Streptomyces pimonensis sp. nov., isolated from the Taklimakan desert in Xinjiang, China

A novel Streptomyces strain, designated TRM 75549T, was isolated from a sample of sand in Pimo reclamation area, Taklimakan desert, Xinjiang, North–West China. Phylogenetic analyses of the 16S rRNA gene sequences placed strain TRM75549T within the genus Streptomyces with the highest similarities to Streptomyces pilosus NBRC 12772T (98.7%). Nonetheless, average nucleotide identity (ANI) value and the digital DNA–DNA hybridization (dDDH) value between strain TRM75549T and S. pilosus NBRC 12772T were, respectively, 88.2% and 44.1%, and well below 95–96% and 70% cutoff point recommended for recognizing genomospecies, respectively. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, recA, rpoB and trpB) and phylogenomic analysis also illustrated that strain TRM75549T should also be assigned to the genus Streptomyces. Strain TRM75549T contained MK-9 (H6) and MK-9 (H8) as predominant menaquinones. The diagnostic diamino acid of cell walls were identified as LL-diaminopimelic acid and meso-diaminopimelic. The whole-cell sugar pattern of strain TRM 75549T consisted of mannose and glucose. The major fatty acids (> 5%) were iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:1H, iso-C16:0. The polar lipids were diphosphatidylglycerol, lysophosphatidylglycerol, phosphatidylethanolamine, phospholipids, phosphatidylglycerol, phosphatidylinositol, phosphatiylinositol mannosides and unidentified phospholipids. Strain TRM75549T could be differentiated from S. pilosus NBRC 12772T, based on physiological and biochemical characteristics. Thus, strain TRM75549T is representative of a novel species of the genus Streptomyces, for which the name Streptomyces pimonensis sp. nov. is proposed. The type strain is TRM75549T (= CCTCC AA 2020054T = LMG 32221T).


Introduction
The genus Streptomyces was first proposed by Waksman and Henrici (1943). Streptomyces species are aerobic actinomycetes with a GC-rich genome, most of which can form branched mycelia and aerial mycelia, which usually differentiate into spore chains (Gadagkar and Kumar 2005). Many members of the genus Streptomyces are known to produce a variety of biologically active metabolites, including antibiotics, enzymes, enzyme inhibitors, vitamins, etc. (Liu et al. 2018). It has always been a popular germplasm resource studied by scientists, which is very important forindustries such as medicine, medicine and agriculture are very important.
The strain TRM75549 T in this paper was isolated from the southern edge of the Taklimakan Desert in Xinjiang, located at the junction of Pishan County and Moyu County in Hotan area, also known as Pimo Reclamation Area; the climate is highly arid, the vegetation is sparse and subject to high solar radiation, and the temperature difference between day and night is large. The environment is an ideal habitat for obtaining new species of actinomycetes bacteria. In the present study, the taxonomic status of strain TRM75549 T was determined using a polyphasic approach. The results show that TRM75549 T represents a novel Streptomyces species for which the name Streptomyces pimonensis is proposed.

Strain isolation and culturing
In July 2020, the strain TRM75549 T was isolated from a sample of sand in Pimo reclamation area, Taklimakan

Antibacterial and antifungal activity
The antimicrobial efficacy of these isolates was tested been tested against various organisms S. aureus ATCC25923, E. faecalis ATCC29212, E. coli ATCC25922 using Kirby-Baur agar diffusion method (Maiti et al. 2020). In brief, lawns of test organisms were prepared on agar medium and 14-day-old colonies were placed on the lawn. The plates were kept at 4 °C for 2 h for a homogenous distribution of antimicrobial compound before the growth of test organisms followed by the incubation at 37 °C for 24 h. After incubation, the zone of inhibitions around the colony was observed and measured.

Chemotaxonomy
The cells, collected by centrifugation, were washed with distilled water, and then freeze-dried. The method proposed by Staneck and Roberts was used to determine cell wall amino acids (Staneck and Roberts 1974). The whole cell sugars were determined by the method proposed by Tang et al. (2009). Polar lipids were extracted and separated by twodimensional TLC and identified by the method proposed by Minnikin et al. (1984). Menaquinones were extracted from freeze-dried biomass according to the method proposed by Collins et al (1977), and subjected to HPLC analysis (Wang et al. 2011). According to the method proposed by Sasser (1990), cellular fatty acids were extracted from fresh cells, and GS chromatographic analysis was performed.

Genome sequencing and phylogenetic analysis
The method of Chun and Goodfellow (1995) was used to extract genomic DNA and PCR amplification of 16S rRNA gene sequence. EzBioCloud (https:// www. ezbio cloud. net/ ident ify, Yoon et al. 2017) was used to calculate the similarity of the 16S rRNA gene sequence with other strains, and then the sequence of the strains with a close relationship were selected to construct phylogenetic trees. These sequences were aligned using MEGA 7.0 software (Kumar et al. 2015) with neighbor-joining (Saitou and Nei 1987), maximum likelihood (Felsenstein 1981), and maximumparsimony (Fitch 1971) algorithms. The topologies of the phylogenetic trees were evaluated by the bootstrap resampling method with 1000 replicates (Felsenstein 1985). A complete genome sequence of strain TRM77549 T was obtained using an Illumina platform and assemble by Velvet 1.2.10. The G + C content of genomic DNA was determined by whole-genome sequence sequencing. The digital DNA-DNA hybridization (dDDH) values were calculated on the GGDC website using formula 2 (http:// ggdc. dsmz. de/ ggdc. php), originally described by Auch et al (2010) and updated by Meier-Kolthoff et al (2013). The genome sequences of strain TRM75549 T (accession no. JAHWZY000000000) and strain S. pilosus NBRC 12772 T (accession no. BMTE00000000) have been submitted to GenBank. Housekeeping genes used for multilocus sequence analysis (MLSA), were atpD (ATP synthase subunit D), gyrB (DNA gyrase B subunit), recA (recombinase A), rpoB (RNA polymerase beta subunit) and trpB (tryp-tophane B, beta subunit). Each locus for each strain was concatenated head to tail in frame as follows: atpD, gyrB, recA, ropB and trpB. The sequences for all loci of other related strains were obtained from the NCBI (https:// www. ncbi. nlm. nih. gov/). AntiSMASH was used topredict the biosynthetic gene clusters of strain TRM75549 T (Kai et al. 2019).

Results and discussion
After the strain TRM75549 T was cultured at 30 °C for 7 days, it was grown on eight kinds of culture medium, and the colors of aerial mycelia and substrate mycelia were recorded. The strain was observed to grow well on ISP 1, ISP 4, ISP 5, ISP 7, Gauze's No. 1 agar, moderately well on ISP 2, Czapek's agar, Patato dextrose agar, with slow growth on ISP 3 and ISP 6, and none on nutrient agar. The results of the antibacterial experiment showed that TRM75549 T inhibits E. coli with an average diameter of 2 cm, inhibits E. faecalis with an average diameter of 1.5 cm, and inhibits S. aureus with an average diameter of 1.3 cm; Streptomyces pilosus DSM 40153 T inhibits E.coli with an average diameter of 1.1 cm, and inhibits E. faecalis, with an average diameter of 1.3 cm, but S.aureus has no obvious transparent circle ( Supplementary Fig. S2). Strain TRM75549 T could be distinguished from S. pilosus DSM 40153 T by some phenotypic characteristics, in particular cultural characteristics (Supplementary Table S1). Morphological characteristics of strain TRM75549 T were observed using SEM (Supplementary Fig. S1). The strain was observed to form an abundant white aerial mycelium, occasionally twisted, which differentiates into spore chains. Each spore was observed to be olivary with a hairy surface. Strains grow at 15-45 °C, pH 6.0-9.0 and 0-9% NaCl, and are best grown at 30 °C and pH 7.0 at 1% (w/v) NaCl.
The G + C content in the draft genome sequence of strain TRM 75549 T was identified as 72.14 mol%. The GenBank login number of the 16S rRNA gene sequence of strain TRM75549 T is MW479154, and the closest phylogenetic neighbor was S. pilosus NBRC 12772 T (GenBank accession no. AB184842). The average nucleotide identity value and the digital DNA-DNA hybridization value between strain TRM75549 T and S. pilosus NBRC 12772 T were 88.20 and 44.10%, respectively, well below 95-96 and 70% cutoff point recommended for delineating species. The phylogenetic tree constructed from the 16S rRNA gene sequence through the neighbor-joining method showed that the strain TRM75549 T formed a unique clade (Fig. 1), which was also restored in the maximum-likelihood trees and maximum-parsimony Fig. 1 Neighbour-joining unrooted tree based on 16S rRNA gene sequences, illustrating the positions of strain TRM75549 T and related taxa. * Branches that were also found using the maximum-parsimony method, + branches that were also found using the maximumlikelihood method, * + Branches that were found using all three methods. Numbers at nodes are percentage bootstrap values based on 1000 resampled datasets; only values above 50% are indicated. Bar, 0.01 substitutions per nucleotide position  Figs. S4, S5). The MLSA with the concatenated atpD, gyrB, recA, rpoB and trpB genes showed that the strain did not cluster with S. pilosus NBRC 12772 T , forming two separate branches and the MLSA distances were much greater than the generally accepted threshold value of 0.007 for species delineation (Fig. 2). A total of 39 secondary metabolites synthetic gene clusters were compared by antiSMASH. The antiSMASH biosynthetic gene clusters of TRM75549 T with a similarity greater than 50% are shown in Table 1. All the data suggested that strain TRM75549 T was a member of the genus Streptomyces. However, on the basis of a combining compare of phylogenetic distinctness and differences in chemotaxonomic and physiological characteristics (Table 2), it is considered that strain TRM75549 T is the representative of a new species of Streptomyces, named Streptomyces pimonensis sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the genome and 16S rRNA gene sequence of strain TRM75549 T is JAHWZY000000000 and MW479154.
Acknowledgements Thanks to Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Tarim Basin of Xinjiang Production and Construction Corps for providing research facilities.
Author contributions PZ participated in the experiment and preparation of the first draft. XL, XL, ZL, ZX, and CW gave guidance during the experiment. LZ contributed to reagents, instrumentation and the financial support for this work.

Conflict of interest
The authors state that there is no conflict of interest.
Ethical approval This article does not contain any research conducted by any author on human participants or animals.