• Molecular characterization of thedprABC operon in Haemophilus influenzae: DNA transformation and the role of dprA, a novel gene involved in DNA processing during transformation

      Karudapuram, Surekha; Barcak, Gerard J. (1998)
      Natural genetic transformation can be defined as a transient physiological state that enables bacteria to bind and internalize DNA from their environment and recombine it into their chromosome. First discovered in the bacterium Streptococcus pneumoniae, DNA transformation is now known to occur across many species of both gram-positive and gram-negative bacteria. The gram-negative bacterium Haemophilus influenzae is known to undergo natural transformation and can bind and internalize large DNA fragments carrying a 29 bp uptake signal sequence. The aim of this dissertation was to characterize at the molecular level, the H. influenzae DNA transformation gene, dprA that had been previously identified through mini-transposon mutagenesis. Cloning and sequencing of a DNA fragment that could complement the transformation defect of strain GBH37F carrying the transposon mutation tfo-37 identified three open reading frames (ORFs) encoding polypeptides of 373, 272 and 193 amino acids. Using subcloning, deletion analysis, and in vivo protein labeling experiments the 373 aa ORF which we named dprA (DNA processing A) was found to be required for efficient DNA transformation. The product of dprA was a 41.6 kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Interestingly, while DprA was conserved across gram-positive and gram-negative bacterial species, its function was unknown. As part of our studies to understand the regulation of dprA during competence, Northern hybridization analysis demonstrated that dprA, dprB (ORF272) and dprC (ORF193) are transcriptionally coregulated and competence-inducible. The use of primer extension analysis to map the transcriptional start site of dprA and of rec-2, another DNA processing gene, led to the identification of a 26 bp dyad symmetry element immediately upstream of the -35 regions of the predicted promoter of dprA, rec-2, and two other transformation genes, comA and pilA. Next, using transcriptional fusions of dprA to the Escherichia coli lacZ gene, it was shown that the expression of dprA::lacZ required tfoX and that the presence of multiple copies of tfoX abolished the temporal regulation of dprA resulting in its constitutive expression. The transcriptional coregulation of the dprABC genes specifically during the development of competence led to the investigation of whether the two downstream genes dprBC are also involved in DNA transformation. When strains carrying a mutation in either dprB or dprC were assayed for transformability, it was found that the dprB and dprC mutations did not affect transformation. Finally, to understand the biochemical function of DprA during transformation, the dprA mutant strain GBH37F was tested in a nucleoside release assay and the DprA protein was purified as a fusion with maltose-binding protein for future use in the production of anti-DprA antiserum. The studies presented in this dissertation have characterized at the molecular level a novel H. influenzae operon, the dprABC operon and have analysed the role of dprA in DNA processing during transformation.
    • 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.