Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur-containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize EGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 µg/100 ml culture (1200 µg/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

Symbiosis between legumes and rhizobia leads to the formation of N₂-fixing root nodules. In soybean, several host genes, referred to as Rj genes, control nodulation. Soybean cultivars carrying Rj4 gene restrict nodulation by specific rhizobia such as Bradyrhizobium elkanii. We previously reported that the restriction of nodulation was caused by the B. elkanii possessing a functional type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria. In the present study, we investigated the molecular basis for the T3SS-dependent nodulation restriction in Rj4-soybean. Inoculation tests revealed that soybean cultivar BARC-2 (Rj4/Rj4) restricted nodulation by B. elkanii USDA61, whereas its nearly isogenic line BARC-3 (rj4/rj4) formed nitrogen-fixing nodules with the same strain. Root-hair curling and infection thread were not observed in the roots of BARC-2 inoculated with USDA61, indicating that Rj4 blocked B. elkanii infection in the early stages. Accumulation of H₂O₂ and salicylic acid (SA) was observed in the roots of BARC-2 inoculated with USDA61. Transcriptome analyses revealed that inoculation of USDA61, but not its T3SS mutant in BARC-2 induced defense-related genes, including those coding for hypersensitive-induced responsive protein, which act in effector-triggered immunity (ETI) in Arabidopsis. These findings suggest that B. elkanii T3SS triggers SA-mediated ETI-type response in Rj4-soybean, which consequently blocks symbiotic interactions. This study revealed a common molecular mechanism underlying both plant–pathogen and plant–symbiont interactions and suggests that establishment of a root nodule symbiosis requires the evasion or suppression of plant immune responses triggered by rhizobial effectors.

Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal.

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Bradyrhizobium japonicum , a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B , pcaG1H1 , and genes for C ₁ compound catabolism was upregulated in B. japonicum USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ. 21: 240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in vanA1B , pcaG1H1 , and mxaF mutants of USDA110. The vanA1B and pcaG1H1 mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of vanA1B and pcaG1H1 are exclusively responsible for vanillate degradation. Mutating mxaF eliminated expression of gfa and flhA , which contribute to glutathione-dependent C ₁ metabolism. The mxaF mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the mxaF mutant and the wild-type strain. These results suggest that the C ₁ pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the mxaF mutant did not, suggesting that the metabolic capability of the C ₁ pathway in B. japonicum extends to methanol oxidation. The mxaF mutant showed normal nodulation and N ₂ fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.

Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B. japonicum USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the soxY ₁ gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Alphaproteobacteria , left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY ₂ gene at sox locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O ₂ respiration was observed only in USDA110 and the soxY ₂ mutant and not in the soxY ₁ mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O ₂ . B. japonicum USDA110 showed ¹⁴ CO ₂ fixation under chemolithotrophic growth conditions. The CO ₂ fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on sox locus I including the soxY ₁ gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the Bradyrhizobiaceae , which phylogenetic analysis showed to be associated with the presence of sox locus I homologues, including the soxY ₁ gene of B. japonicum USDA110.