Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Lee WK

PROVIDER: S-EPMC168813 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

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

Lee W K WK An Y S YS Kim K H KH Kim S H SH Song J Y JY Ryu B D BD Choi Y J YJ Yoon Y H YH Baik S C SC Rhee K H KH Cho M J MJ

Applied and environmental microbiology 19971201 12

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 mai ...[more]

PMID: 9406406

Similar Datasets

Project description:The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which ?-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ? proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ? proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual targeting of these pathways would have the net effect of severely limiting the delivery/transport of components to the OM and preventing the bacterium's ability to infect its human host.

Project description:BackgroundLactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria, which are widely distributed in various diverse natural habitats. These are used in a variety of industrial food fermentations and carry numerous traits with utmost relevance to the food industry. Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions.ResultsThe developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial "pediocin" produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts.ConclusionThe present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets