Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in Azospira suillum PS

Abstract

ABSTRACTAlthough much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism.