Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.     

Helicobacter pylori cadA encodes an essential Cd(II)–Zn(II)–Co(II) resistance factor influencing urease activity

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.     

Effect of host species on recG phenotypes in Helicobacter pylori and Escherichia coli

Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.     

幽门螺杆菌Ⅳ型分泌系统HP0527-C基因的克隆表达及其生物学功能的初步研究

为克隆表达幽门螺杆菌（Helicobacter pylori,H.pylori）NCTC 11637 hp0527-c 基因,探讨其生物学功能。设计引物扩增,T-A克隆,测序正确后构建表达载体pET-28a- hp0527-c,转化大肠杆菌BL21,经IPTG诱导表达后以Ni2＋-NTA柱获得纯度为98％重组蛋白。经Western blot鉴定正确后,免疫新西兰大白兔获得了较高滴度的多克隆抗体;将重组蛋白作用于BGC-823细胞,RT-PCR检测细胞IL-8mRNA表达增高;同时用MTT法来检测重组蛋白对细胞增殖的影响,结果呈现一定量的时间和剂量依赖性。已成功克隆了HP0527-C的重组蛋白,初步探讨了其与细胞之间的相互作用,为进一步阐明H.pylori致病机制奠定了基础。     

Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP0958

Several flagellar genes in Helicobacter pylori are dependent on sigma(54) (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimriderext/common/) revealed interactions between sigma(54) and the conserved hypothetical protein HP0958. To see if HP0958 influences sigma(54) function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two sigma(54)-dependent reporter genes, flaB'-'xylE and hp1120'-'xylE. Levels of sigma(54) in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced sigma(54) levels. Expressing sigma(54) at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of sigma(54) was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type sigma(54) levels in H. pylori by protecting it from proteolysis.     

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.     

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions.

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.     

幽门螺杆菌Cag-PAI hp0525基因的克隆表达及其重组蛋白HP0525对SGC-7901细胞增殖的影响

目的：由于文献报道幽门螺杆菌（Helicobacter pylori）是产生胃溃疡和胃癌的致病菌之一,一个重要的影响因素是由cag致病岛编码的四型分泌系统。Hp0525是Cag致病岛中重要成分,是一种内膜蛋白ATPase。而幽门螺杆菌中Cag蛋白的表达以及Cag PAI编码的各自蛋白功能研究得还很少,为进一步研究幽门螺杆菌的致病机制和研发幽门螺杆菌诊断试剂盒及疫苗,特克隆幽门螺杆菌NCTC 11637hp0525 (caga) 基因,并对其进行测序,构建原核重组质粒,表达HP0525蛋白,初步研究其对SGC-7901细胞增殖的影响。方法：应用PCR技术从H.pylori基因组DNA中扩增hp0525编码基因片段,克隆至pMD18-T载体后,再将其定向插入pET-30a载体中,双酶切鉴定筛选阳性克隆,以DNA自动分析仪进行序列测定。测序分析正确后,经IPTG诱导表达,表达蛋白以Ni2＋-NTA柱进行纯化,并经Western blot和MALDI-TOF鉴定,透析除盐后的蛋白,通过免疫新西兰大白兔和抗体效价的测定,纯化的蛋白作用于SGC-7901细胞,用MTT法检测蛋白对细胞增殖的影响。结果：成功克隆hp0525基因,全长993bp,编码330个氨基酸,与GenBank 公布的其他H.pylori菌株基因序列的核苷酸同源性为97%~99%。工程菌诱导后SDS-PAGE显示新生表达蛋白带,相对分子质量为36 000,与预期一致,经Ni2＋-NTA柱纯化后可获得纯度为98％重组蛋白。蛋白作用于SGC-7901细胞后,结果呈现一定量的时间和剂量依赖性。它是一种ATPase,通过测定具有一定的活性。活性为4.40IU/ml结论：成功克隆hp0525基因,并在大肠杆菌BL21中表达,经过镍柱纯化后得到纯度较高的蛋白, MTT法来检测出重组蛋白抑制细胞增殖;同时具有一定的酶活力,为进一步研究其生物学功能奠定了基础。     

Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.

Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori.     

Different alleles of the flagellin gene hagB in Escherichia coli standard H test strains

Abstract The genes determining flagellar antigen specificities H36, H47 and H53 in the respective E. coli standard H test strains were found to be alleles of the flagellin gene hagB . Until now, only the allele encoding the flagellar antigen H3 has been identified. The chromosomal regions of flagellin genes hagB in E. coli and H2 in Salmonella were non-homologous as these genes integrated at different sites in the E. coli K-12 chromosome and were unable to replace each other. The hagA allele encoding E. coli flagellar antigen H48 was insensitive to the repressor produced by Salmonella gene rhl or by its putative analog in E. coli .     

Protein-protein interaction of HP137 with histidine kinase HP244 does not contribute to flagellar regulation in Helicobacter pylori

Flagellar motility is essential for the ability of Helicobacter pylori to colonize the gastric mucosa. Expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors sigma54 and sigma28 and the anti-sigma28 factor FlgM. The protein-protein interaction map of H. pylori, which is based on a high-throughput two-hybrid screen (Rain et al., 2001. Nature 409, 211-215) indicated a protein-protein interaction between the gene product of ORF hp137 and both the histidine kinase HP244 and the flagellar hook protein HP908. We hypothesized that HP137 might be involved in a feedback regulatory mechanism controlling the activity of histidine kinase HP244. Here we demonstrate that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium Campylobacter jejuni.     

Cloning and expression of tryptophan genes from Brevibacterium lactofermentum in Escherichia coli

A gene bank from the amino acid producer Brevibacterium lactofermentum has been prepared in Escherichia coli using pBR322 as vector. Four clones containing genetic information needed to complement mutations in A,B,C and D genes from E. coli have been isolated. The cloned fragments range between 4.3 kb (pULT61) and 7.9 kb (pULT62). All the four clones contain genetic information that complements trpB gene from E. coli. The cloned trpB gene is very stable and is maintained extrachromosomally in E. coli. It is expressed very efficiently showing high levels of tryptophan synthetase activity.