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Abstract
The Pseudomonas putida flhA-flhF-fleN-fliA cluster encodes a component of the flagellar export gate and three regulatory elements potentially involved in flagellar biogenesis and other functions. Here we show that these four genes form an operon, whose transcription is driven from the upstream PflhA promoter. A second promoter, PflhF, provides additional transcription of the three distal genes. PflhA and PflhF are σN-dependent, activated by the flagellar regulator FleQ, and negatively regulated by FleN. Motility, surface adhesion and colonization defects of a transposon insertion mutant in flhF revealed transcriptional polarity on fleN and fliA, as the former was required for strong surface adhesion and biofilm formation, and the latter was required for flagellar synthesis. On the other hand, FlhF and FleN were necessary to attain proper flagellar location and number for a fully functional flagellar complement. FleN, along with FleQ and the second messenger c-di-GMP differentially regulated transcription of lapA and the bcs operon, encoding a large adhesion protein and cellulose synthase. FleQ positively regulated the PlapA promoter and activation was antagonized by FleN and c-di-GMP. PbcsD was negatively regulated by FleQ and FleN, and repression was antagonized by c-di-GMP. FleN promoted FleQ binding to both PlapA and PbcsD in vitro, while c-di-GMP antagonized interaction with PbcsD and stimulated interaction with PlapA. A single FleQ binding site in PlapA was critical to activation in vivo. Our results suggest that FleQ, FleN and c-di-GMP cooperate to coordinate the regulation of flagellar motility and biofilm development.
Highlights
Bacterial life cycles in the environment are commonly characterized by the alternation of a single cell-based free-living planktonic stage and a sessile stage during which they develop structured highly cooperative surface-associated communities, known as biofilms [1]
Semiquantitative Reverse transcription (RT)-PCR was performed using RNA from the wild-type strain KT2442 and the flhF::miniTn5-Km mutant MRB49 as templates and specific primers amplifying a proximal segment of flhA, the flhA-flhF, flhF-fleN, fleN-fliA and fliA-cheY intergenic regions, and a distal segment of flhF (Fig 1)
Our RT-PCR results showing evidence of transcription across all three intergenic regions indicate that all four genes are co-transcribed as part of an operon (Fig 1)
Summary
Bacterial life cycles in the environment are commonly characterized by the alternation of a single cell-based free-living planktonic stage and a sessile stage during which they develop structured highly cooperative surface-associated communities, known as biofilms [1]. While the planktonic lifestyle allows bacterial cells to colonize new niches and gain access to fresh resources (and escape from unfavourable habitats), biofilm growth has been shown to promote positive interactions between organisms, such as syntrophism and horizontal genetic transfer, while providing a nurturing, protective environment [2,3]. Transition between the planktonic and biofilm lifestyles and the ordered succession of such stages arguably requires a variety of signal transduction and regulatory pathways to connect environmental and physiological signals to the adequate physiological responses [7]
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