Rhodococcus Yananensis Sp. Nov., Isolated From Microbial Fermentation Bed Material From a Pig Farm

An opaque, pink-colored, gram-positive, aerobic bacteria (designated FBM22-1 T ), 0.5 to 1.0 μm in width and 0.5 to 1.5 μm in length, was isolated from microbial fermentation bed material from a pig farm in northwestern China. Optimal growth occurred at 30–37 ℃ , pH 7.0, and 0.5% NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the novel isolate belonged to the family Nocardiaceae of the class Actinomycetia. FBM22-1 T is closely related to Rhodococcus zopi NBRC 100606 T and Rhodococcus rhodochrous NBRC 16069 T , with 16S rRNA gene sequence similarity of 97.95% and 97.73%, respectively. The predominant respiratory quinone in FBM22-1 T was ubiquinone MK-8(H 2 ), and the cellular fatty acids consisted primarily of C 16:1 ω7c/ 16:1 ω6c , C1 6:0, and C 18:0 10-methly1. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and glycolipids. The G+C content of FBM22-1 T was 68.64 mol%. Based on the phenotypic, phylogenetic, and chemotaxonomic characterization, in combination with low values of digital DNA–DNA hybridization between FBM22-1 T and its closest neighbors, FBM22-1 T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus yananensis sp. nov. is proposed; the type strain is FBM22-1 T (=KCTC 49502 T = CCTCC 2020275 T ).

Members of the genus Rhodococcus exhibit diverse metabolic activities, including aliphatic and aromatic hydrocarbon degradation (Goodfellow 2004;Jung-Hoon 2000) and storage compound production (Michael 2008;Bunch 1998). The use of puri ed enzymes and whole-cell biocatalysts is becoming increasingly popular in synthetic organic chemistry. In the present study, a bacterial strain isolated from microbial fermentation bed material was subjected to polyphasic taxonomic analysis and subsequently allocated to the genus Rhodococcus.
The oxidase activity was determined using 1% p-aminodiphenyl-amine-hydrochloride liquor and 1% anaphthol ethanol liquor. Methyl red and Voges-Prokauer test (MR-VP) were measured using glucosepeptone liquid medium. The catalase activity test was performed by the observation of the formation of bubbles using a commercial dropper catalase reagent (bioMérieux). Tween-20, Tween-40, and Tween-80 hydrolysis, amylase, gelatin, and benzpyrole production; nitrate reduction; denitri cation; H 2 S production; and carbon and nitrogen source experiments were evaluated according to multiple classi cation identi cation. Sensitivity to the following antibiotics was tested on PYG plates using antibiotic discs (Changde Beekman Biotechnology Co, Ltd., Hunan, China): penicillin, ampicillin, ceftriaxone, gentamicin, tetracycline, erythromycin, cipro oxacin, lincomycin, cotrimoxazole, and chloramphenicol.

Genome comparison
To support the classi cation of the strain as a novel species within the genus Rhodococcus, the Average Nucleotide Identity (ANI), using BLAST (ANIb) and MUMmer (ANIm) algorithms, was calculated using the JSpecies Web Server (Richter 2016). Digital DNA-DNA hybridization (dDDH) was calculated in silico by the Genome-to-Genome Distance Calculator webserver version 2.1 (http://ggdc.dsmz.de//) (Bhattacharya 2020) using the BLAST method. Results were based on recommended formula 2 (identities/HSP length), which is independent of genome length and is therefore robust against the use of incomplete draft genomes. Total DNA of the samples was sequenced using Illumina NovaSeq platform (MAGIGENE, Guangdong, China). Genome coding gene prediction was achieved using Glimmer3 (Arthur 2007).

Chemotaxonomic analysis
The chemotaxonomic pro le of the isolated actinobacterial strain was investigated to establish whether it has a chemotaxonomic pro le typical of members of the genus Rhodococcus. Standard procedures were used to determine the chemical constituents of FBM22-1 T . The strain was cultured in PYG liquid medium (30℃, 160 rpm) for 7 days. Following this, the culture was centrifuged (4000 x g) for 10 min and washed twice with sterile water. Polar lipids were methylated and analyzed by two-dimensional thin-layer chromatography (TLC silica Gel 60 F254:25 Aluminum Sheets 20 20), using chloroform-methanol-water (65: 25: 4, v/v/v) as the rst solvent and chloroform-methanol-acetic acid-water (40: 6: 7.5: 1, v/v/v/v) as the second. Individual polar lipids were identi ed by spraying with molybdophosphoric acid and molybdenum blue. Fatty acid composition of the strains was analyzed using the Sherlock Microbial Identi cation System (MIDI Inc., DE, USA). Menaquinones were extracted as described by Minnikin et al. (Minnikin 1984) and determined using reversed-phase HPLC as described by Collins (Collins 1979).

Results And Discussion
Morphological and physiological characteristics The bacterial colonies were dry, opaque, light pink. SEM revealed that the cells were short rods, 0.5-1.0 μm in width and 0.5-1.5 μm in length (Fig. S2). These characteristics matched those of the genus Rhodococcus. The FBM22-1 T was identi ed as gram-positive aerobic bacteria. The organism was able to grow at 10-45 °C (optimum, 30-37°C), pH 4.0-10.0 (optimum 7.0). Strain growth rate decreased with an increase in NaCl concentration, with an optimal growth rate at NaCl concentration of 0.5% (Table S2). R.
zop i NBRC100606 T is rod shaped with a size range of 1.10-2.0 μm in length, 0.55-0.80 μm in width, and is light pink in color (Rehfuss 2005). On comparison, it was observed that the two strains were similar in color, and FBM22-1 T was shorter and wider than strain R. zop i NBRC100606 T , presenting a short rod shape. Regarding the physiological characteristics of FBM22-1 T , glycine, L-phenylalanine, tryptophan, methionine, tyrosine, or glutamic acid could be used as sole nitrogen source for the bacteria.
biphenylivorans TG9 T are presented in Table 1.
In terms of the sensitivity to antibiotics test, penicillin, ampicillin, ceftriaxone, and chloramphenicol produced an inhibition zone that exceeded 1.0 cm. Gentamicin, tetracycline, erythromycin, and cipro oxacin produced a smaller zone of inhibition of 0.5-1.0 cm, and the strain was resistant to lincomycin and cotrimoxazole (Table S2).

Phylogenetic analysis
Comparison of 16S rRNA gene sequences of FBN22-1 T and other members of the genus Rhodococcus showed sequence similarities ranging from 96-98%. The results revealed that FBM22-1 T had the closest relationship with R. zop i NBRC 100606 T , with 97.95% similarity, followed by R. rhodochrous NBRC 16069 T (97.73%) and R. biphenylivorans TG9 T (97.66%). The phylogenetic reconstructions revealed that FBM22-1 T formed a distinct phylogenetic link within the Rhodococcus 16S rRNA gene tree (Fig. 1), adjacent to the type strains R. artemisiae YIM 65754 T (Guo 2012) and R. ruber DSM 43338 T (Tsukamura 1985). The pairwise similarities to the sequences of these three strains were 97.58 and 97.29%, respectively. Although the NJ bootstrap support for the placement of this novel Rhodococcus sequence was low, the other two methods showed a similar topology (Fig. S1). It is likely that phylogenetic stability will be achieved when more novel related Rhodococcus strains are described and/or whole genome analyses are performed.
Draft genome sequencing of FBM22-1 T (accession number JAIYEP000000000) yielded a genome of 4,250,953 bp in length after assembly, producing 178 scaffolds, and a N50 and N90 value of 49,062 bp and 11,764 bp, respectively. All scaffolds were > 511 bp and the largest was 16,639 bp. A total of 4303 protein coding genes were predicted. FBM22-1 T had one Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) sequence, and the total length was 393 bp. The G+C mol% content of the FBM22-1 T strain was 68.64 mol%, which falls within the range provided for Rhodococcus species.
According to the data obtained, the ANIb and ANIm between strains FBM22-1 T and R. zop i NBRC 100606 T were 78.88% and 85.57%, respectively; the ANIb and ANIm between the FBM22-1 T and the other two species (R. rhodochrous NBRC 16069 T and R. biphenylivorans TG9 T ) tested were < 86% (Table S3). The species cut-off value for ANIb was selected as 95-96% (Michael 2009). The dDDH between strains FBM22-1 T and R. zop i NBRC 100606 T was 26.8%. The dDDH comparison of FBM22-1 T with the draft genome of the two strains (R. rhodochrous NBRC 16069 T and R. biphenylivorans TG9 T ) yielded low percentages (< 27%) ( Table S3). The species cut-off value for dDDH was 70% (Wayne LG 1987), thus suggesting the FBM22-1 T strain should be considered as a new species of the genus Rhodococcus.
Based on the above characteristics, FBM22-1 T was classi ed as a new species of Rhodococcus in this study, for which the name Rhodococcus yananensis sp. nov. is proposed. We describe the species as follows.
Description of Rhodococcus yananensis sp. nov.
Rhodococcus yananensis (yan.an.en'sis. N.L. masc. adj. yananensis a city in Shaanxi province of China, from where the type strain was isolated).
The type strain FBM22-1 T (=KCTC 49502 T = CCTCC 2020275 T ) was isolated from fermented bedding material from a pig farm, located in a small village in Yan'an, Shaanxi Province (36°61 W) at  Data availability The GenBank accession number for the 16S rRNA gene sequence and Whole Genome Shotgun project of strain FBM22-1 T are OK161026 and JAIYEP000000000, respectively.

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