Formal verification of kLIBC with the WP frama-C plug-in

This paper presents our results in the formal verification of kLIBC, a minimalistic C library, using the Frama-C/WP tool. We report how we were able to completely verify a significant number of functions from and . We discuss difficulties encountered and describe in detail a problem in the implementation of common functions, for which we suggest alternative implementations. Our work shows that it is presently already viable to verify low-level C code, with heavy usage of pointers. Although the properties proved tend to be shallower as the code becomes of a lower-level nature, it is our view that this is an important direction towards real-world software verification, which cannot be attained by focusing on deep properties of cleaner code, written specifically to be verified.This work is funded by ERDF - European Regional Development Fund through the COMPETE Programme (operational programme for competitiveness) and by National Funds through the FCT - Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) within project FCOMP-01-0124-FEDER-020486

DNA Damage Triggers Genetic Exchange in Helicobacter pylori

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen

Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

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

CGAT: a comparative genome analysis tool for visualizing alignments in the analysis of complex evolutionary changes between closely related genomes

BACKGROUND: The recent accumulation of closely related genomic sequences provides a valuable resource for the elucidation of the evolutionary histories of various organisms. However, although numerous alignment calculation and visualization tools have been developed to date, the analysis of complex genomic changes, such as large insertions, deletions, inversions, translocations and duplications, still presents certain difficulties. RESULTS: We have developed a comparative genome analysis tool, named CGAT, which allows detailed comparisons of closely related bacteria-sized genomes mainly through visualizing middle-to-large-scale changes to infer underlying mechanisms. CGAT displays precomputed pairwise genome alignments on both dotplot and alignment viewers with scrolling and zooming functions, and allows users to move along the pre-identified orthologous alignments. Users can place several types of information on this alignment, such as the presence of tandem repeats or interspersed repetitive sequences and changes in G+C contents or codon usage bias, thereby facilitating the interpretation of the observed genomic changes. In addition to displaying precomputed alignments, the viewer can dynamically calculate the alignments between specified regions; this feature is especially useful for examining the alignment boundaries, as these boundaries are often obscure and can vary between programs. Besides the alignment browser functionalities, CGAT also contains an alignment data construction module, which contains various procedures that are commonly used for pre- and post-processing for large-scale alignment calculation, such as the split-and-merge protocol for calculating long alignments, chaining adjacent alignments, and ortholog identification. Indeed, CGAT provides a general framework for the calculation of genome-scale alignments using various existing programs as alignment engines, which allows users to compare the outputs of different alignment programs. Earlier versions of this program have been used successfully in our research to infer the evolutionary history of apparently complex genome changes between closely related eubacteria and archaea. CONCLUSION: CGAT is a practical tool for analyzing complex genomic changes between closely related genomes using existing alignment programs and other sequence analysis tools combined with extensive manual inspection

Using Macro-Arrays to Study Routes of Infection of Helicobacter pylori in Three Families

allowed tracing the spread of infection through populations on different continents but transmission pathways between individual humans have not been clearly described.To investigate person-to-person transmission, we studied three families each including one child with persistence of symptoms after antibiotic treatment. Ten isolates from the antrum and corpus of stomach of each family member were analyzed both by sequencing of two housekeeping genes and macroarray tests. from outside the family appeared to be probable in the transmission pathways. infection may be acquired by more diverse routes than previously expected

Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase

Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer

<p>Abstract</p> <p>Background</p> <p>Persistent colonization of the human stomach by <it>Helicobacter pylori </it>is associated with asymptomatic gastric inflammation (gastritis) and an increased risk of duodenal ulceration, gastric ulceration, and non-cardia gastric cancer. In previous studies, the genome sequences of <it>H. pylori </it>strains from patients with gastritis or duodenal ulcer disease have been analyzed. In this study, we analyzed the genome sequences of an <it>H. pylori </it>strain (98-10) isolated from a patient with gastric cancer and an <it>H. pylori </it>strain (B128) isolated from a patient with gastric ulcer disease.</p> <p>Results</p> <p>Based on multilocus sequence typing, strain 98-10 was most closely related to <it>H. pylori </it>strains of East Asian origin and strain B128 was most closely related to strains of European origin. Strain 98-10 contained multiple features characteristic of East Asian strains, including a type s1c <it>vacA </it>allele and a <it>cagA </it>allele encoding an EPIYA-D tyrosine phosphorylation motif. A core genome of 1237 genes was present in all five strains for which genome sequences were available. Among the 1237 core genes, a subset of alleles was highly divergent in the East Asian strain 98-10, encoding proteins that exhibited <90% amino acid sequence identity compared to corresponding proteins in the other four strains. Unique strain-specific genes were identified in each of the newly sequenced strains, and a set of strain-specific genes was shared among <it>H. pylori </it>strains associated with gastric cancer or premalignant gastric lesions.</p> <p>Conclusion</p> <p>These data provide insight into the diversity that exists among <it>H. pylori </it>strains from diverse clinical and geographic origins. Highly divergent alleles and strain-specific genes identified in this study may represent useful biomarkers for analyzing geographic partitioning of <it>H. pylori </it>and for identifying strains capable of inducing malignant or premalignant gastric lesions.</p

Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity

Unveiling Novel RecO Distant Orthologues Involved in Homologous Recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts