Diversity of restriction-modification gene homologues in Helicobacter pylori

The complete genome sequences of two Helicobacter pylori strains have recently become available. We have searched them for homologues of restriction-modification genes. One strain (26695) carried 52 such homologues, and the other (J99) carried 53. Their sequence alignments were arranged in the form of a phylogenetic tree and compared with the tree based on rRNA. The trees showed that the homologues are scattered among diverse groups of bacteria. They also revealed high polymorphism within the species--there are 42 pairs with high homology, 10 specific to 26695, and 11 specific to J99. Many of the restriction-modification homologues were characterized by a GC content lower than that of the average gene in the genome. Some of the restriction-modification homologues showed a different codon use bias from the average genes. These observations are interpreted in terms of horizontal transfer of the restriction-modification genes.     

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.     

Phenotypic and genotypic variation in methylases involved in type II restriction-modification systems in Helicobacter pylori

To determine relationships between Helicobacter pylori geographical origin and type II methylase activity, we examined 122 strains from various locations around the world for methylase expression. Most geographic regions possessed at least one strain resistant to digestion by each of 14 restriction endonucleases studied. Across all of the strains studied, the average number of active methylases was 8.2 ± 1.9 with no significant variation between the major geographic regions. Although seven pairs of isolates showed the same susceptibility patterns, their cagA/vacA status differed, and the remaining 108 strains each possessed unique patterns of susceptibility. From a single clonal group, 15 of 18 strains showed identical patterns of resistance, but diverged with respect to M.MboII activity. All of the methylases studied were present in all major human population groupings, suggesting that their horizontal acquisition pre-dated the separation of these populations. For the hpyV and hpyAIV restriction-modification systems, an in-depth analysis of genotype, indicating extensive diversity of cassette size and chromosomal locations regardless of the susceptibility phenotype, points toward substantial strain-specific selection involving these loci.     

Cloning and characterization of the MboII restriction-modification system

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.     

Iron trafficking system in Helicobacter pylori

Helicobacter pylori infections are closely associated with peptic ulcers, gastric malignancy and iron deficiency anemia. Iron is essential for almost all living organisms and the investigation of iron uptake and trafficking system is thus important to understand the pathological roles of H. pylori. Up to now, the iron trafficking system of H. pylori is not yet fully clear and merits further efforts in this regards. The available information about iron uptake and regulation has been discussed in this concise review, such as FeoB in ferrous transportation, FrpB2 in hemoglobin uptake, HugZ in heme processing, virulence factors (VacA and CagA) in transferrin utilization, Pfr and NapA in iron storage and Fur in iron regulation. The identified iron trafficking system will help us to understand the pathological roles of H. pylori in the various gastric diseases and iron deficiency anemia and stimulates further development of effective anti-bacterial drugs.     

Purification and characterization of urease from Helicobacter pylori

Urease was purified 112-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori. The urease isolation procedure included a water extraction step, size exclusion chromatography, and anion exchange chromatography. The purified enzyme exhibited a Km of 0.3 +/- 0.1 mM and a Vmax of 1,100 +/- 200 mumols of urea hydrolyzed/min/mg of protein at 22 degrees C in 31 mM Tris-HCl, pH 8.0. The isoelectric point was 5.99 +/- 0.03. Molecular mass estimated for the native enzyme was 380,000 +/- 30,000 daltons, whereas subunit values of 62,000 +/- 2,000 and 30,000 +/- 1,000 were determined. The partial amino-terminal sequence (17 residues) of the large subunit of H. pylori urease (Mr = 62,000) was 76% homologous with an internal sequence of the homohexameric jack bean urease subunit (Mr = 90,770; Takashima, K., Suga, T., and Mamiya, G. (1988) Eur. J. Biochem. 175, 151-165) and was 65% homologous with amino-terminal sequences of the large subunits of heteropolymeric ureases from Proteus mirabilis (Mr = 73,000) and from Klebsiella aerogenes (Mr = 72,000; Mobley, H. L. T., and Hausinger, R. P. (1989) Microbiol. Rev. 53, 85-108). The amino-terminal sequence (20 residues) of the small subunit of H. pylori urease (Mr = 30,000) was 65 and 60% homologous with the amino-terminal sequences of the subunit of jack bean urease and with the Mr = 11,000 subunit of P. mirabilis urease (Jones, B. D., and Mobley, H. L. T. (1989) J. Bacteriol. 171, 6414-6422), respectively. Thus, the urease of H. pylori shows similarities to ureases found in plants and other bacteria. When used as antigens in an enzyme-linked immunosorbent assay, neither purified urease nor an Mr = 54,000 protein that co-purified with urease by size exclusion chromatography was as effective as crude preparations of H. pylori proteins at distinguishing sera from persons known either to be infected with H. pylori or not.     

Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Two restriction-modification systems, S1 and S2, are present in Staphylococcus aureus RN450 (S. Iordanescu and M. Surdeanu, J. Gen. Microbiol., 96:277-281, 1976). System S2 affects phage multiplication after both infection and transfection. Unmodified plasmid and chromosomal DNAs are also not expressed following transduction and transformation into a restrictive host. Restricted phages are, however, capable of conferring phage-mediated competence, although the state of competence does not affect the restriction-modification system. The restricting activity of system S2 is inactivated by heat treatment of the cells. An enzymatic activity that restricts unmodified phage DNA in the presence of ATP, Mg2+, and S-adenosylmethionine was recovered from cell-free extracts of a strain RN450 derivative.     

Structural and functional characterization of Helicobacter pylori DsbG

Dsb proteins play important roles in bacterial pathogenicity. To better understand the role of Dsb proteins in Helicobacter pylori, we have structurally and functionally characterized H. pylori DsbG (HP0231). The monomer consists of two domains connected by a helical linker. Two monomers associate to form a V-shaped dimer. The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to Escherichia coli DsbG. Two polyethylene glycol molecules are bound in the cleft of the V-shaped dimer, suggesting a possible role as a chaperone. Furthermore, we show that H. pylori DsbG functions as a reductase against HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue.     

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The K(m) value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system.     

Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of ≥1 kb.     

Genotypic characterization of clarithromycin-resistant Helicobacter pylori strains

Helicobacterpylori (Hp) resistance to clarithromycin, one of the antibiotics most used to eradicate infection, is connected with the presence of a point mutation on the level of adenine at position 2143 or 2144 of 23S rRNA. AIM: The aim of the study is to evaluate of the presence of these mutation vs control clarithromycin resistant Hp strains present in North Sardinia; to verify the real association between the type of mutation and the resistance-level; to use easier molecular biology methods to quickly locate the resistance-associated mutations beginning with the bioptic material. The clarithromycin susceptibility of Hp isolates was tested by the E-test method (antibiotic assay). Genomic DNA of Hp strains was amplified using specific primers for the domain V. of ribosomic 23S rRNA and sequenced after the reaction with a primer within the fragment 23S. At the same time PCR-RFLP reliability was examined underlining the presence of these mutations with BsaI, BbsI, MboII restriction enzymes. Two mutations in 2143 (A- - G) and 2144 (A- - G) were found by domain V sequencing. The strains with mutation 2143 are characterized by a greater resistance level (MIC>64 g/ml) than those with mutation 2144 (MIC <64 g/ml). Restriction endonucleases BbsI and MboII recognise the site containing the mutation 2143 (A- - G), while BsaI recognise the mutation 2144 (A- - G). These methods might enable us to identify the presence of Hp directly from bioptic material and possible clarithromycin resistance and plan a suitable therapeutic strategy and consequently a better control of the infection.     

Complementation system for Helicobacter pylori

Previously Langford et al. (2006) developed the pIR203C04 complementation system for Helicobacter pylori, which can be used to complement and restore phenotypic effects in H. pylori mutant, and furthermore they used the complementation system in vivo experiments to animals without altering the ability of strain SSI to colonize mice. In their previous study, the pIR203C04 was able to transform 26695, SSI, J99, and 43504 H. pylori strains by an electroporation method. However, in the present study using a natural transformation the pIR203C04 transformed only 26695 H. pylori but not SSI, J99, 7.13, and G27 H. pylori strains. Since the useful complementation system has a limitation of narrow selection among H. pylori strains, we redesigned the complementation system for the improvement. The same intergenic chromosomal site between hp0203 and hp0204 was utilized for the new complementation system because the insertion at the intergenic site didn’t show any polar effects and disruption of other H. pylori genes. The genome sequence analysis showed that the intergenic regions among H. pylori strains may have too low homology to each others to do a homologous recombination. Thus, in addition to the short intergenic region, the fragments of the new complementation system included 3′ conserved parts of hp0203 and hp0204 coding regions. Between the fragments there are a chloramphenicol acetyltransferase cassette and multicloning sites, resulting in pKJMSH. DNA fragment of the interest can be cloned into the multicloning sites of pKJMSH and the fragment can be integrated at the intergenic region of H. pylori chromosome by the homologous recombination. Indeed, by the natural transformation, pKJMSH was able to transform all five H. pylori strains of 26695, SSI, J99, 7.13, and G27, which are common for the investigation of molecular pathogenesis. Thus, the new pKJMSH complementation system is applicable to most H. pylori wild-type stains.     

Functional characterization of Helicobacter pylori DnaB helicase

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5′ to 3′ helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.     

The thioredoxin system of Helicobacter pylori

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.     

Identification and characterization of an aliphatic amidase in Helicobacter pylori

We report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37 746 Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6–urease negative mutant, amidase activity was two- to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.