Genomic Molecular Signatures Determined Characterization of Mycolicibacterium gossypii sp. nov., a Fast-Growing Mycobacterial Species Isolated From Cotton Field Soil Ruirui Huang Nanjing Normal University Shenrong Yang Nanjing Normal University Cheng Zhen Nanjing Normal University Xianfeng Ge Nanjing Normal University Xinkai Chen Nanjing Normal University Zhiqiang Wen Nanjing Normal University Yanan Li Nanjing Normal University Wenzheng Liu ( [email protected] ) Nanjing Normal University https://orcid.org/0000-0003-0710-5748 Research Article Keywords: Mycolicibacterium, fast-growing, cotton eld soil, molecular signatures Posted Date: May 27th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-560868/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Antonie van Leeuwenhoek on August 15th, 2021. See the published version at https://doi.org/10.1007/s10482-021-01638-z. 1 Genomic molecular signatures determined characterization of 2 Mycolicibacterium gossypii sp. nov., a fast-growing mycobacterial 3 species isolated from cotton field soil 4 Authors name：Rui-Rui Huang, Shen-Rong Yang, Cheng Zhen, Xian-Feng Ge, Xin-Kai * 5 Chen, Zhi-Qiang Wen, Ya-Nan Li, Wen-Zheng Liu 6 Author affiliation: School of Food and Pharmaceutical Engineering, Nanjing Normal 7 University, Nanjing 210023, PR China. 8 *Correspondence: Wen-Zheng Liu; E-mail: [email protected] 9 Abstract T 10 A Gram-positive, acid-fast and rapidly growing rod, designated S2-37 , that could form 11 yellowish colonies was isolated from soil samples collected from cotton cropping field located T 12 in the Xinjiang region of China. The draft genome of strain S2-37 was 5.1 Mb in length, with 13 a DNA G+C content of 68.4 mol%. 16S rRNA-directed phylogenetic analysis referred that T 14 strain S2-37 was closely related to bacterial species belonging to the genus Mycolicibacterium 15 and Mycobacterium. Multilocus sequence analysis of three genes (16S rRNA, hsp65 and rpoB) T 16 revealed that strain S2-37 shared high sequence similarities with Mycolicibacterium litorale T T 17 CGMCC 4.5724 (96.5%) and Mycobacterium neglectum CECT 8778 (95.7%). Digital DNA- 18 DNA hybridization (dDDH) and the average nucleotide identity (ANI) presented that strainS2- T 19 37 displayed the highest values of 39.1% (35.7-42.6%) and 81.28% with M. litorale CGMCC T 20 4.5724 , respectively. And characterazition of conserved molecular signatures further T 21 confirmed that strain S2-37 could be well classified into the genus Mycolicibacterium. The 22 main fatty acids were identified as C16:0, C18:0, C20:3ω3 and C22:6ω3. In addition, polar lipids 23 profile was mainly composed of diphosphatidylglycerol, phosphatidylethanolamine and T 24 phosphatidylinositol. Results indicated that strain S2-37 represented genetically and T 25 phenotypically distinct from its closest phylogenetic neighbour, M. litorale CGMCC 4.5724 . 26 Here, we propose a novel species of the genus Mycolicibacterium: Mycolicibacterium gossypii T T T 27 sp. nov. with the type strain S2-37 (=JCM 34327 =CGMCC 1.18817 ). 28 Keywords: Mycolicibacterium, fast-growing, cotton field soil, molecular signatures 29 Declarations 30 Funding: This work was supported by grants from the Natural Science Foundation of Jiangsu 31 Province (grant no. BK20190703) and The Natural Science Research of the Jiangsu Higher 32 Education Institutions of China (grant no. 19KJB530010). 33 Conflicts of interest: The authors declare no conflicts of interest. 34 Availability of data and material: The GenBank/EMBL/DDBJ accession number for the 16S T 35 rRNA gene sequence of the strain S2-37 is MW295419. This Whole Genome Shotgun project 36 has been deposited at DDBJ/ENA/GenBank under the accession JAFEVR000000000. The 37 version described in this paper is version JAFEVR010000000. 38 Authors’ contributions: RRH performed the experiments and finished the draft of the 39 manuscript. RRH, SRY, CZ, XFG and XKC performed strain isolation and phenotypic analyses. 40 RRH and SRY performed genomic analyses. ZQW and YNL assisted to improve the 41 manuscript. WZL designed all the experiments and supervised the manuscript. 42 Ethical approval: This article does not contain any studies with human participants or animals 43 performed by any of the authors. 44 Informed consent: All authors agree to publish this work. 45 Introduction 46 The genus Mycolicibacterium presents the second largest population of Gram-positive, acid- 47 fast and rod-shaped microbes in the family Mycobacteriaceae (Parte et al. 2020). At the time 48 of writing, the genus Mycolicibacterium is composed of 92 recognized species with published 49 names (https://lpsn.dsmz.de) (Parte et al. 2020), which primarily harbors rapid-growing species 50 isolated from diverse range of environments, including river water, marine sediment and soil 51 (Butler et al. 1993; Brown-Elliott et al. 2010; Zhang et al. 2013). There are also relatively high 52 proportion of members in the genus isolated from clinical specimens, showing pathogenic to 53 humans and animals (Shojaei et al. 2000; Brown-Elliott and Wallace 2002). 54 It is well documented that mycobacterial species displayed relatively high diverses in their 55 genomic features at species level (Fedrizzi et al. 2017), which has already remarkably enhanced 56 the difficulty accurately characterizing the taxonomic position of microbial species belonging 57 to this population. Much effort has been devoted to primarily delineate different groups that 58 existes within this meaning fully evolved bacterial branch using well established approaches 59 based on analyses of the 16S rRNA gene, 16S-23S spacer, and housekeeping gene concatenated 60 multilocus sequences (Roth et al. 1998; Mignard and Flandrois, 2008; Magee and Ward, 2012). 61 However, the reliability of these methods used to well distinguish sub-groups (e.g., slow- and 62 fast-growing species) of mycobacterial species remains of concern. Recently, Gupta et al. (2018) 63 have developed a robust method consistently supporting the existence of five distinct 64 monophyletic sub-groups of mycobacterial species, which are designated as the 65 Mycobacterium, Mycolicibacterium, Mycolicibacter, Mycolicibacillus and Mycobacteroides 66 genera. They have identified representative molecular markers in the form of conserved 67 signature indels and proteins, which are uniquely shared by members of the five identified 68 clades. T 69 In this study, a putative novel species (strain S2-37 ) belonging to mycobacterial species was 70 isolated from soil samples of cotton cropping field of Xinjiang in PR China. Multilocus T 71 sequence analysis of three genes (16S rRNA, hsp65 and rpoB) presented that strain S2-37 72 shared high sequence similarities with microbal species belonging to both the genus 73 Mycolicibacterium and Mycobacterium. In the previous work, conserved molecular signatures 74 (indels and proteins) have been clearly identified as specific markers that could be used to 75 distinguish most members belonging to these two genera confidently (Gupta et al. 2018). T 76 Therefore, genome sequence of strain S2-37 was mapped to these documented molecular 77 features specific for each genus, ending up with the conclusion that this strain was 78 taxonomically classified into the genus Mycolicibacterium in combination with following 79 polyphasic approaches. 80 Materials and methods 81 Isolation of the novel strain and cultivation T 82 Strain S2-37 was isolated from a soil sample collected from a cotton cropping field (86°20′ N 83 44°62′ E, Xinjiang, China). The isolation procedures were described in the previous report with 84 some modifications (Hopkins et al. 1991). Soil samples (5 g) were firstly naturally dried, then 85 mixed with 100 ml of sterilized water in 250 ml flasks. In order to efficiently elute microbes 86 firmly attached to soil particles, Sample were placed on a shaker with vibration frequency of 87 160 r/min at 30 °C for 30 minutes, followed by homogenization treatment for 1 min using a 88 sonicator (XO-3200DT, Nanjing Xianou laboratory equipment Co., Ltd) with a frequency of 89 40 kHz. Then, 100 µl serially diluted samples were plated on Gause’s synthetic No.1 agar (G1, 90 20 g soluble starch; 1 g KNO3; 0.5 g K2HPO4; 0.5 g MgSO4·H2O; 0.5 g NaCl; 0.01 g FeSO4; 91 20 g Agar; 1000 ml distilled water; pH 7.2-7.4) for cultivation at 30 °C in the following 14 92 days. Bacterial colonies were sub-cultured on G1 agar until pure isolates were achieved. Strain T 93 S2-37 was maintained aerobically on G1 agar at 30 °C, and stored at -80 °C in G1 broth 94 supplemented with equal volume of 50% (v/v) sterilized glycerol for preservation (Prakash et 95 al. 2013). 96 Phylogenetic analysis based on 16S rRNA gene and multilocus sequences T 97 Genomic DNA of strain S2-37 was extracted and its 16S rRNA gene was achieved by PCR 98 amplification using universal primers 27F and 1492R as described by Fan et al. (2008), 99 followed by sequence alignment in order to preliminarily locate the taxonomic position of the T 100 strain S2-37 using the BLAST function embedded in NCBI database (Federhen 2012). GGDC 101 web server (available at http://ggdc.dsmz.de/) was employed for gene phylogenetic and T 102 similarity analysis between 16S rRNA gene sequences of S2-37 and all published 103 Mycolicibacterium type strains collected from the LPSN database (Parte et al. 2020). 104 Respectively, the phylogenetic trees were reconstructed based on 16S rRNA gene sequences T 105 of strains S2-37 and its 22 closely phylogenetic relatives using the neighbor-joining (NJ) 106 (Saitou and Nei 1987), maximum-parsimony (MP) (Sourdis and Nei 1988) and maximum- 107 likelihood (ML) (Steel and Rodrigo 2008) algorithms supported by the software package T 108 MEGA 7.0 (Kumar et al.