Antibiotic susceptibility of attached and free-floating Helicobacter pylori.

Helicobacter pylori are found attached to mucous cells of the human stomach or under the mucous layer. Models mimicking the in vivo situation may be more suitable for H. pylori MIC determinations than traditional agar dilution methods. Megraud et al. (Antimicrobial Agents and Chemotherapy 1991, 35, 869-72) developed a model for measuring the susceptibility of attached and free-floating H. pylori. We have modified this model so that free-floating and attached H. pylori are treated in a more similar manner, before and after incubation with antibiotic, and performed additional controls to ensure H. pylori and tissue culture cells are not detrimentally affected and maintain their viability during the course of the experiment. We found only 10% of plate-grown H. pylori were competent for attachment to HEp-2 cells; however, all progeny of attached bacteria remained adherent. Killing curves were performed using 0, 0.001, 0.01, 0.1 and 1 mg/L amoxycillin, and 0, 0.0025, 0.0075 and 0.01 mg/L clarithromycin. H. pylori divided at concentrations

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli.

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase.

X-ray crystallographic studies of the Rhodobacter sphaeroides dimethyl sulfoxide (Me2SO) reductase [Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V., & Rees, D. C. (1996) Science, 272, 1615-1620] indicated that the active site is at the bottom of a 25-A funnel. Substrates must travel to the bottom of the funnel for reduction to occur. The homologous DmsA subunit of the trimeric Escherichia coli Me2SO reductase, was subjected to site-directed mutagenesis of residues potentially lining the bottom of the funnel, based on sequence alignment of the E. coli and Rhodobacter Me2SO reductases. Sixteen E. coli DmsA mutants were characterized. Mutants G167N, A178Q, Q179I and R217Q showed functional impairment, as indicated by abnormal anaerobic growth with Me2SO as the sole terminal acceptor, in a recombinant strain deleted for chromosomal dmsABC. The kinetic parameters of the mutant enzymes were examined using the artificial electron donor benzyl viologen and the quinone analogue dimethylnaphthoquinone, with Me2SO and pyridine N-oxide as electron acceptors. Mutants A178Q and R217Q showed dramatic alterations of their electron-acceptor Km, with values at least 35-fold less or greater than wild-type values, respectively, for Me2SO and pyridine N-oxide. T148S showed altered kinetic parameters for pyridine N-oxide and Me2SO, with Km and k(cat) decreasing and increasing approximately fourfold, respectively. Other mutants showed less drastic alterations in kinetic parameters. This analysis has identified amino acids important in substrate binding and catalysis.

Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase.

We have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining Km and Kcat values for 22 different substrates. The enzyme has a very broad substrate specificity. The Km values varied 470-fold, while Kcat values varied only 20-fold, implicating Km as the major determinant of Kcat/Km values. Sulfoxides and pyridine N-oxide exhibited the lowest Km values, followed by aliphatic N-oxides. The Kcat values for these compounds also followed the same pattern. Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on Km while substitution at the 4 position had a greater effect, and increased Km. Negatively charged substrates were poorly accepted. A few compounds that are not S- or N-oxides were also reduced by the enzyme. Most compounds reduced by DmsABC were not toxic to E. coli under anaerobic growth conditions, and E. coli was able to use many of these compounds anaerobically as terminal electron acceptors in the presence of glycerol. Anaerobic growth on sulfoxides is solely due to DmsABC expression. However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.