Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.