Genome mining revealed polyhydroxybutyrate biosynthesis by Ramlibacter agri sp. nov., isolated from agriculture soil in Korea

A white-colony-forming, facultative anaerobic, motile and Gram-stain-negative bacterium, designated G-1-2-2 T was isolated from soil of agriculture field near Kyonggi University, Republic of Korea. Strain G-1-2-2 T synthesized the polyhydroxybutyrate and could grow at 10–35 °C. The phylogenetic analysis based on 16S rRNA gene sequence showed that, strain G-1-2-2 T formed a lineage within the family Comamonadaceae and clustered as a member of the genus Ramlibacter. The 16S rRNA gene sequence of strain G-1-2-2 T showed high sequence similarities with Ramlibacter ginsenosidimutans BXN5-27 T (97.9%), Ramlibacter monticola G-3-2 T (97.9%) and Ramlibacter alkalitolerans CJ661T (97.5%). The sole respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified phospholipid. The principal cellular fatty acids were C16:0, cyclo-C17:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The genome of strain G-1-2-2 T was 7,200,642 bp long with 13 contigs, 6,647 protein-coding genes, and DNA G + C content of 68.9%. The average nucleotide identity and in silico DNA–DNA hybridization values between strain G-1-2-2 T and close members were ≤ 81.2 and 24.1%, respectively. The genome of strain G-1-2-2 T showed eight putative biosynthetic gene clusters responsible for various secondary metabolites. Genome mining revealed the presence of atoB, atoB2, phaS, phbB, phbC, and bhbD genes in the genome which are responsible for polyhydroxybutyrate biosynthesis. Based on these data, strain G-1-2-2 T represents a novel species in the genus Ramlibacter, for which the name Ramlibacter agri sp. nov. is proposed. The type strain is G-1-2-2 T (= KACC 21616 T = NBRC 114389 T).


Introduction
The genus Ramlibacter was first proposed by Hulin et al. with the description of cyst-producing bacteria Ramlibacter tataouinensis and Ramlibacter henchirensis isolated from subdesert soil (Heulin et al. 2003). Later on, the genus description has been emended by Lee et al. regarding colony colour, extracellular polymeric substance production, motility and aesculin hydrolysis (Lee et al.2014). To date, 11 species of the genus Ramlibacter with validly published names have been reported (https:// lpsn. dsmz. de/ genus/ ramli bacter; accessed date: 2022.02.11) (Parte et al. 2020). Members of the genus Ramlibacter are characterized by aerobic, Gram-stain-negative, rodto coccoid-shaped, motile or non-motile and contain ubiquinone-8 (Q-8) as a predominant isoprenoid quinone; C 16:0 , cyclo-C 17:0 , and summed feature 3 (C 16:1 ω6c and/or C 16:1 ω7c) as principal fatty acids; phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol as major polar lipids (Heulin et al. 2003;Lee et al. 2014;Chaudhary and Kim 2017). Most of the members of the genus Ramlibacter have been isolated from soil in addition to few members from aquatic niches (Props et al. 2019;Kim et al. 2021).
Various species of the genus Ramlibacter reported to produce polyhydroxybutyrate (PHB) (Heulin et al. 2003;Lee et al. 2014;Lee and Cha 2017;Props et al. 2019;Zhang et al. 2019). PHB are naturally occurring biodegradable polymers that are synthesized by various bacteria and serve as a carbon and energy storage compound. Bacterial PHB are widely applied for the production of biodegradable plastics (Getachew and Woldesenbet 2016;Mostafa et al. 2020).
In this study, a novel member of the genus Ramlibacter, designated strain G-1-2-2 T was isolated from soil of agriculture field near Kyonggi University and its taxonomic position was determined during the study of diversity of soil microorganisms in agriculture field. Moreover, whole-genome analysis of strain G-1-2-2 T has been explored providing insights into polyhydroxybutyrate (PHB) biosynthesis.

Ecology, isolation and preservation
Strain G-1-2-2 T was isolated from soil of agriculture field, geographically located near Kyonggi University, Suwon, Republic of Korea (37°17′56.9"N and 127°02′23.2"E) and subjected to polyphasic approach for its taxonomic description. A modified culture method with six-well Transwell plates (Corning Inc., NY, USA) was utilized for isolation. Three-gram sieved soil was kept on the bottom of each Transwell plate and 3 ml Reasoner's 2A (R2A) broth (Kisan Bio, Seoul, Korea) was added to each insert. Then, 100 μl soil suspension (1 g soil in 9 ml distilled water; thoroughly stirred and settled) was added to the insert. The Transwell plate was incubated in a shaker at 120 r.p.m. for 4 weeks at 28 °C. After 4 weeks, the culture was serially diluted, and 100 μl of each dilution was spread on R2A agar plates. Isolation, maintenance and preservation of strain G-1-2-2 T was done as described in previous study ).

Reference strains
Based on the 16S rRNA gene sequence similarities and phylogenetic analyses, Ramlibacter ginsenosidimutans KACC 17527 T ; 3, Ramlibacter monticola KACC 19175 T ; 4, Ramlibacter alkalitolerans KACC 19305 T were used as references and analysed under identical conditions for API, biochemical and fatty acid analyses.
For the determination of fatty acids, cells of reference strains and strain G-1-2-2 T were harvested from identical culture condition (at 28 °C on R2A agar plate for 4 days). Fatty acid methyl esters (FAME) of harvested cells were extracted using MIDI protocol technical note #101 (http:// midiinc. com/ pdf/ MIS_ Techn ote_ 101. pdf).
Extracted FAMEs were analysed using a HP 6890 Series GC System (Gas chromatograph; Hewlett Packard; Agilent Technologies) and the FAME compositions (percentage of totals) were identified with TSBA6 database of the Microbial Identification System (Sasser 1990). The polar lipids and isoprenoid quinones were extracted from freeze-dried cells following the protocol of Minnikin et al. (Minnikin et al. 1984). Appropriate reagents for the spot detection were used as described by Komagata and Suzuki (Komagata and Suzuki 1988).

Phylogenetic analysis
The 16S rRNA gene sequence of strain G-1-2-2 T was 1483 bp long and has been deposited at Gen-Bank/EMBL/DDBJ database under the accession MN685325. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain G-1-2-2 T formed a lineage within the family Comamonadaceae and clustered with members of the genus Ramlibacter. The 16S rRNA gene sequences of strain G-1-2-2 T showed high sequence similarities with R. ginsenosidimutans BXN5-27 T (97.9%), R. monticola G-3-2 T (97.9%), and R. alkalitolerans CJ661 T (97.5%). The sequence similarities of strain G-1-2-2 T with all other members of the genus Ramlibacter were < 96.7%. Strain G-1-2-2 T was clustered with R. alkalitolerans, R. monticola, and R. ginsenosidimutans in all three trees i.e. ML, NJ and MP (Fig. 1, S1 and S2). In addition, in all phylogenetic trees, strain G-1-2-2 T formed a distinct lineage. The phylogenetic positions in all three NJ, ML and MP trees strongly supported to assign strain G-1-2-2 T as a novel member within the genus Ramlibacter.

Genomic analysis
The ContEst16S analysis of strain G-1-2-2 T showed that the genome was not contaminated. Whole-genome shotgun sequence has been deposited at DDBJ/ENA/GenBank under the accession JABBFX000000000. The genome size and N50 value of strain G-1-2-2 T were 7,200,642 bp and 3,830,216 bp, respectively. The genome had 13 scaffolds and coverage of 139.0-fold (Table S1). The graphical genomic map revealed the presence of six rRNAs and 52 tRNAs (Fig. 2). The DNA G + C content of strain G-1-2-2 T is 68.9% which is within the range of Ramlibacter species (Heulin et al. 2003;Lee et al. 2014). The ANI threshold for species demarcation is recommended at 95-96% (Richter and Rosselló-Móra 2009) and ANI values between strain G-1-2-2 T and other Ramlibacter members were ≤ 81.2% (Table 1). The dDDH values of ≤ 24.1% was much lower value than the species threshold of 70% recommended for species delineation (Table 1). These values clearly showed that strain G-1-2-2 T represents a novel member within the genus Ramlibacter (Meier-Kolthoff et al. 2013). Furthermore, the phylogenomic position of strain G-1-2-2 T obtained from tree reconstructed using UBCGs (concatenated alignment of 92 core genes) also showed that strain G-1-2-2 T is a novel member of the genus Ramlibacter (Fig.  S3).

Physiological analysis
The cells of strain G-1-2-2 T were rod-shaped (Fig.  S5), facultative anaerobic, catalase and oxidase positive, non-spore-forming, Gram-stain-negative and motile with flagella. Strain G-1-2-2 T formed whitecolony on R2A agar plate and PHB was accumulated in its cells (Fig. S5). In addition, biosynthesis of PHB was confirmed by fluorescence produced by strain G-1-2-2 T on LB agar plate supplemented with Nile red dye when observed under UV light (Fig. S6). Weak growth was observed in anaerobic condition when incubated for 10 days. Strain G-1-2-2 T hydrolysed urea but not CM-cellulose, casein, starch, gelatin, tyrosine, DNA, Tween 80, Tween 60 or Tween 40. The differential physiological characteristics are given in Table 3 along with its closest reference strains.
Description of Ramlibacter agri sp. nov.
Ramlibacter agri (a′gri. L. gen. n. agri of an agriculture field, referring to the source of isolation).