Protein-protein interactions regulate and drive biological processes and understanding the assembly of these interactions is important. The LysR-Type Transcriptional Regulators (LTTRs) are a large family of transcriptional regulators found in prokaryotes. I have used the LTTRs as a model for protein specificity. In order to understand a residue’s contribution to oligomerization, alanine-scanning mutagenesis was used to probe the contribution of residues identified from in silico analysis of two proteins: OxyR and CynR. The contribution of the residues to oligomerization was characterized using lcI repressor fusions. In OxyR, seven residues were identified as hot spots. Moreover, these hot spots are not especially conserved. The interaction surface of OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface. Combined with the in vivo testing, three residues were identified as hot spots. The residues identified in OxyR and CynR do not overlap. To investigate the assembly of the LTTRs I used a negative-dominance assay with lcI repressor fusions. Taken together, I show that the LTTRs in E. coli K-12 are mostly specific in their interactions.

Pseudomonas chlororaphis PA23 inhibits several root pathogens in both the greenhouse and field. A LysR-type transcriptional regulator (LTTR) called PtrA (Pseudomonas transcriptional regulator A) that is essential for Sclerotinia sclerotiorum antifungal activity was discovered through transposon mutagenesis. P. chlororaphis PA23 produces the antibiotics phenazine 1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin, and several additional products that contribute to biocontrol. Phenotypic assays and proteomic analysis have revealed that production of these secondary metabolites are markedly reduced in a ptrA mutant. Most LTTRs regulate genes that are upstream of and divergently transcribed from the LTTR locus. A short chain dehydrogenase (scd) gene lies immediately upstream of ptrA in the opposite orientation. Characterization of an scd mutant, however, has revealed no significant changes in antifungal activity compared to wild-type PA23. Gene expression analysis of the ptrA mutant indicates that ptrA may exert its regulatory effects through the Gac-Rsm network, and may be controlling expression of the scd gene. Collectively these findings indicate that PtrA is an essential regulator of PA23 biocontrol and is connected to other regulators involved in fungal antagonism.

Rhizobiales encompass a diverse group of microbes, ranging from free-living, soil-dwelling bacteria to disease-causing, intracellular pathogens. Although the lifestyle of these organisms vary, many genetic systems are well conserved. One system, named the AbcR/VtlR system, is found throughout rhizobiales, and even extends to bacteria in other orders within the Alphaproteobacteria. The AbcR sRNAs are an example of sibling sRNAs, where two copies of the abcR gene are typically present in the genome. The AbcRs are involved in the negative regulation of ABC-type transport systems, which are important components for nutrient acquisition. Although the AbcRs share several features amongst organisms, major differences can be found in their functional and regulatory redundancy, the targets they regulate and how they regulate them. Specifically, one major difference in the AbcRs lies in the nucleotide sequences utilized by the sRNAs to bind mRNA targets. In the present studies, the regulatory mechanisms of the AbcR sRNAs were further characterized in the mammalian pathogen Brucella abortus, and the full regulatory profiles of the AbcRs were defined in the plant pathogen Agrobacterium tumefaciens. As mentioned above, the AbcR sRNAs are important for the proper regulation of nutrient-acquiring transport systems in the Rhizobiales. Since these sRNAs are critical to the lifestyle of a bacterium, proper regulation of this system is key to survival. A LysR-type transcriptional regulator, named VtlR, was found to be the bonefide transcriptional activator of abcR1 in B. abortus. Furthermore, VtlR has been shown to be a key component in host interactions in several rhizobiales. The preset work has shed light on the evolutionary divergence of this regulator in bacteria, and further defined the regulatory capacity of VtlR in Agrobacterium. Overall, the studies described here have made significant advances in our knowledge of the AbcR/VtlR-regulatory systems in the Rhizobiales, and have further defined this system as being a vital part of host-microbe interactions. / PHD