Teruko Nakazawa

Differences in Genotypes of Helicobacter pylori from Different Human Populations

DNA motifs at several informative loci in more than 500 strains of Helicobacter pylori from five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of the H. pylori cag pathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in the cagA gene and in vacAm1 type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest that H. pylori may have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests that H. pylori infection might have become widespread in people quite recently in human evolution.

Identification of the urease operon in Helicobacter pylori and its control by mRNA decay in response to pH

We investigated the transcription of the urease gene cluster ureABIEFGH in Helicobacter pylori to determine the regulation of gene expression of the highly produced enzyme urease. Northern blot hybridization analysis demonstrated that cells of the wild‐type strain grown in an ordinary broth had transcripts of ureAB, ureABI, ureI, ureIE′ and ure′FGH, but cells of a ureI‐disrupted mutant had only the ureAB transcript. When the wild‐type cells were exposed to pH 8 for 30 min, very little mRNA was detected. However, when exposed to pH 6, a large amount of the ureIE′′ transcript, which was longer than the ureIE′ transcript, together with the additional transcripts ureABIEFGH and ure′EFGH were detected. Rifampicin addition experiments demonstrated that urease mRNAs, and the ureIE′ transcripts in particular, are more stable at pH 5.5 than at pH 7. In accord with these results, urease activity in the crude cell extract of the pH 5.5 culture was twice as much as that of the pH 7 culture, although the amounts of UreA and UreB detected by immunoblot analysis were similar. The transcription start point of ureI was identified by primer extension using a ureA promoter‐deleted mutant, and a consensus sequence of RpoD‐RNA polymerase was found in the ureI promoter. The 3′ end of the ureIE′′ mRNA, determined using S1 nuclease mapping, revealed that the transcript is able to cover the majority of the ureE open reading frame (ORF) that might be sufficient for UreE activity. Based on the above results, we conclude that the urease gene cluster of H. pylori consists of two operons, ureAB and ureIEFGH, and that primary transcripts of the latter as well as the read‐through transcript, ureABIEFGH, are cleaved to produce several species of mRNA. It has been suggested that the ureIEFGH operon is regulated post‐transcriptionally by mRNA decay in response to environmental pH. We are tempted to speculate that the ureE′′ transcript present in acidic pH may contribute to produce an active product that can proceed the nickel incorporation to the active centre, the final step of urease biosynthesis.

Distribution of Chlamydia pneumoniae Infection in the Atherosclerotic Carotid Artery

BACKGROUND AND PURPOSE Chlamydia pneumoniae infection has recently become noteworthy in relation to atherosclerosis. We investigated by immunohistochemistry the distribution of C pneumoniae infection in the atherosclerotic carotid artery. METHODS Twenty carotid atherosclerotic lesions that were resected during carotid endarterectomy were investigated. Parallel sections were stained immunohistochemically with monoclonal antibodies for a C pneumoniae-specific antigen, macrophages, and smooth muscle cells. RESULTS Immunoreactivity for the C pneumoniae-specific antigen was observed in 11 of 20 specimens (55%), and intense immunoreactivity was observed in 7 of 20 (35%). C pneumoniae infection was observed in endothelial cells, macrophages and in smooth muscle cells that had migrated into the atheromatous plaque, as well as in smooth muscle cells and small arteries in the media underlying the atheromatous plaques. C pneumoniae infection was most prominently observed in smooth muscle cells. The severity of the infection as demonstrated by immunohistochemistry was not significantly related to general risk factors for atherosclerosis. CONCLUSIONS C pneumoniae widely infects endothelial cells, macrophages, and smooth muscle cells in the atherosclerotic carotid artery. The results of the present study can help us to understand how C pneumoniae infection contributes to the progression of carotid atherosclerosis.

[64] Pyrocatechase (pseudomonas)☆

Publisher Summary This chapter describes the assay, purification, and properties of pyrocatechase. The assay is based on the measurement spectrophotometrically of the rate of formation of cis,cis-muconic acid. The rate of oxygen uptake can be measured by means of a polarographic technique. One unit of enzyme activity is defined as that amount that catalyzes the formation of 1 micromole of cis,cis-muconic acid per minute at 24°. Protein is determined spectrophotometrically from the absorbance at 280 and 260 mμ. The crude enzyme is stable for long periods of time at 4°. The purified enzyme is less stable; it loses about 15% of its activity during storage for four days at 4 °. The enzyme is most stable at a pH range of 8.0–9.5. Upon freezing, the purified enzyme at -20o for several months, the red enzyme solution changes to a more bluish color with a concomitant complete loss of activity. Sodium dithionite, ascorbic acid, sodium borohydride, mercaptoethanol, cysteine, reduced glutathione, or ferrous ion increase the rate of inactivation.

A positive regulatory gene, pvdS, for expression of pyoverdin biosynthetic genes in Pseudomonas aeruginosa PAO

In response to iron limitationPseudomonas aeruginosa PAO induces production of pyoverdin, a low-molecular-weight siderophore able to capture ferric ion with a very high affinity. Thepvd genes involved in the pyoverdin biosynthesis are organized in a chromosomal region termed thepvd region, and expression of somepvd genes is regulated at the transcriptional level. Two sets of promoter regions for thepvd genes were defined that were transcriptionally derepressed under iron-limiting condition. Analysis of transcription from such promoters inEscherichia coli led to isolation and identification of a positive regulatory gene,pvdS, for expression of thepvd genes, andpvdS was localized in thepvd region. A genomicpvdS mutant of PAO, constructed by allelic exchange mutagenesis, produced no pyoverdin and did not allow transcription from thepvd promoters. Nucleotide sequence analysis revealed thatPvdS shows considerable similarity to FecI ofE. coli, a positive regulator for transcription of thefec (ferric citrate transport system) operon. The promoter region ofpvdS has the sequence that matches well the consensus binding site for theE. coli Fur protein, a global negative regulatory protein that represses the transcription of the ironrepressible genes. Consistent with the presence of such a consensus sequence, addition of iron repressed transcription of thepvdS gene inP. aeruginosa.

The DsbA-DsbB disulfide bond formation system of Burkholderia cepacia is involved in the production of protease and alkaline phosphatase, motility, metal resistance, and multi-drug resistance

In a previous study, we isolated a dsbB mutant of Burkholderia cepacia KF1 and showed that phenotypes of protease production and motility are dependent on DsbB, a membrane‐bound disulfide bond oxidoreductase. We have now isolated a dsbA mutant by transposon mutagenesis, cloned the dsbA gene encoding a periplasmic disulfide bond oxidoreductase, and characterized the function of the DsbA‐DsbB disulfide bond formation system in B. cepacia. The complementing DNA fragment had an open reading frame for a 212‐amino acid polypeptide with a potential redox‐active site sequence of Cys‐Pro‐His‐Cys that is homologous to Escherichia coli DsbA. The dsbA mutant, as well as the previously isolated dsbB mutant, was defective in the production of extracellular protease and alkaline phosphatase, as well as in motility. In addition, mutation in the DsbA‐DsbB system resulted in an increase in sensitivity to Cd2+ and Zn2+ as well as a variety of antibiotics including β‐lactams, kanamycin, erythromycin, novobiocin, ofloxacin and sodium dodecyl sulfate. These results suggested that the DsbA‐DsbB system might be involved in the formation of a metal efflux system as well as a multi‐drug resistance system.

Unique mechanism of Helicobacter pylori for colonizing the gastric mucus.

Helicobacter pylori is a human gastric pathogen causing chronic infection. Urease and motility using flagella are essential factors for its colonization. Urease of H. pylori exists both on the surface and in the cytoplasm, and is involved in neutralizing gastric acid and in chemotactic motility. H. pylori senses the concentration gradients of urea in the gastric mucus layer, then moves toward the epithelial surface by chemotactic movement. The energy source for the flagella movement is the proton motive force. The hydrolysis of urea by the cytoplasmic urease possibly generates additional energy for the flagellar rotation in the mucus gel layer.

Cloning and characterization of the thiD/J gene of Escherichia coli encoding a thiamin-synthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase.

A 1.7 kb DNA fragment isolated from an E. coli genomic library was able to complement the thiamin requirement of strains carrying the thiM, thiJ and thiD mutations. The three genes encode hydroxyethylthiazole kinase, hydroxymethylpyrimidine (HMP) kinase and phosphomethylpyrimidine (HMP-p) kinase, respectively. Sequence analysis revealed that the 1.7 kb fragment contained two ORFs of 708 bp and 801 bp. The former ORF complemented the thiM mutation and the latter ORF both the thiJ and thiD mutations. The latter ORF was cloned into the expression vector pET3a, and the encoded protein was purified through three successive column chromatographies. The purified protein was able to convert HMP to its monophosphate and the monophosphate to its pyrophosphate. These results suggest that the two distinct enzyme activities, HMP kinase and HMP-P kinase, are indeed a bifunctional enzyme encoded by a single gene, designated thiDIJ.

Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases.

Recent studies suggest the association of atherosclerotic cardiovascular diseases with Chlamydia pneumoniae infection in western populations. It is of great interest whether such an association exists in Asians with their distinct genetic background. Symptomatic patients with coronary heart disease (29) or arteriosclerosis obliterans (10) who underwent directional endo-atherectomy were studied. Atherectomy specimens of coronary and iliac arteries were examined for C. pneumoniae by culture, nested PCR and immunohistochemical stain (IHC) with one Chlamydia genus-specific, two C. pneumoniae species-specific, and two C. trachomatis species-specific monoclonal antibodies. Among the 29 patients with coronary artery disease, C. pneumoniae was detected in the coronary arteries of 13 by IHC, 16 by PCR and 20 by IHC or PCR, or both. C. pneumoniae was also found in the iliac arteries of four patients by IHC, three by PCR and five by IHC or PCR, or both, of the 10 patients with arteriosclerosis obliterans. Attempts to isolate C. pneumoniae by culture were unsuccessful. The re-stenotic rate after atherectomy was higher in the C. pneumoniae-positive group than in the negative group, but not significantly so. These findings support the high incidence of C. pneumoniae in atherosclerotic lesions of symptomatic patients with coronary heart disease and arteriosclerosis obliterans in Asians.

Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1☆

Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.