• The role of the dyad symmetry element in the regulation of the late transformation genes dprA and rec-2 in Haemophilus influenzae

      Mudannayake, Malkanthi P.; Barcak, Gerard J. (2002)
      Natural genetic transformation can be defined as a transient physiological state in which the bacteria become competent to bind and internalize naked DNA from the environment and recombine it into the chromosome. Previously, this laboratory had identified a 26-bp dyad symmetry element (DSE) in the promoter region of late competence genes of H. influenzae, which was hypothesized to be the binding site of a competence specific transcriptional activator. The objective of this dissertation was to examine the role of the dyad symmetry element in the regulation of the late transformation genes, dprA and rec-2 of H. influenzae. Sequential deletions from the 5' terminus of the dprA and rec-2 promoter regions showed that a deletion of the 5' half of the DSE resulted in a complete loss of competence-inducible transcription activation of dprA and rec-2. Additionally, a deletion 10 nucleotides upstream of the DSE had a deleterious effect on transcription activation in dprA. This led to the identification of an extended dyad in dprA. A random rearrangement of the 3' half of the DSE or a 6-bp insertion resulting in a phase mutation of the DSE and core promoter elements abolished competence inducible transcription activity or rec-2, while an insertion of 11-bp which brings the DSE back in phase with the core promoter elements restored activity. These results suggested that the activation of the DSE is mediated by a specific sequence not a structural motif per se. The activator which binds the DSE was identified as the catabolite activator protein (CAP) by a gel electrophoretic mobility shift assay. The role of RNA polymerase alpha-carboxy terminal domain (RNAP-alpha-CTD) was examined by expressing wild-type alpha or an alpha-CTD truncation mutant under the control of a tet inducible promoter and determining its effect on transcription induction of rec-2. The results indicate that overexpression of wild-type alpha increases rec-2 expression almost two fold, while overexpression of the alpha-CTD truncation mutant does not increase rec-2 expression. Therefore, we can conclude that the alpha-CTD is required for transcription activation of rec-2. A global analysis of gene expression of the competence regulons of Haemophilus influenzae was performed using DNA microarrays. A wild-type strain KW20 and a cap mutant strain JG87 were examined to identify candidate competence genes. Our microarray data validates previous findings as all the known competence genes and approximately 60 other genes were upregulated in strain KW20, during competence induction. The involvement of CAP in transcription activation was also validated by the fact that virtually all the genes upregulated in KW20, were not upregulated in a cap mutant strain. Our microarray analyses have confirmed the inducible expression patterns of previously known late competence genes and identified several new competence inducible genes which may be involved in the transformation pathway.