Investigating AmrZ-mediated activation of Pseudomonas aeruginosa twitching motility and alginate production

The Gram negative bacterium Pseudomonas aeruginosa is ubiquitous in the natural environment and responsible for various human infections. Successful P. aeruginosa infections require many virulence factors such as flagella, type IV pilus and alginate, whose expression is under tight control by transcription factors such as AmrZ. AmrZ activates type IV pilus-mediated twitching motility and alginate production while repressing the flagella machinery. However, molecular mechanisms of AmrZ-mediated activation of twitching motility and the alginate biosynthesis operon promoter ( algD promoter) remain elusive and are the emphases of this study.;Specifically, DNA microarray analyses were performed in order to identify AmrZ-dependent gene(s) that is required for twitching motility. The AmrZ regulon contains 112 genes, many of which are involved in virulence. However, this regulon does not contain a gene that is known to be necessary for twitching motility. In addition, we used multiple biochemical methods to show that AmrZ tetramerizes via its C-terminal domain, the loss of which results in diminished DNA binding and reduced efficiency during AmrZ-mediated activation and repression. Demonstration of AmrZ tetramerization also assists in understanding the activation mechanism of the algD promoter. This complex promoter contains a number of transcription factor binding sites with many in the far upstream region. It is unclear how those transcription factors from far upstream contribute to transcription activation. This study identified four AmrZ binding sites within this promoter and showed that each is required for algD transcription activation. We also illustrated phase-dependent activation of this promoter, suggesting interactions between upstream and downstream factors. Furthermore, in vitro fluorescence resonance energy transfer experiments indicate the formation of trans DNA-AmrZ complexes, suggesting that two DNA molecules are held together by AmrZ, likely via AmrZ oligomerization. These results thus provide new support for the DNA looping model of the algD promoter.;Taken together, the work described in this document has significantly enhanced our knowledge of AmrZ-mediated regulation, full understanding of which may provide insights into discovering new means of interfering this regulation and successful control of P. aeruginosa infections.