Whole genome sequence analysis of Methylorubrum thiocyanatum VRI7-A4: a versatile sulfur-oxidizing bacterium with environmental stress resilience

Abstract

Whole-genome sequencing and analysis of Methylorubrum thiocyanatum VRI7-A4, a plant-associated methylotroph, revealed key genomic features underlying its ecological adaptability, sulfur oxidation and plant growth-promoting potential. VRI7-A4 exhibited the mixotrophic growth with oxidation of thiosulfate to sulfate in the presence of methanol or succinate and exhibited elevated rhodanese (334.20 nmol min⁻¹ mg⁻¹) and sulfite oxidase (34.60 nmol min⁻¹ mg⁻¹) activities. The genome was sequenced using Illumina platform and assembled into 26 high-quality contigs, totalling 5.34 Mb with a GC content of 69.53%. RASTtk annotation revealed 5152 protein-coding genes and 46 tRNAs, with 68% of genes functionally annotated. Phylogenetic analyses placed VRI7-A4 within the Methylobacteriaceae family, closely related to M. populi and M. thiocyanatum JCM 10893^T. Comparative genomics revealed both conserved core regions and structural rearrangements, indicative of niche-specific adaptations. Functionally, the genome encodes 1893 genes involved in diverse metabolic pathways, including amino acid biosynthesis, energy metabolism, and membrane transport. KEGG and Gene Ontology (GO) analyses highlighted genes linked to nitrogen and phosphate metabolism, abiotic stress tolerance, and plant hormone biosynthesis, specifically indole-3-acetic acid (IAA), cytokinins, and ACC deaminase. Notably, genes implicated in sulfur metabolism, including sulfite oxidase and rhodanese, were identified, indicating a possible function in the sulfur cycle. Core enzymes essential for methylotrophic metabolism and C1 carbon utilization such as methanol dehydrogenase and crotonyl-CoA carboxylase/reductase were identified. Additional genes associated with stress resilience (heat, salinity, and oxidative stress), rhizosphere and phyllosphere competence (flagellar biosynthesis, chemotaxis, and secretion systems) were present. Phenotypic assays confirmed that VRI7-A4 tolerates up to 8% NaCl and 20% PEG-induced osmotic stress also observed. Overall, the genome of M. thiocyanatum VRI7-A4 reflects a highly adaptable and functionally diverse bacterium, well-suited for beneficial plant–microbe interactions. These traits position it as a strong candidate for bioinoculant development in salt- and drought-stressed also sulfur deficient agroecosystems.