Zhongming Ge

Cloning and Heterologous Expression of an α1,3-Fucosyltransferase Gene from the Gastric PathogenHelicobacter pylori

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel α1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian α1,3/1,4-fucosyltransferases. The ∼52-kDa protein encoded byHpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to α1,3-fucosyltransferase activity but neither α1,4-fucosyltransferase nor α1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40–45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken α1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial α1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic α1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.

Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, oRF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611‐amino‐acid sequence of ORF1, a P‐type ATPase (designated hpCopA), had striking homology (29‐38%) with several bacterial P‐type ATPases and contained the potential functional domains conserved in P‐type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion‐transporting protein, and contains the heavy metal‐binding motif. Disruption of ORF1 with a chloramphenicol‐resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alternation of susceptibility to Ni2+, Cd2+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation‐transporting system which is associated with copper export out of the H. pylori cells.

Cloning and functional characterization of Helicobacter pylori fumarate reductase operon comprising three structural genes coding for subunits C, A and B

In this study, we cloned and sequenced the Helicobacter pylori genes encoding fumarate reductase (FRD). H. pylori frdA, frdB and frdC specify polypeptides of 715, 245 and 254 aa, respectively. The deduced aa sequences of FrdA and FrdB are highly homologous to those of the corresponding subunits of Wolinella succinogenes FRD and also exhibit a significant sequence identity with other bacterial FRD and succinate dehydrogenase subunits A and B. However, H. pylori FrdC shares a striking degree of sequence identity only with W. succinogenes FrdC, which is a cytochrome b with two haem groups. The products encoded by H. pylori frdA, frdB and frdC were overproduced in maxicells and H. pylori FrdA was characterized using an anti-E. coli FrdA serum. H. pylori FRD activity, which was measured as fumarate-dependent benzyl viologen oxidation, is membrane-associated. Inactivation of frdA led to the loss of such activity and the mutant H. pylori cells were delayed (approx. 10-20 h behind their parent cells) in entering the mid-log phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H. pylori cells in vitro. H. pylori FRD contains three subunits, of which FrdA and FrdB appear to form the catalytic dimer, whereas FrdC serves as a membrane anchor.

H. pylori DNA Transformation by Natural Competence and Electroporation

Helicobacter pylori is an important etiological pathogen of human stomach diseases, such as gastritis, peptic ulcer, and gastric carcinoma (1). In the past few years, great progress has been made in the cloning and characterization of H. pylori genes. Success of these studies stems in part from the finding that chromosomal and recombinant plasmid DNA are able to be efficiently transformed into H. pylori cells by natural competence (2-4) and electroporation (3,5). Such techniques allow the transfer of cloned H. pylori genes, manipulated in vitro, which can then shed light on the structural and functional relationships of the genes of interest. In this chapter, we describe the protocols for the isolation of H. pylori chromosomal and plasmid DNA, natural transformation, and electroporation.

Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations.

Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains.

Conservation and Diversity of the Helicobacter pylori Copper-Transporting ATPase Gene (copA) Sequence Among Helicobacter Species and Campylobacter Species Detected by PCR and RFLP

Background Helicobacter pylori is a causative pathogen of such human stomach diseases as chronic type B gastritis, ulcer, and possibly gastric carcinoma. As a co‐factor in various redox enzymes and an essential trace metal required for the synthesis of metalloproteins, copper might play a role in the pathogenesis of H. pylori. A gene, copA, associated with copper transport, has been isolated from H. pylori UA802. In this study, conservation and diversity of this gene were analyzed among some Helicobacter and Campylobacter species.