C. L. Clayton

Genetic analysis of a tetracycline resistance element from clostridium difficile and its conjugal transfer to and from bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in Gram-positive bacteria

A 1.5kb cryptic plasmid was isolated from Helicobacter pylori. Low‐stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication‐initiation proteins commonly found in small plasmids endogenous to Gram‐positive bacteria which replicate by the ‘rolling‐circle’ mechanism. Sequence motifs corresponding to the origins‐of‐replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram‐negative bacterium to show replication determinants characteristic of the ‘rolling‐circle’ group of plasmids from Gram‐positive bacteria. The cloned plasmid will be used to develop a shuttle‐vector for H. pylori.

Molecular cloning and genetic analysis of a chloramphenicol acetyltransferase determinant from Clostridium difficile.

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. Images

Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K 12

Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits. A gene bank of C. difficile DNA in Escherichia coli was constructed by cloning Sau3A‐cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified. One of these clones λtA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A. The size of the λtA5 insert DNA was 14.3 kb.

Evaluation of fingerprinting methods for identification of Helicobacter pylori strains

Variation amongst strains ofHelicobacter pylori was examined by35S-methionine-labelled protein SDS-PAGE (Radio-PAGE), immunoblot and DNA fingerprinting techniques. These methods allowed discrimination amongst isolates and showed total correlation. Pig and baboon gastricHelicobacter pylori-like organisms were found to be very similar toHelicobacter pylori by both Radio-PAGE and immunoblotting, whereas aHelicobacter mustelae isolate was markedly different. TheHindIII restriction endonuclease digest patterns ofHelicobacter pylori DNA illustrated the considerable genomic variation of this organism. The Radio-PAGE and immunoblot typing methods both gave precise identification ofHelicobacter pylori strains, but Radio-PAGE was found to give higher resolution and represents a standardised universally applicable fingerprinting method forHelicobacter pylori.

Molecular Cloning and Sequencing of Helicobacter pylori Urease Genes and Detection of H. pylori Using the Polymerase Chain Reaction Technique

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.

Molecular Cloning and Nucleotide Sequence Determination of htrA, a Gene Encoding a 48-kDa Stress Protein of Helicobacter pylori

The importance of the outer membrane and its pathogenesis of Helicobacter pylori in gastroduodenal disease has not fully determined. Surface-exposed proteins or those that may be exported to the surface may play an important role in the organisms’ colonization, maintenance and pathogenic action on gastric epithelial cells. It has previously been reported that H. pylori possesses three major outer memb 31 kDa [6] and which have all been shown to be highly immunogenic [2]. Acid extractable preparations and 125 I surface labelling of H. pylori has demonstrated several variable proteins, including a 48-kDa antigen [6, 8]. Genotypic diversity, already described for this organism, may allow variation in antigentic determinants on the cell surface [7]. Such a phenomenon may not only explain the presence of variable proteins on the membrane but may play a role in assisting the organism in evading the host’s immune system. This led us to investigate membrane-associated proteins from H. pylori. The colning of genes encode these proteins in Escherichia coli should provide information not only on virulence, but both phenotypic and genotypic interstrain variation and possible regulatory sequences. We isolated a crude membrane prepration to which polyclonal antibody was prepared and used to screen a lambda (λ) EMBL 3 H. pylori genomic library.

Molecular Fingerprinting of Helicobacter pylori: An Evaluation of Methods

In order to fully investigate the role of Helicobacter pylori in the aetiology and pathogenesis of peptic ulcer disease sensitive methods of identifying and fingerprinting isolates of this organism are required. Conventional typing methods for bacteria, such as phage typing and serotyping have proven largely ineffective in typing H. pylori. Limited success has been achieved using systems based upon preformed enzymes such as the API-Zym system; however, the biotypes defined by such systems are limited and a large proportion of isolates fall into one biotype [1]. The more successful methods of identifying and fingerprinting H. pylori make use of molecular biological methods such as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), restriction endonuclease digestion of genomic DNA and ribosomal RNA gene hybridisation patterns [2, 3].

DNA Probes and PCR Analysis in the Detection of Clostridium difficile and Helicobacter pylori

We have undertaken the molecular characterisation of a variety of species-specific antigens and potential virulence determinants from the enteric pathogens Clostridium difficile and Helicobacter pylori. The cloning and sequencing of these determinants has allowed the construction of nucleic acid probes and the synthesis of oligonucleotide primers used in PCR analysis for the sensitive and specific detection of these organisms.