Understanding The Molecular Mechanism Of Manganese Oxidation In Leptothrix Discophora Ss1

Abstract

The purpose of this research is to understand the molecular mechanism of manganese oxidation in Leptothrix discophora SS1 which until now has been hampered by the lack of a genetic system. Leptothrix discophora SS1 is an important model organism that has been used to study the mechanism and consequences of biological manganese oxidation. In this study we report on the development of a genetic system for L. discophora. First, the antibiotic sensitivity of L. discophora 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 (transconjugant/donor). Genetic manipulation of Leptothrix was demonstrated by disrupting pyrF via chromosomal integration of a plasmid with an R6Kɣ ori through homologous recombination. This resulted in resistance to fluoroorotidine which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS-5. This genetic system was further used to disrupt five genes in Leptothrix discophora SS1, which were considered to be the best candidates for the enzyme encoding the manganese oxidizing activity in this bacterium. All of the disrupted mutants continued to oxidize manganese, suggesting that these genes may not play a role in manganese oxidation, as hypothesized. MofA a putative muticopper oxidase, identified from the oxidizing fraction of Leptothrix discophora SS1 supernatant to encode the manganese oxidizing activity, was deleted from the genome and the cells lacking mofA did not lose the ability to oxidize manganese. This finding suggests that mofA is dispensable to Mn oxidation in Leptothrix. Transposon mutagenesis performed on a [INCREMENT]mofA Leptothrix strain resulted in the isolation of white, non-manganese oxidizing mutants. Mapping of the transposon insertions identified insertions in 4 genes located in 2 regions on the chromosome. One of the genes, named mnxG2 is a putative multicopper oxidase similar to the manganese oxidizing enzyme in Bacillus sp SG-1. The other three genes with insertions encode a hypothetical protein, a putative cytochrome c next to a putative copper metallochaperone (Sco1/SenC/PrrC) involved in the biogenesis of cytochrome oxidase. Further analysis of the nonmanganese oxidizing mutants identified through transposon mutagenesis coupled with the draft genome sequence of Leptothrix discophora SS1 should provide information about the number and the nature of proteins involved in manganese oxidation in this bacterium. The draft genome of Leptothrix discophora SS1 contains 4.2Mb with 3,791 identified protein coding sequences. In contrast with previous information of Leptothrix as an obligate aerobic heterotroph, functional analysis of the draft genome revealed the potential for a diverse metabolism such as fermentation, anaerobic respiration with nitrate and arsenate, sulfur oxidation and carbon fixation. The information provided by the draft genome about the metabolism of L. discophora SS1 as well as genomic context information about the genes identified to be important in manganese oxidation represent an important addition to the genetic system developed for Leptothrix, and together with the new metabolic information should expand our understanding of the manganese oxidation in Leptothrix discophora SS1