Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O 2 for both growth and viability. While the wild-type strain is routinely grown at 12% O 2 , growth of the mutant strains is severely inhibited at above 5 to 6% O 2 . The effect of O 2 on viability was determined by subjecting nongrowing cells to atmospheric levels of O 2 and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H 2 O 2 , and this sensitivity is exacerbated by increased O 2 concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.
To assess the importance of two separate antioxidant activities in Helicobacter pylori, we tested the abilities of strains with mutations in either tpx (encoding thiolperoxidase) or ahpC (encoding alkyl hydroperoxide reductase [AhpC]) to colonize the stomachs of mice. The tpx strain was clearly more sensitive than the parent strain to both oxygen and cumene hydroperoxide. The strain colonized only 5% of the inoculated mice. Two different classes of oxygen-sensitive ahpC mutants in the type strain (ATCC 43504) were recently described (A. A. Olczak, J. W. Olson, and R. J. Maier, J. Bacteriol. 184:3186-3193, 2002). The same two classes of mutants were recovered upon ahpC mutagenesis of the mouse-adapted strain, SS1. Neither of these mutants was able to colonize mouse stomachs, whereas 78% of the mice inoculated with the parent strain became H. pylori positive.
SummaryDuring Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon (cotJA and cotJC) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA. Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.
We have demonstrated that the bldB gene of Streptomyces coelicolor is required for the formation of aerial hyphae and the synthesis of antibiotics. We also found that BldB forms a higher-order complex (most likely a dimer) and that amino acid residues 20 to 78 are important for this interaction. This region is conserved in the BldB family, suggesting that dimer formation may be a common feature of these proteins.The life cycle of the filamentous bacterium Streptomyces coelicolor begins with spore germination and the growth of filamentous, branching cells called substrate hyphae. After 24 to 36 h, a second cell type, the aerial hyphae, appears on the colony surface and grows up into the air. The two cell types have completely different fates: substrate hyphae produce secondary metabolites, including two pigmented antibiotics, and the aerial hyphae produce spores (1,6,14,18).Antibiotic synthesis and the formation of aerial hyphae commence at approximately the same time during colony development, and there is evidence that these events are coordinated at the molecular level. For example, several genes that are dispensable for cell viability are required for both of these developmental events to occur (5,17,23). Among these, mutations in the gene bldB have the most severe consequences (5,17). While the developmental phenotypes of most bld mutants can be at least partially restored by growth on minimal medium containing the carbon source mannitol, bldB mutations block both the formation of aerial hyphae and the synthesis of antibiotics under all growth conditions (5, 17). bldB mutants are also defective in catabolite control (22) and do not fit into the hierarchy of extracellular complementation exhibited by many other bld mutants (19,20,21,29,30).The bldB gene has been cloned and shown to encode a 98-amino-acid protein with a molecular mass of 10,899 Da (23) (Fig. 1). There are numerous homologues of bldB in the S. coelicolor genome (11), including abaA and whiJ, which are required for antibiotic synthesis and spore formation, respectively (9,11,24). At this time, no convincing bldB homologue has been detected in a nonactinomycete.The biochemical roles of BldB and its homologues are unknown. The remarkable pleiotropy of bldB mutants could suggest a role in controlling gene expression, and indeed, analysis of the BldB polypeptide sequence suggested that it might include a helix-turn-helix DNA binding motif (23). bldB expression, which is normally low during vegetative growth and increases at the time that aerial hyphae appear, is constitutive in bldB mutants, suggesting that BldB might regulate its own synthesis, like the developmental transcription factor BldD (8).No interaction between BldB and the bldB promoter has been detected in our laboratories, however, suggesting that BldB may affect bldB expression indirectly. The sequence of BldB provides no other clues to its function.We have constructed a chromosomal deletion of the bldB open reading frame in S. coelicolor strain M145. The phenotype of this null mutant w...
Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students’ understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students’ explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.
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