The dprA gene is required for natural transformation of Helicobacter pylori

Genetic recombination in Helicobacter pylori is believed to be involved in host adaptation of this gastric pathogen and uptake of DNA by natural transformation can result in changes in virulence factors as well as antigenic variation. To elucidate the mechanisms involved in natural transformation we tested two genes with homology to known competence genes (dprA and traG) for their role in this process. Insertion mutants in these genes were constructed in two different H. pylori strains and their competence by natural transformation was compared to the wild-type. Mutation of the traG homolog did not reduce competence. Mutation of the dprA gene, however, severely impaired natural transformation both with plasmid and chromosomal DNA. Our data indicate that dprA and comB3 are essential parts of a common pathway for chromosomal and plasmid transformation.     

Functional characterization of the competence protein DprA/Smf in Escherichia coli

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.     

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

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.     

comH, a novel gene essential for natural transformation of Helicobacter pylori

Helicobacter pylori is naturally competent for transformation, but the DNA uptake system of this bacterium is only partially characterized, and nothing is known about the regulation of competence in H. pylori. To identify other components involved in transformation or competence regulation in this species, we screened a mutant library for competence-deficient mutants. This resulted in the identification of a novel, Helicobacter-specific competence gene (comH) whose function is essential for transformation of H. pylori with chromosomal DNA fragments as well as with plasmids. Complementation of comH mutants in trans completely restored competence. Unlike other transformation genes of H. pylori, comH does not belong to a known family of orthologous genes. Moreover, no significant homologs of comH were identified in currently available databases of bacterial genome sequences. The comH gene codes for a protein with an N-terminal leader sequence and is present in both highly competent and less-efficient transforming H. pylori strains. A comH homolog was found in Helicobacter acinonychis but not in Helicobacter felis and Helicobacter mustelae.     

Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer

Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.     

DprB facilitates inter- and intragenomic recombination in Helicobacter pylori

For naturally competent microorganisms, such as Helicobacter pylori, the steps that permit recombination of exogenous DNA are not fully understood. Immediately downstream of an H. pylori gene (dprA) that facilitates high-frequency natural transformation is HP0334 (dprB), annotated to be a putative Holliday junction resolvase (HJR). We showed that the HP0334 (dprB) gene product facilitates high-frequency natural transformation. We determined the physiologic roles of DprB by genetic analyses. DprB controls in vitro growth, survival after exposure to UV or fluoroquinolones, and intragenomic recombination. dprB ruvC double deletion dramatically decreases both homologous and homeologous transformation and survival after exposure to DNA-damaging agents. Moreover, the DprB protein binds to synthetic Holliday junction structures rather than double-stranded or single-stranded DNA. These results demonstrate that the dprB product plays important roles affecting inter- and intragenomic recombination. We provide evidence that the two putative H. pylori HJRs (DprB and RuvC) have overlapping but distinct functions involving intergenomic (primarily DprB) and intragenomic (primarily RuvC) recombination.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.     

Identification of genes associated with natural competence in Helicobacter pylori by transposon shuttle random mutagenesis.

To identify genes involved in DNA transformation, we generated 1500 insertion mutants of a Helicobacter pylori strain by transposon shuttle mutagenesis. All mutant strains were screened for their frequency of natural transformation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic loci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknockout of HP1326 revealed no impairment in natural transformation, while the other 5 mutants showed the same defective in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels compared with competent strains. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Therefore, our data indicate that HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation.     

Natural transformation of Gallibacterium anatis

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.     

Characteristics of Helicobacter pylori natural transformation

For Helicobacter pylori, which exhibits substantial genetic diversity, many strains are naturally competent for transformation by exogenous DNA. To better understand the mechanism of natural transformation and its role in the generation of diversity, we sought to systematically identify factors important for natural transformation in H. pylori. We now show that the highest frequency of H. pylori transformation occurs when DNA is introduced prior to exponential phase growth, and that it is a saturable phenomenon. That transformation can be inhibited by DNA from Helicobacter (H. pylori and Helicobacter bilis) but not Escherichia coli suggests specificity based on DNA source. Finally, the cag island was determined to be unnecessary for high-frequency transformation.     

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

rpe, a cis-acting element from the strA region of the Haemophilus influenzae chromosome that makes plasmid establishment independent of recombination.

Plasmids that share homology with the Haemophilus influenzae chromosome transform wild-type cells more efficiently than they transform recombination-defective mutants. A 5.2-kilobase-pair chromosomal fragment containing the strA gene of H. influenzae was found to promote efficient plasmid establishment in recombination-defective mutants. A cis-acting element in the insert, called rpe for rec-less plasmid establishment, promoted plasmid transformation in rec-1 and rec-2 mutants without suppressing the recombination defects of these strains. The rpe locus increased plasmid transformation in wild-type cells without interfering with the pathway of plasmid establishment that is dependent on recombination functions.     

Transformation of Haemophilus influenzae by plasmid RSF0885.

Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximum, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.     

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.     

Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA and Mediates Annealing of Complementary Strands Coated by SsbB and SsbA

Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.