No 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...

EcoRII restriction sites [5'-CC(A/T)GG] in phage T3 and T7 DNA are refractory to cleavage by EcoRII, but become sensitive to cleavage in the presence of DNAs which contain an abundance of EcoRII sensitive sites (e.g. pBR322 or lambda DNA). Studies using fragments of pBR322 containing different numbers of EcoRII sites show that the susceptibility to EcoRII cleavage is proportional to the number of sites in the individual fragment. We postulate that EcoRII is the prototype of restriction endonucleases which require at least 2 simultaneously bound substrate sites for their activation. EcoRII sites are refractory when they occur at relatively low frequency in the DNA. The restriction enzyme can be activated by DNA with a higher frequency of sites.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-TnlOkan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformationdeficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10-2 to 106. Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.In the late logarithmic growth phase or in response to environmental stimuli such as a nutritional shift down and/or a decrease in oxygen tension (15, 16), Haemophilus influenzae cells undergo a transient physiological change (35) which results in a state of competence (that is, the ability of cells to bind, take up DNA, and transform). Three changes have been documented as being associated with competence development and transformation. The first is modification of the cellular envelope. This includes the synthesis of new polypeptides (11,12,42,49), an alteration in the lipopolysaccharide composition (50), and the appearance at the cell surface of vesicle-like structures, or transformasomes, which presumably are the sites of DNA binding and entry (20). The second is an enhanced recombination capacity of the cells, measured as increased frequencies of phage and plasmid recombination (3,7,27). The third is the appearance of single-stranded gaps and tails in the chromosome (24,29). Although relief of catabolite repression is thought to be an important factor in the regulation of the process (31, 47), the physiological link(s) between the different environmental stimuli and the onset of these changes (48) and the exact nature of the involvement of essential and inhibitory metabolites (31,33,34) have yet to be determined.The genetic and biochemical characterization of transformation-deficient mutants generated by nitrosoguanidine mutagenesis (5, 9, 22, 37) led to the identification of steps involved in DNA uptake and transformation (1, 3, 4). As a result, a model for chromosomal transformation of competent H. influenzae cells was proposed (17)(18)(19). Despite these efforts, a systematic genetic analysis of the transformation pathway has not been achieved, in part due to th...