Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid). These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.

Aerial plant surfaces are colonized by diverse bacteria such as the ubiquitous Methylobacterium spp. The specific physiological traits as well as the underlying regulatory mechanisms for bacterial plant colonization are largely unknown. The purpose of this study was to identify proteins produced specifically in the phyllosphere by comparing the proteome of Methylobacterium extorquens colonizing the leaves either with that of bacteria colonizing the roots or with that of bacteria growing on synthetic medium. We identified 45 proteins that were more abundant in M. extorquens present on plant surfaces as compared with bacteria growing on synthetic medium, including 9 proteins that were more abundant on leaves compared with roots. Among the proteins induced during epiphytic growth, we found enzymes involved in methanol utilization, prominent stress proteins, and proteins of unknown function. In addition, we detected a previously undescribed type of two-domain response regulator, named PhyR, that consists of an N-terminal sigma factor (RpoE)-like domain and a C-terminal receiver domain and is predicted to be present in essentially all Alphaproteobacteria . The importance of PhyR was demonstrated through phenotypic tests of a deletion mutant strain shown to be deficient in plant colonization. Among PhyR-regulated gene products, we found a number of general stress proteins and, in particular, proteins known to be involved in the oxidative stress response such as KatE, SodA, AhpC, Ohr, Trx, and Dps. The PhyR-regulated gene products partially overlap with the bacterial in planta -induced proteome, suggesting that PhyR is a key regulator for adaptation to epiphytic life of M. extorquens .

Bacteria have evolved regulatory traits to rapidly adapt to changing conditions. Two principal regulatory mechanisms to modulate gene expression consist of regulation via alternative sigma factors and phosphorylation-dependent response regulators. PhyR represents a recently discovered protein family combining parts of both systems: a sigma factor-like domain of the extracytoplasmic function (ECF) subfamily linked to a receiver domain of a response regulator. Here we investigated the mode of action of this key regulator of general stress response in Methylobacterium extorquens . Our results indicate that PhyR does not act as a genuine sigma factor but instead controls gene expression indirectly through protein-protein interactions. This is evident from the analysis of additional proteins involved in PhyR-dependent gene regulation. We demonstrated that the ECF sigma factor-like domain of PhyR interacts with a protein, designated NepR, upon phosphorylation of the PhyR receiver domain. Using transcriptome analysis and phenotypic assays, we showed that NepR is a negative regulator of PhyR response. Furthermore, we provide biochemical and genetic evidence that NepR exerts this inhibitory effect through sequestration of the ECF sigma factor σ ^EcfG1 . Our data support an unprecedented model according to which PhyR acts as a mimicry protein triggering a partner-switching mechanism. Such a regulation of general stress response clearly differs from the two known models operating via σ ^S and σ ^B . Given the absence of these master regulators and the concomitant conservation of PhyR in Alphaproteobacteria, the novel mechanism presented here is most likely central to the control of general stress response in this large subclass of Proteobacteria.