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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.

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.