Two-component systems (TCS) are broadly employed by bacteria to appropriately sense and respond to stimuli. TCS are typically thought of as insulated, linear pathways. However, emerging data provide evidence that complex cellular decisions requiring integration of multiple signals can use interconnected networks of TCS proteins. Many bacteria utilize TCS to regulate their transition to a surface-associated community lifestyle, known as a biofilm. While a surface-associated lifestyle can have advantages, shifts in the physiochemical state of the environment may result in conditions in which attachment has a negative fitness impact. Therefore, bacteria employ numerous mechanisms to control the complex surface attachment decision. The Alphaproteobacterium Caulobacter crescentus secretes a polar polysaccharide adhesin known as the holdfast, which enables permanent attachment to surfaces. The small protein, HfiA, is a potent developmental inhibitor of holdfast synthesis. Multiple environmental cues influence expression of hfiA, but mechanisms of hfiA regulation remain largely undefined. The TCS LovK/LovR has previously been shown to repress hfiA transcription, which results in an increase in holdfast development. However, LovR lacks a DNA-binding output domain, suggesting that regulation of hfiA transcription by LovK/LovR is indirect. Through a forward genetic selection, I sought to identify other regulatory proteins that play a role in LovK/LovR-dependent regulation of hfiA transcription. I have discovered a multi-gene network encoding a suite of two-component system (TCS) proteins and transcription factors that coordinately control hfiA transcription, holdfast development and surface adhesion. The hybrid HWE-family histidine kinase, SkaH, is central among these regulators and forms heteromeric complexes with the kinases, LovK and SpdS. The response regulator SpdR indirectly inhibits hfiA expression by activating two XRE-family transcription factors that directly bind the hfiA promoter to repress its transcription. My thesis provides evidence for a model in which a consortium of environmental sensors and transcriptional regulators integrate environmental cues at the hfiA promoter to control the attachment decision.




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