Y. pestis, the etiologic agent of plague, is a significant human pathogen because of its historic role as the cause of three major pandemics and its current role as a potential bioterrorism agent. Gene regulation in this organism is complex and multiple microarray studies have shown that the expression of several genes is affected by quorum sensing as well as temperature. Maltose utilization genes are of particular interest because they are regulated by both of these systems. In order to perform genetic studies on mal genes, and to better understand gene expression in general in Y. pestis, we developed a number of new tools that comprise the majority of the work in this thesis.

To take advantage of recombineering as a useful system for constructing new mutants, we improved the protocol to promote a higher efficiency of survival and recombination in electroporated Y. pestis cells. We also designed a ligase-independent cloning system to build lacZ gene fusions more quickly and efficiently. We then took advantage of this system to design a method for building single-copy lacZ fusions that are conveniently transferred to Y. pestis by conjugation. These methods were used in combination to examine temperature regulation and quorum sensing effects on malK expression. We determined that a malK-lacZ translational gene fusion is strongly downregulated at 37°C in Y. pestis and slightly downregulated in similar conditions in E. coli. Fusions to a constitutively expressed promoter, however, did not exhibit this kind of expression. In contrast, the gene fusion did not show significant differences in a quorum sensing negative strain, suggesting that AHL quorum sensing does not contribute to regulation of the expression of mal genes under the conditions of the experiment. When these fusions were assayed in strains deleted for malT but complemented with malT expressed on low-copy plasmids, we observed that Y. pestis MalT produced a more significant change in response to temperature than did E. coli MalT. Because the amino acid sequences are mostly similar between both species, future work will focus on what differences in MalT produce such dissimilar temperature regulation effects in these bacteria. The systems we have developed will improve future work with this organism to determine the molecular mechanisms of gene regulation in this species.