Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli

During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than 100 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-lacZ operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an rpoS/rpoS+ merodiploid (rpoS background transformed with an rpoS-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an rpoS background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in Escherichia coli do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to otsA, katE, ecnB, and osmY-genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely, gabP, yhiUV, o371, o381, f186, and o215.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using promoter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present within the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

In vitro effect of imipenem on Acinetobacter baumannii

In Salmonella Typhimurium, the stationary phase sigma factor RpoS regulates the expression of genes associated with adaptation, survival, and virulence. Expression of rpoS is known to be under environmental control and yet, to date, there have been no studies that assess the expression of this global regulator in real food systems. Skim milk represents a useful model food; using an spvRA::luxCDABE reporter construct, we have determined RpoS availability from an inoculum of Salmonella Typhimurium LT2. We report that RpoS activity increases exponentially at the end of the logarithmic phase of growth, consistent with data from nutrient media.     

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.     

Rapid, high-resolution HPLC separation of peptides using small particles at elevated temperatures

Practical advantages are described for operating small-particle columns at elevated temperatures to increase the peak capacity, resolution or speed for separating peptides. Columns of small, highly-purified porous silica microspheres with a dense covalently bonded, bulky alkylsilane stationary phase permit continuous operation at temperatures of at least 90 degrees C. Operation at elevated temperatures decreases mobile phase viscosity and enhances solute diffusion, resulting in increased column plate number and separation resolution. Higher temperatures also decrease column back pressure, permitting longer columns of small particles for separating complex mixtures requiring large plate numbers. Use of a sterically protecting di-isobutyl-n-octadecylsilane stationary phase ensures stable and reproducible columns for operation at high temperatures, with the aggressive low pH mobile phase preferred for separating peptides. The monomeric nature of this phase ensures rapid mass transfer and high column efficiency.