The genus Methylobacterium is Gram-staining-negative, aerobic and rod-shaped bacteria that are commonly found on the phyllosphere (Jahan and McDonald, 2023). These bacteria have the ability to establish epiphytic (surface), endophytic (inside) or even symbiotic associations with plants (Sy et al., 2001), although they are not known to cause plant diseases. Methylobacterium species primarily utilize methanol, a volatile organic compound (VOC) released by plants during growth through stomatal pores (Galbally and Kirstine, 2002), but can also rely on other plant-derived carbon compounds for colonization (Sy et al., 2005; Delmotte et al., 2009).

Methylobacterium species, referred to as pink-pigmented facultative methylotrophs (PPFMs), are able to utilize methanol as their sole carbon and energy source (Corpe and Rheem, 1989). In addition to methanol metabolism, these bacteria are capable of producing plant growth-promoting substances, such as cytokinins, auxins, and vitamin B₁₂ (Ivanova et al., 2000, 2001). These promote seed germination, root development, and crop yields (Meena et al., 2012). The genus consists of approximately 50 species, which have been identified in a diverse environment, including plant tissues, soil, and water (Kelly et al., 2014). Several species have exhibited capabilities in nitrogen fixation, phosphate solubilization, and the prevention of plant pathogens (Madhaiyan et al., 2006; Kumar et al., 2016). Due to their wide distribution and metabolic versatility, Methylobacterium species are increasingly recognized as promising candidates for microbial biofertilizers, offering a sustainable alternative to the use of agrochemicals and contributing to improvements in soil fertility (Grossi et al., 2020). In this study, we report the whole genome sequence of strain HMF5984, a member of the genus Methylobacterium with plant growth promoting potential.

Methylobacterium sp. HMF5984 was isolated from the leaves of the Korean fir (Abies koreana) collected in mountain Jiri, Gurye, Republic of Korea (GPS, 35°18'19.2"N 127°30'41.3"E). The collected leaves (0.5 g) were finely chopped and added to a solution of sterile distilled water containing 0.5% NaCl (w/v). This solution was then vortexed for five minutes to prepare a suspension. Serial dilution was then employed to produce a solution of the appropriate concentration, which was subsequently spread on R2A agar (Difco). The plates were then incubated at 25°C for three days. The pink colored colonies were picked and cultivated periodically in R2A at 25°C.

The whole-genome sequence of strain HMF5984 was obtained using a combination of the PacBio Sequel II (Pacific Biosciences) and NovaSeq (Illumina) platforms. Genomic DNA extraction and sequencing for the genome analysis were performed at Macrogen (Korea). The PacBio HiFi long reads were assembled using the Improved Phased Assembler (IPA) (Pacific Biosciences, 2020), and the assembly was further corrected and improved with Pilon using short reads from the Illumina system (Walker et al., 2014). The assembled genome sequences were deposited in NCBI GenBank under the accession number JBJFLN000000000. The 16S rRNA gene of HMF5984, extracted from the genome and 1,483 bp in length, showed 98.59% sequence similarity to M. cerastii C44ᵀ, as determined using the EzBioCloud server (www.ezbiocloud.net). To further assess the genomic relatedness between strain HMF5984 and the closely related species M. cerastii DSM 23679^T, analyses of average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) were performed. The ANI values were calculated using an ANI Calculator provided by EzBioCloud tools (Yoon et al., 2017), and dDDH values were calculated using the Genome-to-Genome Distance Calculator (Meier-Kolthoff et al., 2013). The ANI and dDDH values between strain HMF5984 and M. cerastii DSM 23679^T were 89.2% and 37.5%, respectively, which were below the thresholds of 95–96% ANI and 70% dDDH (Goris et al., 2007). The genome-based phylogeny was reconstructed using the Type Strain Genome Server (TYGS) (Meier-Kolthoff and Goker, 2019). In the phylogenomic tree, strain HMF5984 was formed a robust clade with M. cerastii DSM 23679^T (Supplementary data Fig. S1).

Gene prediction and genome annotation were carried out using the rapid annotation tool Prokka v1.14.6 (Seemann, 2014). Functional prediction of protein-coding genes was performed by searching against clusters of orthologous groups (COGs) using the eggNOG database (Huerta-Cepas et al., 2017), and whole-genome functional annotation was completed using eggNOG-mapper v2. The metabolic pathways of strain HMF5984 were reconstructed using BlastKOALA (Kanehisa et al., 2016). Completeness assessment of genome assembly was examined using BUSCO 4.1.4 (Simão et al., 2015). The potential contamination of the assembled sequence was evaluated using the contEst16S web server (Lee et al., 2017).

The draft genome of strain HMF5984 comprises five contigs, with a total length of approximately 5.6 Mbp (5,616,623 bp), an N50 value of 5,448,379 bp and a final assembly coverage of 331.10×. The genome sequences of strain HMF5984 were found to contain a total of 5,488 genes, of which 5,410 were protein-coding genes, 66 were tRNA genes, and 12 were rRNA genes. The genomic G + C content was 69.6% (Table 1). In the COGs functional categories, the predominant categories observed in strain HMF5984 were function unknown (S, 28.76%), general function prediction only (R, 12.35%), replication, recombination and repair (L, 6.29%), amino acid transport and metabolism (E, 5.71%), signal transduction mechanisms (T, 5.69%) and cell wall/membrane/envelope biogenesis (M, 5.45%). The distribution of genes into COG functional categories were presented in Supplementary data Table S1. The genome of strain HMF5984 encoded various functional genes, including those related to cell motility, such as bacterial chemotaxis and flagella assembly. Additionally, the genome contained genes associated with carbon fixation (mdh, ppc, maeB, and ppdK), Vitamin B₁₂ biosynthsis, methanol oxidation (xoxF), and formaldehyde assimilation via the serine pathway (glyA, AGXT, hprA, qck, eno, ppc, mdh, mtkAB, and mcl). The genome encoded genes for both the tetrahydrofolate (H₄F) and tetrahydromethanopterin (H₄MPT) pathways involved in C1 oxidation, including methenyl-H4F cyclohydrolase (fch) for H₄F pathway, and formaldehyde-activating enzyme (fae), methylene-H₄MPT dehydrogenase (mtdB), methenyl-H₄MPT cyclohydrolase (mch) and the formyltransferase/hydrolase complex genes (fhcA, fhcB, fhcC, and fhcD) for the H₄MPT pathway. Genes encoding NAD(P)+ transhydrogenase (pntA, pntB) maintain redox balance. The strain also utilizes the ethylmalonyl-CoA (EMC) pathway, with genes encoding β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), crotonyl-CoA carboxylase/reductase (ccr), ethylmalonyl-/methylmalonyl-CoA epimerase (epi), ethylmalonyl-CoA mutase (ecm), mesaconyl-CoA hydratase (mcd), and malyl-CoA thioesterase (mcl2) contributing to acetyl-CoA assimilation. Genes related to the glycine cleavage system (gcv) and the phosphoserine pathway, including phosphoglycerate dehydrogenase (serA), phosphoserine phosphatase (serB), and phosphoserine aminotransferase (serC), support amino acid and nucleotide biosynthesis. Additional genes involved in C3 metabolism include those for PEP carboxykinase (pckA), malic enzyme (dme), pyruvate kinase (pyk), pyruvate phosphate dikinase (ppdK), and the pyruvate dehydrogenase complex (pdhA, pdhB, pdhC, and lpd). The genome also encodes formate dehydrogenase components, including dehydrogenases (fdhA), molybdopterin-binding aldehyde oxidase/xanthine dehydrogenase (fdhC, fdhD, and fdhE), both necessary for formate dehydrogenase activity. Genes encoding 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase; accD) were also identified. Furthermore, the genome harbored genes involved in aromatic amino acid metabolism, including those of the shikimate pathway (aroABCEFKQ) and L-tryptophan biosynthesis (trpABCDEFG). Genes related to indole-3-acetic acid (IAA) biosynthesis (amiE, nthAB, and nitrilase) were present, along with those involved in assimilatory nitrate reduction (nasC, nirA, nasABDE) and assimilatory sulfate reduction (cysCDHIJN). Additionally, genes responsible for inorganic phosphate solubilization via pyrroloquinoline quinone (PQQ) synthesis (pqqABCDEL) were identified. The urease gene cluster (comprising ureABCDEFGJ) is also present and plays a role in urea hydrolysis and nitrogen metabolism. The bacterial secondary metabolite biosynthetic gene cluster was analysed using antiSMASH version 7.1.0, which revealed that the genome of strain HMF5984 contains six biosynthesis gene clusters (BGCs), including the genes for terpene (one of the three regions is the carotenoid-associated BCG), thiopeptide, homoserine lactone (hserlactone), polyketide synthase type I (T1PKS), redox-cofactor (such as PQQ) and non-alpha poly-amino acids (NAPAA).

Table 1

General genomic features of Methylobacterium sp. HMF5984

Genome features	Value
Genome size (bp)	5,616,623
No. of contigs	5
Contig N50 values	5,448,379
Total no. of genes	5,488
No. of protein coding genes	5,410
No. of tRNA genes	66
No. of rRNA genes	12
GenBank accession number	JBJFLN000000000

Strain HMF5984 exhibits several potential characteristics that could promote plant growth, including the presence of genes that enhance plant development and soil fertility. Under phyto-stimulation, the strain supports plant growth by producing indole-3-acetic acid (IAA), a phytohormone involved in root elongation and cell division. Additionally, it encodes 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which mitigates plant stress by reducing ethylene levels, a hormone associated with stress responses. The presence of genes for L-tryptophan biosynthesis further supports IAA production, enhancing the bacterium’s ability to stimulate plant growth. In terms of phyto-fertilization, HMF5984 plays a vital role in nutrient acquisition. It facilitates inorganic phosphate solubilization through the production of pyrroloquinoline quinone (PQQ), making phosphorus more accessible to plants. Moreover, the strain contributes to nitrogen availability by encoding genes involved in nitrate reduction, converting nitrate into forms usable by plants. Thus, these features suggest that HMF5984 has the potential to be an effective plant growth-promoting bacterium (PGPB).

Strain and nucleotide sequence accession numbers

Methylobacterium sp. HMF5984 has been deposited at the Honam National Institute of Biological Resources (HNIBR) under the accession number HNIBRBA12172. The strain HMF5984 is available from the Bank of Bioresources from Island and Coast (BOBIC). The draft genome accession number of strain HMF5984 is JBJFLN000000000. The associated BioProject and BioSample accession numbers are PRJNA449135 and SAMN44066587, respectively

Methylobacterium sp. HMF5984는 구상나무(Abies koreana) 잎에서 분리된 호기성, 분홍색 색소를 가진 그람음성 박테리아입니다. HMF5984 균주의 초안 유전체는 5개의 컨티그로 구성되어 있으며, 총 길이는 약 5.6 Mbp이고 G + C 함량은 69.6%입니다. HMF5984 균주의 유전체에는 5,410개의 단백질이 암호화된 유전자, 66개의 운반RNA (tRNA), 12개의 리보솜 RNA (rRNA)가 포함되어 있습니다. Methylobacterium sp. HMF5984의 유전체는 인산 용해, 인돌아세트산(IAA) 합성 및 1-아미노사이클로프로판카르복실산 탈아민 효소 활성을 포함한 여러 식물 성장 촉진과 관련된 유전자들이 존재함을 보여줍니다. 또한, 요소 대사, 황 및 질산염 환원과 관련된 유전자들도 확인되었습니다. 이 유전자들은 Methylobacterium sp. HMF5984가 식물 성장 촉진 박테리아로서의 잠재력을 가지고 있음을 나타내며, 식물 생장 향상 및 스트레스 내성 개선에 도움이 될 것입니다.

This work was supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (HNIBR202101111).

The authors declare that there are no conflicts of interest.