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...

We previously showed that dprA is required for efficient processing of linear DNA during cellular transformation in Haemophilus influenzae. In this study the transcriptional regulation of dprA and two downstream genes, dprB and dprC, is examined. We demonstrate by Northern blot analysis that the dprABC genes are transcriptionally coregulated and competence inducible. We used primer extension analysis to map the transcriptional start site of dprA and of rec-2, another transformation gene involved in DNA processing. Based upon these results, we were able to identify a 26-bp dyad symmetry element immediately upstream of the ؊35 regions of the predicted promoters of dprA, rec-2, and two other transformation genes, comA and pilA. Finally, using transcriptional fusions of dprA to the Escherichia coli lacZ gene, we show that expression of dprA::lacZ requires tfoX and that the presence of multiple copies of tfoX abolishes the temporal regulation of dprA, resulting in its constitutive expression.Fifty-three years ago Avery and coworkers reported the observation that the bacterium Streptococcus pneumoniae could be phenotypically transformed by DNA (1), and to date as many as forty species of bacteria are known to be capable of natural DNA transformation (18). Most of our understanding of this complex process comes from studies of Bacillus subtilis and S. pneumoniae among the gram-positive bacteria and of Haemophilus influenzae and Neisseria gonorrhoeae among the gram-negative bacteria (18). The basic steps of linear DNA transformation in both gram-positive and gram-negative bacteria are similar and involve DNA binding, internalization, translocation, and recombination with the host chromosome. In H. influenzae, the exact environmental trigger for competence remains unknown; however, competence can be induced in the laboratory by a shift from aerobic to anaerobic growth or when cells reach stationary phase or by the widely used method of incubation in chemically defined M-IV minimal medium (14).The analysis of H. influenzae strains deficient in transformation has contributed a great deal to our understanding of the process of DNA transformation at the molecular level. Our prior studies of the transposon-induced transformation mutant strain GBH37F (26) led to the identification of dprA, a gene encoding a 41.6-kDa protein, which is required for efficient chromosomal, but not plasmid, DNA transformation (15). A dprA mutant strain transforms at frequencies less than 0.1% of that of the wild type, in spite of its capacity to bind and internalize DNA like the wild type, suggesting that the dprA gene product is involved in DNA translocation into the cytoplasmic compartment and/or recombination. DNA sequence analysis and protein expression studies of the dprA locus identified two additional open reading frames (ORFs) (dprB [ORF272] and dprC [ORF193]) of unknown function downstream of dprA (15).Here, using Northern blot analysis and primer extension mapping, we further characterize the transcriptional regulation of the dprABC genes...