key: cord-0032165-jwyd9umr
authors: Liu, Qian; Fan, Guoying; Wu, Kui; Bai, Xiangning; Yang, Xi; Song, Wentao; Chen, Shengen; Xiong, Yanwen; Chen, Haiying
title: Description of Corynebacterium poyangense sp. nov., isolated from the feces of the greater white-fronted geese (Anser albifrons)
date: 2022-05-25
journal: J Microbiol
DOI: 10.1007/s12275-022-2089-9
sha: ce0b9e8b154cc7d170a0366bd1d93582fe658aff
doc_id: 32165
cord_uid: jwyd9umr

Two novel Gram-positive, non-spore-forming, facultatively anaerobic, non-motile, and short rods to coccoid strains were isolated from the feces of the greater white-fronted geese (Anser albifrons) at Poyang Lake. The 16S rRNA gene sequences of strains 4H37-19(T) and 3HC-13 shared highest identity to that of Corynebacterium uropygiale Iso10(T) (97.8%). Phylogenetic and phylogenomic analyses indicated that strains 4H37-19(T) and 3HC-13 formed an independent clade within genus Corynebacterium and clustered with Corynebacterium uropygiale Iso10(T). The average nucleotide identity and digital DNA-DNA hybridization value between strains 4H37-19 and 3HC-13 and members within genus Corynebacterium were all below 95% and 70%, respectively. The genomic G + C content of strains 4H37-19(T) and 3HC-13 was 52.5%. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine, and phosphatidyl inositol mannosides (PIM) were the major polar lipids, with C(18:1)ω9c, C(16:0), and C(18:0) as the major fatty acids, and MK-8 (H(4)), MK-8(H(2)), and MK-9(H(2)) as the predominant respiratory quinones. The major whole cell sugar was arabinose, and the cell wall included mycolic acids. The cell wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The polyphasic taxonomic data shows that these two strains represent a novel species of the genus Corynebacterium, for which the name Corynebacterium poyangense sp. nov. is proposed. The type strain of Corynebacterium poyangense is 4H37-19(T) (=GDMCC 1.1738(T) = KACC 21671(T)). Supplemental material for this article may be found at 10.1007/s12275-022-2089-9.

Corynebacterium is the type genus of the family Corynebacteriaceae, order Corynebacteriales, class Actinomycetia, phylum Actinomycetota (Ludwig et al., 2015; Salam et al., 2020; Oren and Garrity, 2021) . The genus Corynebacterium is composed of Gram-positive, non-motile, non-spore-forming, rod-or club-shaped, catalase-positive bacteria with a high G + C content (Bernard and Funke, 2015; Nouioui et al., 2018) . As of 21 February 2022, the genus Corynebacterium included 136 species with validly published and correct names (https://lpsn.dsmz.de/genus/corynebacterium) (Parte et al., 2020) . Species of Corynebacterium have been recovered from a variety of samples, such as humans, animals, soil, water, and food (Bernard and Funke, 2015) . The type species, Corynebacterium diphtheriae, is a well-known human pathogen that causes diphtheria by multiplying and secreting diphtheria toxin (Sharma et al., 2019) . However, Corynebacterium glutamicum, a non-pathogenic species, is commonly used as biochemical industrial producers of amino acids (Yu et al., 2021) . The greater white-fronted geese (Anser albifrons) belong to migratory birds which hold long-distance migration every year and might spread emerging and re-emerging pathogens across the world (Samuel et al., 2005; Boros et al., 2018; Xiang et al., 2019; Fukuda et al., 2021; Zhu et al., 2021) . In the previous study, a novel bacterial genus (Nanchangia) and two novel species of genus Corynebacterium, i.e., C. anserum, and C. heidelbergense, were identified from feces of migratory birds (Braun et al., 2018; Liu et al., 2021a Liu et al., , 2021b . In this study, we isolated two strains 4H37-19 T and 3HC-13, belonging to undescribed species within the genus Corynebacterium, from the feces of the greater white-fronted geese, and depicted the taxonomic characteristics of the two strains.

Fecal samples of the greater white-fronted geese (Anser albifrons) were obtained from Poyang Lake in China. The specimens were homogenized and serially diluted (10 -4 -10 -1 ) in sterile phosphate-buffered saline. The diluted samples were spread on tryptone soya agar (TSA) and incubated at 37°C. Pure colonies were obtained by repeated subcultivations and stored at -80°C in 30% (v/v) glycerol stocks for further identification. The representative isolates were deposited at Guangdong Microbial Culture Collection Center (GDMCC) of China and Korean Agricultural Culture Collection (KACC) under the accession numbers GDMCC 1.1738 and KACC 21671, respectively.

For phylogenetic analyses, 16S rRNA gene sequences of strains 4H37-19 T and 3HC-13 were amplified using primers 27F and 1492R, and then sequenced through the Sanger sequencing (Zhu et al., 2022) . The obtained sequences were searched against the quality-controlled databases of 16S rRNA sequences using EzBioCloud service . Phylogenetic trees were constructed using the MEGA-X program based on neighbor-joining (NJ), maximum-likelihood (ML), and minimum-evolution (ME) algorithms with 1,000 bootstrap replicates (Kumar et al., 2018) . Mycobacterium tuberculosis H37Rv T was used as the outgroup.

Genomic DNA was extracted from pure culture using the Wizard Genomic DNA Purification kit (Promega). To obtain the complete genome of strain 4H37-19 T , a combination of PacBio Sequel platform and Illumina NovaSeq platform was used. The draft genome of strain 3HC-13 and C. uropygiale Iso10 T were sequenced on the Illumina NovaSeq platform. After filtering out the low-quality reads, the SPAdes optimizer Unicycler v0.4.8 (Wick et al., 2017) was used for de novo assembly. Multiple rounds of polishing were performed with Pilon 1.23 (Walker et al., 2014) in the Unicycler pipeline to correct small sequence errors. To further validate the taxonomic status of the two strains in the genus Corynebacterium, up-to-date bacterial core gene (UBCG, https:// www.ezbiocloud.net/tools/ubcg) trees (Na et al., 2018; Kim et al., 2021) were constructed using the FastTree program with Mycobacterium tuberculosis H37Rv T as the outgroup (Price et al., 2010) . To evaluate the genomic relatedness, the digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) values were calculated using the Genome-to-Genome Distance Calculator (GGDC) 2.1 (http:// ggdc.dsmz.de/) (Meier-Kolthoff et al., 2022) and OrthoANI tool (Lee et al., 2016; , respectively. Gene annotation was performed using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Li et al., 2021) and Rapid Annotation using Subsystem Technology (RAST) server (https:// rast.nmpdr.org/) (Brettin et al., 2015) . The secondary metabolite biosynthesis gene clusters were predicted using anti-SMASH 6.0 (Blin et al., 2021) . Carbohydrate-active enzyme features were analyzed in the dbCAN2 meta server, using DIAMOND, HMMER and eCAMI tools, respectively (https:// bcb.unl.edu/dbCAN2/index.php) (Yin et al., 2012; Zhang et al., 2018) .

Based on the 16S rRNA gene similarities, the four closely related type strains (C. uropygiale Iso10 T , C. choanae 200CH T , C. jeikeium NCTC 11913 T , and C. falsenii DSM 44353 T ) purchased from three culture collections (JCM, ATCC, and CCUG) were used as the reference strains for phenotypic, biochemical, and chemotaxonomic comparisons with strains 4H37-19 T and 3HC-13. Comparative genome analyses and pairwise comparisons of ANI and dDDH values were also performed with genomic data of representative strains within the genus Corynebacterium publicly available from NCBI database. Whole-genome orthologous gene annotations and comparisons, including the genetic ontogeny of all predicted protein-coding genes, were conducted using OrthoVenn2 (Xu et al., 2019) .

To determine the optimal growth conditions, strains 4H37-19 T and 3HC-13 were cultured under various conditions. The growth temperatures were tested in tryptone soya broth (TSB) at 4, 10, 15, 20, 25, 30, 37, 45, 50 , and 55°C, respectively. The salt tolerance was determined by culturing strains 4H37-19 T and 3HC-13 in the presence of different NaCl concentrations (0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, and 12%, w/vol) in TSB. Growth was also evaluated at different pH values (4.0-11.0, at 1.0 pH unit intervals) using TSB. The pH values were re-adjusted after sterilization (121°C, 15 min) if necessary. The optimal growth conditions were determined by measuring the turbidity at 600 nm using Varian Cary 50 spectrophotometer (CARY-50, Agilent Technologies). The cells cultured under optimum conditions in TSB was used for following analyses, unless otherwise mentioned. Oxygen tolerance was evaluated in an anaerobic chamber in the presence of N 2 (90%), H 2 (5%), and CO 2 (5%) for 1 week. Cell morphologies were observed under a transmission electron microscope (HT7700, Hitachi). Gram staining reactions and spore formation were observed under a light microscope (Eclipse 50i, Nikon) using a Gram staining kit (bioMérieux) and the malachite green staining method (Schaeffer and Fulton, 1933) . Semi-solid medium containing 0.4% agar was used for motility testing. Catalase and oxidase activity were detected as previously described (Liu et al., 2021a) . Antibiotic resistance was determined using K-B method (Bauer et al., 1966) .

The biochemical characteristics of strains 4H37-19 T and 3HC-13 were tested using API 50 CH (combined with API 50 CHB/E), the API ZYM system and API Coryne kits following the manufacturer's instructions (bioMérieux). Cellular fatty acids of strains 4H37-19 T and 3HC-13, and four reference strains were extracted, analyzed, and identified according to the previous studies (Sasser, 1990; Kim et al., 2021) . The respiratory quinones of strain 4H37-19 T was extracted and analyzed by HPLC as previously reported (Collins et al., 1977; Oh et al., 2020) . The polar lipids of the isolate was analyzed by two-dimensional thin-layer chromatography (TLC) as previously described by Minnikin et al. (1984) . Whole-cell sugars were obtained by hydrolyzing the cell harvests in 0.5 M sulfuric acid (100°C, 2 h), as described previously (Komagata and Suzuki, 1988) . The cell wall peptidoglycan was analyzed as described previously (Schumann, 2011) . The mycolic acids were extracted as previously described (Guerrant et al., 1981) , then detected by gas chromatograph (HP 6890, Agilent) using an Ultra-2 chromatographic column (25 m by 0.2 mm inside diameter and 0.33 μm liquid film thickness). The temperature of the injector and detector were 250°C and 300°C, respectively. The flow rate of the carrier gas (hydrogen) was 300 ml/min.

The DDBJ/ENA/GenBank accession numbers for the 16S rRNA gene sequences of strains 4H37-19 T and 3HC-13 are MN611115 and MN611764, respectively. The DDBJ/ENA/ GenBank accession numbers for the whole-genome sequences of strain 4H37-19 T , strain 3HC-13 and C. uropygiale Iso10 T are CP046884, WWCB00000000, and JAKGSI000000000, respectively.

Based on almost full-length 16S rRNA gene sequences comparisons against the EzBioCloud database, strains 4H37-19 T and 3HC-13 were identified to be members of the genus Corynebacterium within family Corynebacteriaceae, and most -Tamisier et al., 2015) , suggesting that the two isolates could represent a novel species of the genus Corynebacterium. In addition, the phylogenetic tree based on 16S rRNA gene sequences showed that strains 4H37-19 T and 3HC-13 formed a single clade and clustered with C. uropygiale Iso10 T ( Fig. 1 ; Supplementary data Figs. S1 and S2). Whole-genome analyses showed that strains 4H37-19 T and 3HC-13 contained 2,617,997 bp with a 52.5% DNA G + C content, and 2,559,826 bp with a 52.5% DNA G + C content, respectively. The genome of strain 4H37-19 T contained 2,465 genes and 2,401 CDSs, which were different with the number of genes 2,386 and CDSs 2,331 of strain 3HC-13 genome.

To compare strain 4H37-19 T with strain 3HC-13 in detail, the conservation and variation were compared using MAUVE software (Darling et al., 2011) with strain 4H37-19 T as the reference (Supplementary data Fig. S3 ). Result showed that these two genomes have a high content of homologous regions, including a total of 11 locally collinear blocks (LCBs) with minimum weight of 2,881 were generated. A total of 13,460 SNPs were located within genomes, and some regions of strain 3HC-13 displays inversion, translocation, and Tran+Inver, revealing different syntenial relationships to type strain 4H37-19 T . Strains 4H37-19 T and 3HC-13 may have the same genes related to the physiological and fatty acid characteristics. However, MAUVE result confirmed that genome of strains 4H37-19 T was different from that of strain 3HC-13. It also should be noted that genome of strain 4H37-19 T was a complete genome, but genome of strain 3HC-13 was a draft genome. (Supplementary data Fig. S3 ) The genomic characteristics of the two isolates were compared with their closely related type species (Table 1) . Compared with these four related species, strains 4H37-19 T and 3HC-13 had medium genome sizes, lower GC content genomes, and less number of tRNA genes. To infer the UBCG tree, 92 housekeeping core genes of our isolates and their related type strains were extracted and concatenated. Phylogenomic result (Fig. 2) showed that strains 4H37-19 T and 3HC-13 clustered closely with C. uropygiale Iso10 T , consistent with result based on 16S rRNA gene sequences (Fig. 1) . To determine the genome relatedness, dDDH and ANI values between our isolates and the closely related species of genus Corynebacterium were calculated and pre-sented in Table 2 . These values were below the threshold values for species demarcation (70% for dDDH value and 95-96% for ANI value) (Luo et al., 2014; Chun et al., 2018) . Thus, the two isolates 4H37-19 T and 3HC-13 belonged to different species from these reference strains (Jackman et al., 1987; Sjöden et al., 1998; Braun et al., 2016; Busse et al., 2019) . The dDDH and ANI values between stains 4H37-19 T and 3HC-13 were 95.5% and 99.4%, indicating that the two isolates belonged to the same species.

Using the RAST server, the genome of strain 4H37-10 T was annotated, 731 genes (29%) were further clustered into 24 subsystems. The most represented subsystem features were carbohydrates (201), amino acids and derivatives (193), protein metabolism (166), cofactors, vitamins, prosthetic groups, pigments (100), nucleosides and nucleotides (61), and DNA metabolism (46) (Fig. 3A) . Screening the genes coding secondary metabolites showed that genome of strain 4H37-10 T contained four (Regions 1-4) different genes clusters of secondary metabolites (Fig. 3B ). Region 1 (649,498-670,421 nt, total 20,924 nt) and region 2 (2,183,106-2,193,576 nt, total 10,471 nt) displayed terpene and an unspecified ribosomally synthesised and post-translationally modified peptide product (RiPP) cluster types, respectively. Both region 1 and 2 were unable to identify the most similar known gene cluster. Region 3 (2,247,432-2,281,292 nt, total 33,861 nt) and region 4 (2,436,578-2,481,374 nt, total 44,797 nt) showed 5% and 8% similarity to pyrrolomycin A/pyrrolomycin B/pyrrolomycin C/pyrrolomycin D genes (BGC0000130) and stambomycin A / stambomycin B / stambomycin C / stambomycin D genes (BGC0000151), respectively. The OrthoVenn2 analysis assigned 2,358 protein sequences from strain 4H37-19 T to 2,255 orthologous clusters with 90 singletons, while 2,294 proteins from strain 3HC-13 were assigned to 2,234 clusters with 55 singletons. The Venn diagram (Supplementary data Fig. S4 ) showed 1,076 gene clusters shared by strains 4H37-19 T and 3HC-13, and their closely related type strains. In addition, the six strains had 91 unique gene clusters, with strains 4H37-19 T and 3HC-13 having two and zero unique gene cluster, respectively.

According to the results from dbCAN2 meta server, we identified a total of 88 genes encoding glycosil transferases (GT), 75 genes encoding glycosil hydrolases (GH), 22 genes encoding carbohydrate esterase (CE), 12 genes encoding carbohydrate-binding module (CBM), and 2 genes encoding auxiliary activities (AA) (Supplementary data Table S1). 

Strains 4H37-19 T and 3HC-13 were Gram-stain-positive, facultatively anaerobic, non-motile and short rods to coccoid (0.2-0.4 × 0.6-0.9 μm; Supplementary data Fig. S5 ). Cells were catalase-positive and oxidase-negative. Colonies were creamy whitish, circular colonies with rough edges on TSA. Strains 4H37-19 T and 3HC-13 grew at 15-45°C, pH 6.0-9.0 and in the presence of 0-7.5% (w/vol) NaCl in TSB, with optimal growth at 37°C, pH 7.0 and in the presence of 0.5-1.5% (w/vol) NaCl. Antibiotic testing indicated that strains 4H37-19 T and 3HC-13 were susceptible to amikacin, ampicillin, cefazolin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, kanamycin, penicillin G, streptomycin, sulfanilamide, tetracycline, and vancomycin. The detailed biochemical characteristics of our isolates were described in the species descriptions, and the differential characteristics between strains 4H37-19 T and 3HC-13 and their closely related Corynebacterium type strains were summarized in Table 3 . The cell morphology of strains 4H37-19 T and 3HC-13 were short rods to coccoid which were different with rod-club shape of C. uropygiale Iso10 T , C. jeikeium NCTC 11913 T , and C. falsenii DSM 44353 T , coccoid or irregular rod shape of C. choanae 200CH T . Strains 4H37-10 T and 3HC-13, C. uropygiale Iso10 T , C. jeikeium NCTC 11913 T , and C. falsenii DSM 44353 T were non-spore-forming strains, except that C. choanae 200CH T was not determined (Table 3) . Biochemical results indicated that strains 4H37-19 T and 3HC-13 differed from their closely related neighbors by being positive for arbutin, L-arabinose, trypsin, α-glucosidase, and βglucuronidase (Table 3) . Strains 4H37-19 T and 3HC-13, C. jeikeium NCTC 11913 T , and C. falsenii DSM 44353 T were positive for alkaline phosphatase, while other related strains were negative. Corynebacterium uropygiale Iso10 T and C. choanae 200CH T were positive for the reduction of nitrates, while the other related strains were negative, including our two isolates in this study. Furthermore, strains 4H37-19 T and 3HC-13, and C. uropygiale Iso10 T could utilize D-fructose, while the other related strains couldn't. Corynebacterium choanae 200CH T , C. jeikeium NCTC 11913 T (weakly), and C. falsenii DSM 44353 T were positive for galactose, while our isolates and C. uropygiale Iso10 T were negative for this sugar.

The detailed fatty acid profiles of our isolates and their closely related type strains were showed in Table 3 . The major fatty acids (> 10%) of strains 4H37-19 T and 3HC-13 were C 18:1 ω9c (both were 53.4%), C 16:0 (22.8% and 22.9%, respectively), and C 18:0 (20.8% and 20.4%, respectively) ( Table 4) . Strain 4H37-19 T contained MK-8 (H 4 ) (38.3%), MK-8(H 2 ) (36.4%), and MK-9(H 2 ) (11.7%) as major respiratory quinones, which possessed the unique MK-8 (H 4 ) that was absent in their closely related type strains, such as C. choanae 200CH T , and revealed different proportions of MK-8(H 2 ) and MK-9(H 2 ). (Bernard and Funke, 2015; Busse et al., 2019) . The polar lipid profile of strain 4H37-19 T was composed of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylinositol mannosides (PIM), two unidentified phospholipids (PL), and two unidentified glycolipids (GL) (Supplementary data Fig.  S6 ), which was similar to those of their closely related strains (Bernard and Funke, 2015) . The whole-cell sugar of strain 4H37-19 T consisted of arabinose (Supplementary data Fig.  S7 ). The strain 4H37-19 T included mycolic acids in cell wall and contained meso-diaminopimelic acid (meso-DAP) in the peptidoglycan.

Taken together, the overall phylogenetic, genomic, physiological, biochemical, and chemotaxonomic findings distinguished strains 4H37-19 T and 3HC-13 from their closely related species and suggested that they represent a novel species within the genus Corynebacterium. We propose the name Corynebacterium poyangense sp. nov. for strains 4H37-19 T and 3HC-13.

Corynebacterium poyangense (po.yang.en'se. N.L. neut. adj. poyangense, of or belonging to Poyang Lake from where the type strain was isolated) Cells are Gram-stain-positive, non-spore-forming, facultatively anaerobic, non-motile and short rods to coccoid (0.2-0.4 × 0.6-0.9 μm). Colonies are creamy whitish, circular colonies with rough edges on TSA at 37°C after 48 h. Cells grow at 15-45°C and pH 6.0-9.0 and in the presence of 0-7.5% (w/vol) NaCl. Optimal growth occurs at 37°C and pH 7.0 and in the presence of 0.5-1.5% (w/vol) NaCl. Cells are positive for acid phosphatase, alkaline phosphatase, esterase (C4), naphthol-AS-BI-phosphohydrolase, trypsin, α-glucosidase, β-galactosidase, β-glucosidase, and β-glucuronidase, but negative for cystine arylamidase, hydrolysis, leucine arylamidase, lipase (C8), N-acetyl-β-glucosaminidase, reduction of nitrates, pyrazinamidase, urease, valine arylamidase, α-chymotrypsin, αfucosidase, α-galactosidase, α-glucosidase, and α-mannosidase. Cells can assimilate arbutin, esculin ferric citrate, Dfructose, D-glucose, D-maltose, D-mannose, D-ribose, D-trehalose, D-turanose, gentiobiose, gluconate, L-arabinose, and surcose, but not assimilate amygdalin, erythritol, dulcitol, D-adonitol, D-arabinose, D-arabitol, D-cellobiose, D-fucose, D-lyxose, D-melezitose, D-melibiose, D-raffinose, D-tagatose, D-sorbitol, inositol, inulin, mannitol, methyl α-D-glucopyranoside, methyl α-D-mannopyranoside, methyl-βD-xylopyranoside, N-acetylglucosamine, galactose, glycerol, glycogen, lactose, L-arabitol, L-fucose, L-rhamnose, L-sorbose, salicin, starch, xylitol, or xylose. The major fatty acids are C 18:1 ω9c, C 16:0 , and C 18:0 , while MK-8 (H 4 ), MK-8(H 2 ), and MK-9(H 2 ) are predominant respiratory quinones. The major polar lipids are DPG, PG, PI, PC, and PIM. The major whole cell sugar was arabinose, and the cell wall included mycolic acids. The cell wall peptidoglycan contained meso-DAP. The genomic DNA G + C content is 52.5%. The type strain is 4H37-19 T (= GDMCC 1.1738 T = KACC 21671 T ), isolated from the feces of the greater white-fronted geese (Anser albifrons) at Poyang Lake, China. The GenBank/ EMBL/DDBJ accession numbers for the 16S rRNA gene and genome sequences of strains 4H37-19 T strain 3HC-13 are MN611115 and MN611764 (16S rRNA gene), and CP-046884 and WWCB00000000 (genome), respectively. 

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This work was supported by grants from the National Science and Technology Major Project (2018ZX10301407-002), the Major Science and Project of Jiangxi Province (20201BBG-71010), and the Independent Research Project of State Key Laboratory of Infectious Disease Prevention and Control (2019SKLID311). We express our sincere gratitude to the Jiangxi Province Department of Forestry for organizing the rescue of migratory birds and sampling. We are also grateful to the staff at Nanchang Center for Disease Prevention and Control who contributed to the sampling.

The authors declare that there are no conflicts of interest.

The migratory birds were live captured in Jiangxi province, China. All animals were subjected to non-invasive sampling (feces) and then released. We only collected feces for relevant microbiological studies. The animal welfare practices associated with this study were reviewed and approved by the Jiangxi Province Department of Forestry (No. 20181030).