DNA sequence and characterization of Haemophilus influenzae dprA+, a gene required for chromosomal but not plasmid DNA transformation

Abstract

Natural genetic transformation in Haemophilus influenzae involves DNA binding, uptake, translocation, and recombination. In this study, we cloned and sequenced a 3.8-kbp H. influenzae DNA segment capable of complementing in trans the transformation defect of an H. influenzae strain carrying the tfo-37 mutation. We used subcloning, deletion analysis, and in vivo protein labeling experiments to more precisely define the gene required for efficient DNA transformation on the cloned DNA. A novel gene, which we called dprA ؉ , was shown to encode a 41.6-kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Analysis of the deduced amino acid sequence of DprA suggested that it may be an inner membrane protein, which is consistent with its apparent role in DNA processing during transformation. Four other open reading frames (ORFs) on the cloned DNA segment were identified. Two ORFs were homologous to the phosphofructokinase A (pfkA) and alpha-isopropyl malate synthase (leuA) genes of Escherichia coli and Salmonella typhimurium, respectively. Homologs for the two other ORFs could not be identified.Natural genetic transformation in bacteria is a complex genetically programmed process involving DNA binding, uptake, translocation, and recombination. Haemophilus influenzae is a gram-negative bacterium that in growing cultures can be induced to become competent for transformation by a temporary shift to anaerobic conditions, by physiological change occurring during the late-log phase growth, or by a transfer of cells to a nutrient-poor chemically defined medium such as MIV (10).Only recently has the molecular cloning of transformation genes allowed identification of several structural and regulatory components of the transformation apparatus (4-6, 16, 17, 26, 39). To identify the genes involved in transformation, Tomb and coworkers (35) performed minitransposon mutagenesis using H. influenzae chromosomal DNA and isolated 24 mutant strains that were defective in transformation. These mutant strains were characterized for DNA binding and uptake and for their transformability with H. influenzae chromosomal DNA. Among the 24 strains analyzed, only 2 bound and took up radiolabeled DNA in a manner similar to that of the wild type but transformed at frequencies less than 0.1% of that of the wild type. This suggested that the two strains were defective in events occurring subsequent to DNA uptake, i.e., DNA processing. This processing may involve DNA translocation and/or recombination.In this study, we examine the nature of the defect in one of the two DNA uptake-proficient mutant strains, JG37. The mutated locus derived from strain JG37 was cloned and used to isolate the wild-type locus. The DNA sequence of a 3.8-kbp portion of the locus was determined and shown to encode several polypeptides. At least one of these, DprA, is essential for efficient chromosomal DNA transformation but is not required for plasmid transformation. MATERIALS AND METHODSBacterial strains, plasmids, and culture cond...

Population subdivision and the detection of recombination in non-typable Haemophilus influenzae

“…The precise mechanism of competence in Hi remains incompletely understood, although many proteins important for competence have been identified, including those that make up structures implicated in uptake on the surface of the cells (ComE, PilA), those that are in the periplasm/inner membrane (ComF, ComC, Rec-2) and the cytoplasm (ComA, DprA, ComM) (Gwinn et al, 1998;Karudapuram et al, 1995;Karudapuram & Barcak, 1997;Larson & Goodgal, 1991;McCarthy, 1989;Tomb et al, 1991). Following internalization, the DNA can be inserted into the chromosome by homologous recombination.…”

Multiple interactions among the competence proteins of Bacillus subtilis

“…DprA (Smf) and SsbB (YwpH) (Berka et al, 2002;Lindner et al, 2004) are single-stranded DNA (ssDNA) binding proteins (Mortier-Barrière et al, 2007). dprA (smf) was identified in Haemophilus influenzae and H. pylori as a gene needed for transformation (Karudapuram et al, 1995;Smeets et al, 2000), and in Streptococcus pneumoniae, DprA was shown to protect incoming transforming DNA from degradation (Berge et al, 2003). YjbF (CoiA) is a protein of unknown function, needed for optimal transformation in B. subtilis (Eschevins, 2003).…”

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial GeneraCampylobacterandHelicobacter

“…C. jejuni DprA has been shown to be required for uptake of plasmid DNA but, interestingly, is expendable during uptake of chromosomal DNA (589). This function is in direct contrast to DprA of H. influenzae, which is implicated in natural transformation of chromosomal but not plasmid DNA (317). The fact that C. jejuni carries proteins with similarity to those of known DNA uptake systems, as well as less conserved proteins that are also involved in transformation, perhaps suggests that this bacterium has evolved alternative strategies other than the prototypical T4P/T2SS to facilitate DNA uptake from the environment.…”

“…DprA in H. pylori is homologous to DprA of H. influenzae, which is essential for natural transformation of chromosomal DNA (317). In H. pylori, the function of DprA is unclear; however, the fact that an H. pylori dprA mutant does not completely lose the ability to be naturally transformed suggests that DprA may function to enhance the transformation process (19).…”