Transposon mutagenesis and characterisation of the selenite reduction pathway in Methylosinus trichosporium

This study aimed to identify genes responsible for remediation of selenite pollution in the methane-oxidising bacterium Ms. trichosporium OB3b. It was proposed to achieve this by making a library of mutants inactivated in different genes and screening these mutants to see which were deficient in the remediation reactions and hence identify genes that are involved. The pSAM_Rl plasmid, which contains the mariner transposon and was developed for use in the Rhizobiaceae, was introduced into Ms. trichosporium by conjugative transfer of the plasmid from E. coli SM10 λpir. The progeny from the conjugation were kanamycin (Km) resistant (strongly suggesting they contain the plasmid) and resistant to nalidixic acid (confirming removal of the E. coli donor strain). The presence of the transposon and its distribution around the chromosome were confirmed by genome sequencing of 18 randomly selected clones. To make a transposon library suitable for screening, single colonies were picked from the conjugation plates onto a regular 6  8 grids on fresh plates to allow transfer of the library into 96-well plates with a Microplate Replicator. The individual clones of the library were stored in NMS medium with 30% glycerol at -80ºC in 96-well plates. 5,500 clones prepared in this way were screened on NMS agar with Km and selenite, to identify any mutants that were unable to reduce selenite to red elemental selenium. Screening of the library identified a mutant with liminished selenite-reduction activity. This mutant was inactivated in one of two gene copies of pmoB, encoding the largest subunit of the particulate methane monooxygenase involved in oxidizing methane to methanol. Hence, it may be impaired in selenite reduction due to a general deficiency of reducing equivalents within the cell. Parallel experiments investigating the cellular localization of selenite-reducing activity indicated that the cytoplasmic selenite-reducing activity is likely to be due to a small molecule, because when the proteins of cytoplasm were digested the cytoplasm still reduced selenite.