In this study with the model organism Agrobacterium tumefaciens , we used a combination of lacZ gene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of As~(III) oxidation. A deletion mutation that removed the RpoN binding site from the aioBA promoter and an aacC3 (gentamicin resistance) cassette insertional inactivation of the rpoN coding region eliminated aioBA expression and As~(III) oxidation, although rpoN expression was not related to cell exposure to As~(III). Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters for aioB , phoB1 , pstS1 , dctA , glnA , glnB , and flgB that were examined by using qualitative RT-PCR and lacZ reporter fusions to assess the relative contribution of RpoN to their transcription. The expressions of aioB and dctA in the wild-type strain were considerably enhanced in cells exposed to As~(III), and both genes were silent in the rpoN :: aacC3 mutant regardless of As~(III). The expression level of glnA was not influenced by As~(III) but was reduced (but not silent) in the rpoN :: aacC3 mutant and further reduced in the mutant under N starvation conditions. The rpoN :: aacC3 mutation had no obvious effect on the expression of glnB , pstS1 , phoB1 , or flgB . These experiments provide definitive evidence to document the requirement of RpoN for As~(III) oxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by As~(III) or by this sigma factor.
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