Deciphering control of Mechano-Transcription Activators of σ54-RNA polymerase

Sammanfattning: To survive and proliferate, bacteria have to respond to a plethora of fluctuating signals within their habitats. Transcriptional control is one crucial entry point for such signal-responsive adaption responses. In this thesis I present new insights into the signal-responsive control of two specific transcriptional regulators that belong to a specialized class of mechano-transcriptional regulators. These regulators employ ATP-hydrolysis to engage and remodel σ54-RNA polymerase, which allows transcriptional initiation from the promoters they control. In the first part of my thesis I present findings on DmpR – the obligate activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR is a sensory-regulator that can only transition to its active multimeric form upon binding a phenolic compound and ATP. Previous work has established that binding of phenolic effectors by the N-terminal domain of DmpR relieves inter-domain repression of its central ATPase domain and further that a structured inter-domain linker between the phenolic- and ATP-binding domains is involved in coupling these processes. However, the mechanism underlying this coupling remained enigmatic. Here I present evidence that a tyrosine residue of the inter-domain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and phenolic-responsive transcriptional activation by DmpR. A model is presented in which binding of phenolics relocates Y233 from the ATP-binding site to synchronise signal-reception with multimerisation to provide appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family. The finding that an alanine substitution of Y233 enhances transcriptional responses adds a new approach to manipulating the sensitivity of this class of proteins and thereby generate hyper-sensitive detectors of aromatic pollutants for use in safe guarding the environment.The second part of my thesis concerns VCA0117 – a master regulator of the type VI contractile nanomachinery of Vibrio cholerae, which it utilizes to introduce toxic proteins into both bacterial and eukaryotic cells. These type VI-mediated properties enable V. cholerae to establish infections and to thrive in niches co-occupied by predators and competing bacteria. VCA0117 is strictly required for functionality of the type VI system through its role in controlling production of a key type VI structural protein called Hcp, which is encoded within two small s54-dependent operons. This regulatory role is conserved in both pandemic and non-pandemic V. cholerae strains. However, while some strains come pre-equipped with a functional system, others do not, and require specific growth conditions of low temperature and high osmolarity for type VI expression. Within this work, integration of these regulatory growth signals was traced to the activity of the promoter controlling a large operon in which many components of the machinery and VCA0117 is itself encoded. This in turn elevates the levels of VCA0117, which is all that is required to overcome the need for the specialized growth conditions of low temperature and/or high osmolarity. A model is presented in which signal integration via the activity of the large operon promoter to elevate levels of VCA0117 ultimately dictates a sufficient supply of the missing Hcp component required for completion of a functional type VI machine. Repercussions of the proposed quantity-based regulatory circuit of VCA0117 for generating bacterial sub-populations that are differentially “fit” for different environmental eventualities are discussed.