Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mosaic DNA imports with interspersions of recipient sequence after natural transformation of Helicobacter pylori.

ABSTRACT: Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen.

SUBMITTER: Kulick S

PROVIDER: S-EPMC2582958 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori.

Project description:Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.

| S-EPMC6877725 | biostudies-literature

Natural transformation of helicobacter pylori involves the integration of short DNA fragments interrupted by gaps of variable size.

Project description:Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950-1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus.

| S-EPMC2650093 | biostudies-literature

Genome-wide analysis of chromosomal import patterns after natural transformation of Helicobacter pylori.

Project description:Recombination plays a dominant role in the evolution of the bacterial pathogen Helicobacter pylori, but its dynamics remain incompletely understood. Here we use an in vitro transformation system combined with genome sequencing to study chromosomal integration patterns after natural transformation. A single transformation cycle results in up to 21 imports, and repeated transformations generate a maximum of 92 imports (8% sequence replacement). Import lengths show a bimodal distribution with averages of 28 and 1,645 bp. Reanalysis of paired H. pylori genomes from chronically infected people demonstrates the same bimodal import pattern in vivo. Restriction endonucleases (REases) of the recipient bacteria fail to inhibit integration of homeologous DNA, independently of methylation. In contrast, REases limit the import of heterologous DNA. We conclude that restriction-modification systems inhibit the genomic integration of novel sequences, while they pose no barrier to homeologous recombination, which reconciles the observed stability of the H. pylori gene content and its highly recombinational population structure.

| S-EPMC4917963 | biostudies-literature

Characterization of a ComE3 homologue essential for DNA transformation in Helicobacter pylori.

Project description:To find genes involved in natural competence in Helicobacter pylori, we used a bioinformatics database search and found two transformation-related open reading frames (ORFs): a comE3 homologue (HP1361 ORF) of Bacillus subtilis and a comL homologue (HP1378 ORF) of Neisseria gonorrhoeae. We failed to obtain an HP1378 ORF knockout mutant, while an HP1361 ORF knockout mutant was obtained by transposon shuttle mutagenesis. The DNA transformation abilities of both natural transformation and electroporation were severely impaired (frequency, <10(-9)) in the HP1361(-) mutant. Complementation with a pHel2 vector carrying the HP1361 ORF restored the capabilities of natural competence (to a frequency of 4.21 x 10(-7)) and electroporation (to 3.62 x 10(-7)). The HP1361(-) mutant showed impairment in DNA binding and uptake. The results suggest that HP1361 is a comE3 homologue and is required for DNA binding and uptake during DNA transformation.

| S-EPMC187363 | biostudies-literature

Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases.

Project description:Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.

| S-EPMC3434758 | biostudies-literature

HP0333, a member of the dprA family, is involved in natural transformation in Helicobacter pylori.

Project description:Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.

| S-EPMC94075 | biostudies-literature

Environmental determinants of transformation efficiency in Helicobacter pylori.

Project description:Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O2 (5% O2, 7.6% CO2, 7.6% H2) and high O2 (15% O2, 2.9% CO2, 2.9% H2) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O2 tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O2 concentration for these initial experiments also changed the concentrations of CO2 and H2, transformations were repeated under conditions where O2, CO2, and H2 were each varied individually. The results showed that the increase in transformation efficiency under high O2 was largely due to a decrease in CO2. An increase in pH similar to that caused by low CO2 was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.

| S-EPMC3911246 | biostudies-literature

DNA sequence analysis of South African Helicobacter pylori Vacuolating Cytotoxin Gene (vacA).

Project description:Sequence diversity and population structures can vary widely among pathogenic bacteria species. In some species, all isolates are highly similar, whereas in others most of the isolates are distinguished easily. H. pylori is known for its wide genetic diversity amongst the various strains most especially in the genes involved in virulence. The aim of this study was to evaluate by PCR and sequence analysis, the genetic profile of H. pylori vacA gene (s1, s2, m1 and m2). We sequenced small DNA segments from 13 vacAs1, 10 vacAm2, 6 vacAm1 and 6 vacAs2 strains which were amplified with amplicon size of 259/286 bp, 290 bp and 352 bp for vacAs1/s2, m1 and m2 respectively. Based on similarities among our strains accession numbers were provided for seven vacAs1 (HQ709109-HQ709115), six vacAs2 (JN848463-JN848468), six vacAm1 (JN848469-JN848474) and six vacAm2 (HQ650801-HQ650806) strains. Amongst the strains studied, 98.07%, 98.58%, 97.38% and 95.41% of vacAs1, vacAs2, vacAm1 and vacAm2 of the strains were conserved respectively. Findings of this study underscores the importance of understanding the virulence composition and diversity of H. pylori in South Africa for enhanced clinico-epidemiological monitoring and pathophysiology of disease.

| S-EPMC3233416 | biostudies-literature

Characterization of Helicobacter pylori factors that control transformation frequency and integration length during inter-strain DNA recombination.

Project description:Helicobacter pylori is a genetically diverse bacterial species, owing in part to its natural competence for DNA uptake that facilitates recombination between strains. Inter-strain DNA recombination occurs during human infection and the H. pylori genome is in linkage equilibrium worldwide. Despite this high propensity for DNA exchange, little is known about the factors that limit the extent of recombination during natural transformation. Here, we identify restriction-modification (R-M) systems as a barrier to transformation with homeologous DNA and find that R-M systems and several components of the recombination machinery control integration length. Type II R-M systems, the nuclease nucT and resolvase ruvC reduced integration length whereas the helicase recG increased it. In addition, we characterized a new factor that promotes natural transformation in H. pylori, dprB. Although free recombination has been widely observed in H. pylori, our study suggests that this bacterium uses multiple systems to limit inter-strain recombination.

| S-EPMC3075595 | biostudies-literature

The nucleotide excision repair (NER) system of Helicobacter pylori: role in mutation prevention and chromosomal import patterns after natural transformation.

Project description:BACKGROUND: Extensive genetic diversity and rapid allelic diversification are characteristics of the human gastric pathogen Helicobacter pylori, and are believed to contribute to its ability to cause chronic infections. Both a high mutation rate and frequent imports of short fragments of exogenous DNA during mixed infections play important roles in generating this allelic diversity. In this study, we used a genetic approach to investigate the roles of nucleotide excision repair (NER) pathway components in H. pylori mutation and recombination. RESULTS: Inactivation of any of the four uvr genes strongly increased the susceptibility of H. pylori to DNA damage by ultraviolet light. Inactivation of uvrA and uvrB significantly decreased mutation frequencies whereas only the uvrA deficient mutant exhibited a significant decrease of the recombination frequency after natural transformation. A uvrC mutant did not show significant changes in mutation or recombination rates; however, inactivation of uvrC promoted the incorporation of significantly longer fragments of donor DNA (2.2-fold increase) into the recipient chromosome. A deletion of uvrD induced a hyper-recombinational phenotype. CONCLUSIONS: Our data suggest that the NER system has multiple functions in the genetic diversification of H. pylori, by contributing to its high mutation rate, and by controlling the incorporation of imported DNA fragments after natural transformation.

| S-EPMC3438104 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data