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Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in Campylobacter jejuni and Campylobacter coli. Karen T. Elvers, 2004.Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins . One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins . A Cgb-deficient mutant of C . jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate) . The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type . Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress . In contrast, the expression of Cgb was independent of aeration and the presence of superoxide . In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared . However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant . Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species . We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents . Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival of Mycobacterium smegmatis within Macrophages. T. Douglas, 2004.Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine . Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria . To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination . The msrA mutant strain of M . smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart . Furthermore, immunofluorescence and immnunoblotting of phagosomes containing M . smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67phox of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain . In addition, the msrA mutant strain of M . smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro . These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M . smegmatis . Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae. Karin Elbing, 2004.The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen . In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced . The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production . Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics . The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production . Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S . cerevisiae does not control glycolytic flux during exponential batch growth . In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree . Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S . cerevisiae and will provide new tools for the control of industrial fermentations . Purification and Characterization of the Flagellar Basal Body of Rhodobacter sphaeroides. K. Kobayashi, 2003.Flagellar hook-basal body (HBB) complexes were purified from Rhodobacter sphaeroides . The HBB was more acid labile but more heat stable than that of Salmonella species, and protein identification revealed that HBB components were expressed only from one of the two sets of flagellar gene clusters on the R . sphaeroides genome, under the heterotrophic growth conditions tested here . Multiple Light Inputs Control Phototaxis in Synechocystis sp . Strain PCC6803. Wing-On Ng, 2003.The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp . strain PCC6803 was studied with a glass slide-based phototaxis assay . Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis . We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm . This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm) . Previous work demonstrated that a taxD1 mutant (Cyanobase accession no . sll0041; also called pisJ1) lacked positive but maintained negative phototaxis . Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis . Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light . This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm . A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm . A  taxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm) . These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp . strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors .
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