Biomedical subjects

D Schwan

Publications and source records attributed to D Schwan.

5 recordsLinked to original sources

PubMed2000

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier.

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.

Acids↗

PubMed1999

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins.

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori.

Bacterial Outer Membrane Proteins↗

PubMed1999

Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing urease activity.

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.

Adenosine Triphosphatases↗

PubMed1977

Lecithin/spingomyelin ratio procedure by thin-layer chromatography.

We assessed several modifications of thin-layer chromatography for evaluation of amniotic fluid lecithin/sphingomyelin ratios. For a procedure which is reliable, economical, and easy to perform, we preferred laboratory-prepared plates using SI-LICAR TLC-7GF. For spot detection, we preferred the iodine vapor method.

Amniotic Fluid↗

PubMedDate not supplied

Genetic competence in Helicobacter pylori: mechanisms and biological implications.

Helicobacter pylori is naturally competent for genetic transformation. The comB locus, consisting of the open reading frames orf2, comB1, comB2, and comB3, is involved in natural transformation competence. Homologies of the ComB proteins with components of the type IV secretion apparatus (VirB9 and VirB10) from the Ti plasmid of Agrobacterium tumefaciens, as well as proteins involved in conjugation of plasmids RP1 and RP4, suggest a similar organization of DNA import (transformation) in H. pylori with well-known DNA export systems.

Bacterial Proteins↗