Browsing School, Graduate by Author "Karudapuram, Surekha"
Molecular characterization of thedprABC operon in Haemophilus influenzae: DNA transformation and the role of dprA, a novel gene involved in DNA processing during transformationKarudapuram, 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.