Joanne Simala-Grant

Multiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide Reductase of Escherichia coli

Dimethyl sulfoxide (Me2SO) 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 Me2SO 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-Me2SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamineN-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.

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.

High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli

Using a wide variety of N- and S-oxide compounds we have shown by kinetic analysis that only two N-oxides, trimethylamine-N-oxide and 4-methylmorpholine-N-oxide, can be considered good substrates for trimethylamine-N-oxide (TMAO) reductase on the basis of their kcat/Km ratio. This result demonstrates that TMAO reductase possesses a high substrate specificity. Induction of the torCAD operon using the same S- and N-oxide compounds was also analyzed. We demonstrate that there is no correlation between the ability for a compound to be reduced by TMAO reductase and to induce TMAO reductase synthesis.

Molecular biology methods for the characterization of Helicobacter pylori infections and their diagnosis.

Simala‐Grant JL, Taylor DE. Molecular biology methods for the characterization of Helicobacter pylori infections and their diagnosis. APMIS 2004;112:886–97.

Characterization of the DNA adenine 5'-GATC-3' methylase HpyIIIM from Helicobacter pylori.

The effect of inactivation of the 5′-GATC-3′ methylase HpyIIIM in Helicobacter pylori (H. pylori) on mismatch repair, adherence, and in vitro fitness was examined. Chromosomal DNA from 90 H. pylori strains was isolated, and restriction enzyme digestion indicated all strains examined possess HpyIIIM. Wild-type H. pylori and a strain with an inactive HpyIIIM were found to have rifampicin mutation frequencies of 2.93 × 10−7 and 1.05 × 10−7 (p > 0.05), respectively, indicating that HpyIIIM does not appear to be important in mismatch repair. Adherence of H. pylori in an in vitro model cell system was also unaffected by inactivation of HpyIIIM. Inactivation of HpyIIIM did not result in a decrease in fitness, as determined by liquid in vitro competition experiments.

Antimicrobial Resistance in Helicobacter and Campylobacter

Helicobacter and Campylobacter are Gram-negative spiral flagellated bacteria that inhabit and cause diseases of the gastrointestinal tract. Despite early microscopic observations of “Vibrio-like” organisms in blood, stool, and gastric contents, the role of these two genera in infectious disease was established in relatively recent times.