Aestuariimicrobium ganziense sp. nov., a new Gram-positive bacterium isolated from soil in the Ganzi Tibetan autonomous prefecture, China

A novel Gram-stain positive, oval-shaped, and non-flagellated bacterium, designated YIM S02566T, was isolated from alpine soil in Shadui Towns, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, PR China. Growth occurred at 23–35 °C (optimum, 30 °C) in the presence of 0.5–4% (w/v) NaCl (optimum, 1%) and at pH 7.0–8.0 (optimum, pH 7.0). The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain YIM S02566T was most closely related to the genus Aestuariimicrobium, with Aestuariimicrobium kwangyangense R27T and Aestuariimicrobium soli D6T as its closest relative (sequence similarities were 96.3% and 95.4%, respectively). YIM S02566T contained LL-diaminopimelic acid in the cell wall. MK-9(H4) was the predominant menaquinone. The major fatty acid patterns were anteiso-C15:0 (60.0%). The major polar lipid was DPG. The genome size of strain YIM S02566T was 3.1 Mb, comprising 3078 predicted genes with a DNA G + C content of 69.0 mol%. Based on these genotypic, chemotaxonomic and phenotypic evidences, strain YIM S02566T was identified as a novel species in the genus Aestuariimicrobium, for which the name Aestuariimicrobium ganziense sp. nov. is proposed. The type strain is YIM S02566T (= CGMCC 1.18751 T = KCTC 49,477 T).


Introduction
The genus Aestuariimicrobium, first described by Jung et al. (2007), is a member of the family Propionibacteriaceae within the order Propionibacteriales, class Actinomycetia (Stackebrandt 2014;Stackebrandt et al. 1997Stackebrandt et al. , 2002. Up to now, this genus comprises only two recognized species (http:// www. bacte rio. net/), Aestuariimicrobium kwangyangense (found in tidal flat sediment) and Aestuariimicrobium soli (found in farmland soil). In this study, a yellow bacterial strain YIM S02566 T was isolated from alpine soil in Shadui Towns, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, PR China, and was identified as the third strain of the genus Aestuariimicrobium (Chen et al. 2018) by means of a polyphasic taxonomic study. On the basis of the data obtained, we propose that isolate YIM S02566 T represents a novel species in the genus Aestuariimicrobium.

Bacterial isolation and cultivation
Strain YIM S02566 T was isolated from a soil sample in the Shadui Towns, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, PR China (31° 49′ 24.19″ N, 100° 15′ 28.90″ E). The altitude of the sample collection was 4643.88 m. 2 g of soil sample was serially diluted (10 -1 dilution), and approximately 100 µl of the diluted culture suspension was spread on Luria-Bertani (LB) agar. The plates were then incubated at 30 °C for 10 days. Strain YIM S02566 T was one of the isolates that appeared on the LB

Phylogenetic analyses and genome sequencing
Genomic DNA for Polymerase Chain Reaction (PCR) amplification was extracted using the method described by Feng et al. (2020). The 16S rRNA gene was amplified by PCR with the universal primers PA (5′-CAG AGT TTG ATC CTG GCT-3′) and PB (5′-AGG AGG TGA TCC AGC CGC A-3′) (Yang et al. 2020). The amplicon was cloned into PEASY-Blunt (TRANSGEN Biotech) and then sequenced by the Tsingke Company (Beijing, PR China). Comparison of 16S rRNA with related strains was conducted by EzTaxon server (https:// www. ezbio cloud. net/) (Yoon et al. 2017). A total of 15 species form 13 genera were used in this study.
Terrabacter tumescens was selected as the outgroup. All sequences used for phylogenetic analysis were obtained from GenBank, and accession numbers were listed in Fig. 1.
Multiple alignments with corresponding sequences of the most closely relatives were executed using the CLUSTAL X 1.8 program (Thompson et al. 1997). Phylogenetic analyses were performed by MEGA version 7.0 software (Kumar Asterisks (*) indicate that the corresponding branches were also recovered in trees generated with the maximum parsimony and maximum likelihood methods. Terrabacter tumescens JCM 1365 T was used as an outgroup. Bar, 1% sequence divergence et al. 2016) using neighbor-joining (NJ) (Saitou and Nei 1987), Maximum Parsimony (MP) (Fitch 1971) and Maximum-Likelihood (ML) (Felsenstein 1981) methods, with bootstrap values based on 1000 replications (Felsenstein 1985). The genome sequences of YIM S02566 T were determined using a PacBio + Illumina Hiseq at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). The sequenced reads were assembled using SOAPdenovo software version 2.04 (https:// soap. genom ics.org.cn/soap. enovo.html). The DNA G + C mol% value was obtained from the genomic sequences. A genome tree was constructed using RAxML (Stamatakis 2014), and fast bootstrapping (Stamatakis et al. 2008) was used to generate the support values in the tree.

Chemotaxonomic characteristics
The strain biomass for chemotaxonomic characterization was obtained from 5-days old cultures grown on medium R2A with 1% NaCl at 30 °C. The isomer type of the diaminopimelic acid of the cell wall was analyzed according to the method described by Lechevalier (Lechevalier and Lechevalier 1971). The respiratory quinones were isolated using the method of Collins et al. (1977), and analyzed by HPLC (Agilent Technologies 1260 Infinity) (Groth et al. 1996). Polar lipids profiles were analyzed as described by twodimensional TLC (Minnikin et al. 1984;Toru et al. 1983), and the different spots were observed by spraying with the proper detection reagents (molybdophosphoric acid, molybdenum blue, ninhydrin, D reagent, and α-naphthol). The cellular fatty acids were extracted and analyzed according to the standard MIDI protocol (Microbial Identification) and Sherlock Microbial Identification System (Sherlock version 6.1; midi database: TSBA6).

Morphological and physiological characteristics
The colonies of the strain YIM S02566 T was Gram positive, oval-shaped, non-flagellated, yellow and 0.5-1.0 μm in diameter after 5 days of incubation at 30 °C (Fig. S1). Other phenotypic and physiological characteristics of YIM S02566 T and its related members are given in Table 1,  Table S1 and in the species description.

Phylogenetic analysis
The almost complete 16S rRNA gene sequence (1509 bp) of strain YIM S02566 T was generated and displayed 96.3% 16S rRNA gene sequence identity with Aestuariimicrobium kwangyangense R27 T . The next highly related species was Aestuariimicrobium soli D6 T , with pairwise similarities of 95.4%. The NJ tree, MP tree, and ML tree for the 16S rRNA shared the same topology and were presented in Fig. 1, Fig.  S2 and Fig. S3, respectively. The phylogenetic tree indicated that strain YIM S02566 T clustered with Aestuariimicrobium kwangyangense R27 T and Aestuariimicrobium soli D6 T .
The draft genome sequence of strain YIM S02566 T consisted of 68 scaffolds with the N50 value of 449,246 and contained 3078 coding sequences (CDSs), 3 complete rRNA genes, 44 tRNA genes. A phylogenetic tree based on genome sequences was reconstructed using RaxML to confirm the relationships already displayed in the 16S rRNA gene-based tree, with YIM S02566 T as a part of the genus Aestuariimicrobium (Fig. S4).
The combination of phenotypic (Table 1), phylogenetic ( Fig. 1 and Fig. S4), and chemotaxonomic characteristics indicates that strain YIM S02566 T represents a novel species within the genus Aestuariimicrobium, for which the name Aestuariimicrobium ganziense is proposed.