Efficient counterselection for Methylococcus capsulatus (Bath) using a mutated pheS gene

ABSTRACT

Methylococcus capsulatus (Bath) is a representative gamma-proteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is considerably low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introducing two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the alpha-subunit of phenyl alanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, that we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated the successful disruption of the xoxF gene in M. capsulatus (Bath). The difference of counterselection efficiencies by the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).

Importance Methanotrophs have long been considered a promising strain for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenesis efficiency remains lower than that of tractable strains such as Escherichia coli. Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.