Characterization of Brucella abortus sigma factor σ54 (rpoN): Genetic complementation of Sinorhizobium meliloti ntrA mutant

The intracellular pathogen Brucella abortus has an alternative sigma factor σ54 (RpoN) highly similar to Sinorhizobium meliloti NtrA. RpoN was described to be required for the transcription of a wide range of genes involved in diverse physiological functions including the regulation of virulence-related factors in both plants and animal pathogens. B. abortus rpoN gene restored the normal growth of an S. meliloti ntrA mutant in minimal media with succinic acid as a sole carbon source as well as the formation of functional nodules in alfalfa, thus revealing that the gene is functional. B. abortus rpoN mutant and B. abortus wild-type strain harboring a multicopy plasmid coding for a wild-type rpoN gene displayed reduced survival under stationary-phase conditions suggesting that expression of RpoN must be tightly regulated. Real-time PCR analysis revealed that B. abortus rpoN expression is downregulated during the stationary phase of growth. This regulation is absent in the rpoN mutant background, indicating that RpoN regulates its own expression. Intracellular multiplication in HeLa or J774 cells, and survival in BALB/c mice of the rpoN mutant, are not affected. However 2 weeks postinfection survival of rpoN mutant complemented with a multicopy plasmid containing a wild-type rpoN gene is reduced, thus suggesting that overexpression of rpoN may misregulate the expression of genes involved in this stage of infection.