Application of mxaF functional gene sequence to determine genetic relatedness among environmental Methylobacterium strains (PPFMs)

Abstract

Pink-pigmented, facultatively methylotrophic bacteria (PPFMs) of the Methylobacterium genus utilize plant-derived methanol as a carbon and energy source when growing on plant roots. To accomplish this, they employ the methanol dehydrogenase (MDH) enzyme, for which mxaF is the structural gene. Changes in mxaF sequence can result in decreased enzyme efficiency. Environmental conditions such as soil pH can alter MDH efficiency and impact bacterial proliferation in soil by affecting the solubility and chemical speciation of metals and nutrients. Soil pH can be influenced by location, soil type, ecosystem type and plant cover. Phenotypic analyses as well as 16S rRNA and mxaF functional gene phylogeny and denaturing gradient gel electrophoresis (DGGE) were performed on nine New Jersey agricultural soil PPFM isolates to compare gene characteristics and to determine the feasibility of using mxaF as a genetic tool to distinguish between PPFM species. 16S rRNA and mxaF functional gene phylogeny of 114 New Jersey, South Carolina, and California forest and agricultural soil Methylobacterium isolates were examined in order to apply mxaF sequence as a tool for describing genetic relatedness among newly acquired soil Methylobacterium strains. The pH of soil sample source was considered as a potential influencing factor for PPFM species distribution and thus mxaF sequence in soil environments. Nucleotide sequences of PPFM 16S rRNA gene fragments were too similar to be useful for species characterization. DGGE separation of mxaF fragments revealed species-specific banding patterns for PPFM strains whereas 16S rRNA patterns were nearly identical for multiple strains. pH, location and plant type were found not to be a selecting factor for mxaF sequence and PPFM species distribution in soil, suggesting the robust and versatile nature of the mxaF gene in Methylobacterium.