucleic Acids Res 43:D261-269. https://doi.org/10.1093/nar/gku1223
García-Fraile P, Velázquez E, Mateos PF, et al (2008) Cohnella phaseoli sp. nov., isolated from root nodules of Phaseolus coccineus in Spain, and emended description of the genus Cohnella. Int J Syst Evol Microbiol 58:1855–1859. https://doi.org/10.1099/ijs.0.65468-0
Gonzalez C, Gutierrez C, Ramirez C (1978) Halobacterium vallismortis sp. nov. an amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24:710–715. https://doi.org/10.1139/m78-119
Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29:319–322.
Jiang F, Dai J, Wang Y, et al (2012) Cohnella arctica sp. nov., isolated from arctic tundra soil. Int J Syst Evol Microbiol 62:817–821. https://doi.org/10.1099/ijs.0.030247-0
Jiang L, Pheng S, Lee KC, et al (2019) Cohnella abietis sp. nov., isolated from Korean fir (Abies koreana) rhizospheric soil of Halla mountain. J Microbiol 57:953–958. https://doi.org/10.1007/s12275-019-9136-1
Johnson ET, Dowd PF, Skory CD, Dunlap CA (2021) Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures. Antonie van Leeuwenhoek 114:37–44. https://doi.org/10.1007/s10482-020-01495-2
Kämpfer P, Rosselló-Mora R, Falsen E, et al (2006) Cohnella amylolytica gen. nov., sp. nov., and classification of ‘Paenibacillus hongkongensis’ as Cohnella hongkongensis sp. nov. Int J Syst Evol Microbiol 56:781–786. https://doi.org/10.1099/ijs.0.63985-0
Kanehisa M, Goto S. (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28(1), 27-30. https://doi.org/10.1093/nar/28.1.27
Khianngam S, Tanasupawat S, Akaracharanya A, et al (2012) Cohnella cellulosilytica sp. nov., isolated from buffalo faeces. Int J Syst Evol Microbiol 62:1921–1925. https://doi.org/10.1099/ijs.0.032607-0
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581
Kudryashova EB, Karlyshev AV, Ariskina EV, et al (2018) Cohnella kolymensis sp. nov., a novel bacillus isolated from Siberian permafrost. Int J Syst Evol Microbiol 68:2912–2917. https://doi.org/10.1099/ijsem.0.002919
Kumar S, Stecher G, Li M, et al (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Larkin MA, Blackshields G, Brown NP, et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. https://doi.org/10.1093/bioinformatics/btm404
Lee Y, Jeon CO (2017) Cohnella algarum sp. nov., isolated from a freshwater green alga Paulinella chromatophora. Int J Syst Evol Microbiol 67:4767–4772. https://doi.org/10.1099/ijsem.0.002377
Li R, Zhu H, Ruan J, et al (2010) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 20:265–272. https://doi.org/10.1101/gr.097261.109
Lim HJ, Lee E-H, Yoon Y, et al (2016) Portable lysis apparatus for rapid single-step DNA extraction of Bacillus subtilis. J Appl Microbiol 120:379–387. https://doi.org/10.1111/jam.13011
Mayilraj S, Ruckmani A, Kaur C, et al (2011) Cohnella ferri sp. nov. a novel member of the genus Cohnella isolated from haematite ore. Curr Microbiol 62:1704–1709. https://doi.org/10.1007/s00284-011-9917-1
Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. https://doi.org/10.1186/1471-2105-14-60
Meng L-J, Tuo L, Yan X-R (2019) Cohnella endophytica sp. nov., a novel endophytic bacterium isolated from bark of Sonneratia apetala. Int J Syst Evol Microbiol 69:2004–2009. https://doi.org/10.1099/ijsem.0.003417
Minnikin D, Odonnell A, Goodfellow M, et al (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241.
Mise K, Iwasaki W (2020) Environmental atlas of prokaryotes enables powerful and intuitive habitat-based analysis of community structures. iScience 23:101624. https://doi.org/10.1016/j.isci.2020.101624
Moss CW, Dees SB (1975) Identification of microorganisms by gas chromatographic-mass spectrometric analysis of cellular fatty acids. J Chromatogr 112:594–604. https://doi.org/10.1016/s0021-9673(00)99988-6
Oostu K, Matsumoto T, Harada S, Kishi T (1975) Antitumor and immunosuppressive activities of lankacidin-group antibiotics: structure-activity relationships. Cancer Chemother Rep 59:919–928.
Ootsu K, Matsumoto T (1973) Effects of lankacidin group (T2636) antibiotics on the tumor growth and immune response against sheep erythrocytes in mice. Gan 64:481–492.
Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proceedings of the National Academy of Sciences 106:19126–19131. https://doi.org/10.1073/pnas.0906412106
Saitou N, Nei M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4(4), 406-425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Shiratori H, Tagami Y, Beppu T, Ueda K (2010) Cohnella fontinalis sp. nov., a xylanolytic bacterium isolated from fresh water. Int J Syst Evol Microbiol 60:1344–1348. https://doi.org/10.1099/ijs.0.014605-0
Tamaoka J (1986) Analysis of bacterial menaquinone mixtures by reverse-phase high-performance liquid chromatography. Methods Enzymol 123:251–256. https://doi.org/10.1016/s0076-6879(86)23028-1
Tang J, Zhao J-Y, Li L-L, et al (2022) Cohnella mopanensis sp. nov., a new Gram-negative bacterium isolated from soil in Xinping Mountain National Forest Park. Arch Microbiol 204:492. https://doi.org/10.1007/s00203-022-03050-1
Tang S-K, Wang Y, Chen Y, et al (2009) Zhihengliuella alba sp. nov., and emended description of the genus Zhihengliuella. Int J Syst Evol Microbiol 59:2025–2031. https://doi.org/10.1099/ijs.0.007344-0
Tuo L, Yan X-R, Li F-N, et al (2019) Amnibacterium flavum sp. nov., a novel endophytic actinobacterium isolated from bark of Nerium indicum Mill. Int J Syst Evol Microbiol 69:285–290. https://doi.org/10.1099/ijsem.0.003156
Wang L-Y, Wang T-S, Chen S-F (2015) Cohnella capsici sp. nov., a novel nitrogen-fixing species isolated from Capsicum annuum rhizosphere soil, and emended description of Cohnella plantaginis. Antonie Van Leeuwenhoek 107:133–139. https://doi.org/10.1007/s10482-014-0310-5
Wayne LG (1988) International committee on systematic bacteriology: announcement of the report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Zentralbl Bakteriol Mikrobiol Hyg A 268:433–434. https://doi.org/10.1016/s0176-6724(88)80120-2
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703. https://doi.org/10.1128/jb.173.2.697-703.1991
Won M, Lee SA, Hong SB, Han BH, Kwon SW (2022) Cohnella herbarum sp. nov., isolated from wild grass fermentation broth. Int J Syst Evol Microbiol.;72(8). https://doi.org/10.1099/ijsem.0.005485
Yoon S-H, Ha S-M, Lim J, et al (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286. https://doi.org/10.1007/s10482-017-0844-4
Zhang H, Yohe T, Huang L, et al (2018) dbCAN2: a meta server for automated carbohydrate-active enzyme annotation. Nucleic Acids Res 46:W95–W101. https://doi.org/10.1093/nar/gky418

Table 1 General genome chatacteristics of strains YIM B05605^T and C. fontinalis YT-1101^T.

	YIM B05605^T	*C. fontinalis* YT-1101^T
Sequence information
Genome length (bp)	5,510,416	6,658,403
Number of contigs	55	161
G + C content (mol%)	58.76	57.78
N50 (bp)	243,336	161,101
L50	7	14
Annotation information
CDS	4,957	5,690
tRNA genes	45	56
rRNA genes	4	7
Protein features
Proteins with GO assignments	1,626	2,258
Proteins with COG assignments	4,131	5,019
Proteins with KEGG assignments	2,606	2,763
Specialty genes/source
Antibiotic Resistance/CARD	314	350
Pathogen Host Interactions/PHI	1,005	1,089
Virulent Factor/VFDB	552	622

Table 2 Differential characteristics between strains YIM B05605^T and C. fontinalis YT-1101^T. All data shown were obtained in this study. +, Positive; - Negative.

Characteristic	YIM B05605^T	C. fontinalis YT-1101^T
Growth pH range (optimum)	6.0-8.0 (7.0)	6.0-9.0 (7.0)
Temperature range for growth (optimum) (°C)	37-50 (45)	25-45 (37)
NaCl tolerance range (optimum) (%, w/v)	0-3 (0.5)	0-2.5 (0.5)
Nitrate reduction	-	+
Urease	-	+
Hydrolysis of:
Starch	+	-
Gelatin	+	-
Carbon source utilization (API 20NE):
tryptophan	-	+
glucose	-	+
D-mannitol	-	+
gluconate	-	+
API ZYM:
esterase lipase C8	-	+
leucine arylamidase	-	+
acid phosphatase	+	-
naphthol-AS-BI-phosphohydrolase	+	-
β-glucosidase	+	-
α-fucosidase	-	+
DNA G + C content (mol%)	58.76	57.78

No competing interests reported.

Download PDF

Journal Publication

published 08 Jan, 2024

Read the published version in Antonie van Leeuwenhoek →

Editorial decision: Major revision
12 Jul, 2023
Submission checks completed at journal
11 Jul, 2023
Editor assigned by journal
11 Jul, 2023
First submitted to journal
07 Jul, 2023

You are reading this latest preprint version

Description and genomic characterization of Cohnella amylolytica sp. nov., isolated from Hot Springs in Yunnan province, south-west China

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods and Materials

Result and discussion

Conclusions

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1