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The gene encoding a 29-kDa flagellar sheath protein was cloned and found to be similar to hpaA, reported to encode an N-acetylneuraminyllactose-binding fibrillar hemagglutinin (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993). The transcriptional start was mapped by primer extension from Helicobacter pylori mRNA, indicating an active consensus promoter at a location different from that suggested by Evans et al. Immunogold labelling of the flagellar sheath with a monoclonal antibody to HpaA was demonstrated in four strains, contrary to previous reports of a surface (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993) or a cytoplasmic (P. W. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. J. Trust, J. Bacteriol. 177:6049-6057, 1995) locale. Agglutination of erythrocytes and adherence to AGS cells by a delta hpaA mutant were no different from those of the parent strain, confirming a recent finding of O'Toole et al.

A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.

Analysis of the derived amino acid sequences of toxins A and B from Clostridium difficile has identified an extraordinarily large number of repeat amino acid units in the C-terminal regions of the proteins. Nearly one third of each of the proteins consist of repeating units which appear, at least in the case of toxin A, to be responsible for carbohydrate binding. Similar repeat units are also found in the C-terminal region of four glucosyltransferases from Streptococcus mutans and Streptococcus downei, and in four lytic enzymes from Streptococcus pneumoniae and its bacteriophages (HB-3, Cp-1 and Cp-9). In each case the repeats constitute the ligand-binding portion of the respective enzymes. A glucan-binding protein from S. mutans, which lacks enzymatic activity, has similar repeats spanning almost the entire molecule. This family of ligand-binding proteins appears to be of modular design, with one module consisting of a repetitive ligand-binding domain located in the C-terminal region and the other module(s) providing enzymatic functions.

In August 1997 the complete annotated genome sequence of Helicobacter pylori was published,1 just 15 years after the organism was first cultured.2 This is an important milestone in gastroenterology research as H pylori is the first enteropathogen to be fully sequenced. The availability of a complete genetic data set heralds a new era in H pylori research as it will provide a framework for global studies of virulence and other aspects of the organism’s biology. In this article we outline the genomics approaches that can now be applied and highlight the potential practical benefits of this research in terms of eradication therapy and disease prevention. Scientists at The Institute for Genomic Research (TIGR) (http://www.tigr.org) have determined the order of the 1 667 867 nucleotides that constitute the circular chromosomal content of H pylori strain ACTC 26695.1 Initial computer analysis suggests the presence of 1590 open reading frames (ORFs or genes), of which nearly 70% can be matched to genes encoding proteins of known function. This immense achievement gives us unequalled opportunities to relate gene sequence to biology and will provide insight into many aspects of the organism’s biology, such as replication, DNA repair, metabolism, ion and protein transport, cell wall structure, and virulence. The most intriguing findings are as follows

Ganglioside mimicry by Campylobacter jejuni lipo-oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain-Barré and Miller-Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1-like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside-mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal beta-1,3-linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a beta-1,3 galactosyltransferase responsible for converting GM2-like LOS structures to GM1-like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside-mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1-like LOS structure as well as the ganglioside GM2-like LOS structure proposed in the literature.

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.

The aim of this study was to determine whether 16 epidemic methicillin-resistant strains of Staphylococcus aureus (EMRSA) could be killed by the light-activated antimicrobial agent aluminium disulphonated phthalocyanine (AIPcS(2)). EMRSA suspensions were irradiated with light from a laser diode in the presence of ArPcS2 and survivors enumerated. All strains were susceptible to killing, the bactericidal effect being dependent on the AlPcS, concentration and the light dose. AlPcS, rendered the bacteria light-sensitive almost immediately and killing was unaffected by the growth phase of the organism. Scavengers of singlet oxygen and free radicals protected the bacteria from killing. These results imply that light-activated antimicrobial agents may be useful in eliminating EMRSA from wounds or carriage sites.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.

Department of Medical Microbiology, St. Bartholomew's Hospital Medical College, London, EC1A 7BE, UK The polymerase chain reaction (PCR) holds great potential for the rapid and sensitive detection of pathogenic microorganisms. (1~ There has been much interest in detecting and preventing falsepositive PCR results owing to product carry-over(2); however, less attention has been paid to false-negative reactions. These can result from defective PCR reagents, mistakes in setting up the PCR, or from inhibitory substances present in the sample. To distinguish true negative reactions from failed PCRs, we have devised a simple method in which an additional artificial template is incorporated into the amplification mixture. This template is constructed by engineering a small deletion in the usual PCR product. The control template is amplified by the same primers as the test fragment, but can be distinguished from it by mobility on electrophoresis. Using such an approach, there are three possible outcomes to a PCR: (1) no DNA is amplified in the PCR (the assay has failed); (2) the control, but not the test, fragment is obtained (the assay has worked, and the sample is negative); (3) the test fragment, with or without the control fragment, is obtained (the assay has worked, and the sample is positive). We have applied this technique to two different PCR assays--one for the detection of toxigenic Corynebacterium diphtheriae), (3~ the other for the identification of pathogenic yersinias. (4~ We constructed the control template in both cases by creating a deletion in the central portion of the natural PCR product (see Fig. 1).

A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits. Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide. A polyclonal antibody against the glucan-binding protein, which cross-reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. We recently identified toxin A and the glucan-binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules. This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

The genes encoding the two structural subunits of the Helicobacter(Campylobacter) pyloriurease enzyme were cloned and sequenced. The derived amino acid sequences of the two H. pylori urease genes showed marked homology to both jack bean and soybean ureases. Almost 57% of residues were identical in the bacterial and plant ureases. This degree of similarity between a eucaryotic protein and a bacterial protein is almost unprecedented. The close similarity of the bacterial protein to the plant enzymes is an evolutionary anomaly that is perhaps best explained by horizontal gene transfer. The polymerase chain reaction technique using oligonucleotide primers to the H. pylori urease DNA sequence was found to sensitively detect H. pylori cells and may allow determination of the source and route of infection of the organism.

Introduction: Previous studies have suggested that soluble water extracts of H.pylori contain a 15kDa protein (NapA) which directly activates neutrophils (PMNs) and may thus be a factor in the aetiology of active gastritis. Aim: to establish the role of NapA by disruption and characterisation of a napA isogenic mutant Methods: napA gene fragments were isolated from H.pylori (strain N6) using degenerate primers and the full sequence of each gene fragment obtained from a H.pylori lambda(I)ZAP library. A defined mutant was constructed by insertional activation using a kanamycin cassette followed by allelic replacement (N6:nap-). Isolated clones were expanded and water extracts of parent and mutant strains prepared as previously described, pMN activation was assessed by flow cytometric shape change assay, expression of cdl lb/cdl8 and NBT dye reduction assay. Results: In comparison to parent strain N6, SDS-PAGE of N6:nap" showed loss of a 15 kDa protein indicating absence of NapA; disruption of nap was also confirmed by Southern blotting. In addition PAGE profiles revealed the differential expression of other proteins in N6:nap'. Serial dilutions (1:10) of water extracts from N6 and N6:napwere equally able to activate PMNs as assessed by flow cytometry, cdllb/18 expression and NBT dye reduction assay. After bioinformatic re-analysis suggested close homology between nap and the regulatory DNA binding protein of E.coli (dps), the survival of N6 and N6:napunder acid (ph4) and oxidative (H2O 2, 20 and 120 mM) stress was determined (as colony forming units) and urease + catalase activities and protein profiles assessed. Triplicate assays were performed at least 5 times, and p values determined by Mann Whitney U. *=p

A gene bank from a clinical isolate of Clostridium difficile expressing high chloramphenicol acetyltransferase activity was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the plasmid vector pUC13. Among 1,020 clones tested, 11 were resistant to chloramphenicol; 1 of these, with an insert size of 1.9 kilobases (pPPM9), was studied further. The clone pPPM9 was mapped using a variety of restriction enzymes, and a 0.27-kilobase EcoRV-TaqI restriction fragment was shown to be within the chloramphenicol resistance (Cmr) gene by using transposon (Tn1000) mutagenesis. The 0.27-kilobase fragment and the 1.9-kilobase insert were radiolabeled and used as DNA probes in hybridization studies. Southern blot analysis with the gene probes against chromosomal DNA from Cmr strains of C. difficile obtained from five distinct geographical locations revealed that at least two copies of the same chloramphenicol acetyltransferase gene were present for each strain. Hybridization of the gene probes against Cmr strains of Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella edwardsii, Escherichia coli, and to four other clostridial species revealed no homology even under conditions of low stringency.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.

HindIII restriction enzyme digests of genomic DNA from nine distinct strains of Clostridium difficile were undertaken, and the results were related to those of a previously established typing method based on [35S]methionine-labeled protein profiles. Each of the typed strains identified by its protein profile could also be distinguished by its unique DNA digestion pattern. Analysis of strains isolated from 10 patients during a hospital outbreak of antibiotic-associated colitis revealed identical DNA profiles, confirming a single strain as the source of cross-infection. Characterization of isolates from worldwide sources revealed similar digestion patterns within the same strain type. Restriction endonuclease DNA analysis provides a sensitive and useful technique for studying the epidemiology of C. difficile.

The chloramphenicol resistance determinant from a nonconjugative strain of Clostridium perfringens was cloned and shown to be expressed in Escherichia coli. Subcloning and deletion analysis localized the resistance gene, catQ, to within a 1.25-kilobase (kb) partial Sau3A fragment. The catQ gene contained internal HindII, HaeIII, and DraI restriction sites and was distinct from the catP gene, which was originally cloned (L. J. Abraham, A. J. Wales, and J. I. Rood Plasmid 14:37-46, 1985) from the conjugative C. perfringens R plasmid, pIP401. Hybridization studies were carried out with a 0.35-kb DraI-P fragment of pJIR260 as an internal catQ-specific probe and a 0.38-kb EcoRV-HinfI fragment of pJIR62 as an internal catP-specific gene probe. The results showed that the catP and catQ genes were not similar and that neither probe hybridized with cat genes from other bacterial genera. However, the catP gene was similar to the cloned catD gene from Clostridium difficile. Comparative studies with both catP and catD probes showed that these genes had significant restriction identity. We therefore suggest that these genes were derived from a common source.

DNS bildet unter γ-Bestrahlung bei 77 K zu gleichen Teilen positive und negative Ladungszentren in Guanin-(G+) bzw. Thyminbasen (T").