Staphylococcus aureus nitrosative stress resistance is due in part to flavohemoprotein (Hmp). Although hmp is present in all sequenced S. aureus genomes, 37% of analyzed strains also contain nor, encoding a predicted quinol-type NO reductase (saNOR). DAF-FM staining of NO-challenged wild-type, nor, hmp, and nor hmp mutant biofilms suggested that Hmp may have a greater contribution to intracellular NO detoxification relative to saNOR. However, saNOR still had a significant impact on intracellular NO levels, and complemented NO detoxification in a nor hmp mutant. When grown as NO-challenged static (low-oxygen) cultures, hmp and nor hmp mutants both experienced a delay in growth initiation, whereas the nor mutant's ability to initiate growth was comparable to the wild-type strain. However, saNOR contributed to cell respiration in this assay once growth had resumed, as determined by membrane potential and respiratory activity assays. Expression of nor was upregulated during low-oxygen growth and dependent on SrrAB, a two-component system that regulates expression of respiration and nitrosative stress resistance genes. High-level nor promoter activity was also detectable in a cell subpopulation near the biofilm substratum. These results suggest that saNOR contributes to NO-dependent respiration during nitrosative stress, possibly conferring an advantage to nor+ strains in vivo.
Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight, host factors that could contribute to oral diseases such as caries and periodontitis. However, the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study, high aspect ratio vessel S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles, H2O2 resistance, and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures, but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated ≥2-fold and 94 genes down-regulated ≥2-fold during simulated microgravity high aspect ratio vessel growth. These included a number of genes located on extrachromosomal elements, as well as genes involved in carbohydrate metabolism, translation, and stress responses. Collectively, these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions.
Thermoacidophilic archaea belonging to the order Sulfolobales thrive in extreme biotopes, such as sulfuric hot springs and ore deposits. These microorganisms have been model systems for understanding life in extreme environments, as well as for probing the evolution of both molecular genetic processes and central metabolic pathways. Thermoacidophiles, such as the Sulfolobales, use typical microbial responses to persist in hot acid (e.g. motility, stress response, biofilm formation), albeit with some unusual twists. They also exhibit unique physiological features, including iron and sulfur chemolithoautotrophy, that differentiate them from much of the microbial world. Although first discovered more than 50 years ago, it was not until recently that genome sequence data and facile genetic tools have been developed for species in the Sulfolobales. These advances have not only opened up ways to further the probe novel features of these microbes, but have also paved the way for potential biotechnological applications. Discussed here are the nuances of the thermoacidophilic lifestyle of the Sulfolobales, including their evolutionary placement, cell biology, survival strategies, genetic tools, metabolic processes, and physiological attributes together with how these characteristics make thermoacidophiles ideal platforms for specialized industrial processes.
Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression.
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