Transcriptional profiling of Pseudomonas aeruginosa grown under steady-state hyperosmotic stress conditions showed an up-regulation of genes associated with osmoprotectant synthesis, putative hydrophilins, and the type III secretion system with associated cytotoxins. A large number of regulatory genes, including several two-component systems not previously known to be influenced by osmolarity, were differentially expressed by P. aeruginosa in immediate response to hyperosmotic shock.Changes in osmolarity are likely an inescapable reality for microbes colonizing any environment (22), and bacteria must be able to both respond immediately to osmotic shock, which might occur during transitions between growth environments, and sustain growth under lasting conditions of osmotic stress. As an opportunistic pathogen of humans, Pseudomonas aeruginosa employs adaptive responses that originally evolved for survival in diverse and often stressful environmental conditions (21,22). Hence, genes differentially expressed in response to osmotic stress may play a key role in the prevalence and persistence of P. aeruginosa in osmotically stressful infection sites (9). Aside from the accumulation of osmoprotectants, very little is known about osmoadaptive processes in P. aeruginosa (3,5). In this study we used Affymetrix GeneChip microarrays to investigate the transcriptional responses of P. aeruginosa to both osmotic up-shock and growth under steady-state osmotic stress conditions.Bacterial growth conditions and genetic manipulations. P. aeruginosa PA14 was grown in modified minimal A medium (14) containing 60 mM K 2 HPO 4 , 30 mM KH 2 PO 4 , 7.5 mM (NH 4 ) 2 SO 4 , 1 mM MgSO 4 , 10 M FeSO 4 , and 17 mM glucose, with or without 0.3 M NaCl or 0.7 M sucrose, pH 7.1. A 40-ml volume of medium was inoculated with 0.3 ml of washed cells from an overnight culture to yield a starting optical density at 600 nm (OD 600 ) of ϳ0.03. Log-phase cells used for DNA microarray analysis under steady-state conditions were harvested at an OD 600 of 0.25. For the osmotic up-shock experiments, 3.9 ml of prewarmed minimal medium containing 3 M NaCl (pH 7.1) was added to 35 ml of log-phase culture (OD 600 , 0.25) to yield a final NaCl concentration of 0.3 M. As a control, 3.9 ml of prewarmed minimal medium without NaCl was added to 35 ml of log-phase culture. Cells were harvested for gene expression analysis 15 to 60 min after up-shock. All incubations were performed in a 37°C water bath with shaking at 200 rpm.RNA isolation, removal of contaminating DNA, cDNA synthesis, and fragmentation of cDNA were performed as previously described (16). Processing of the P. aeruginosa GeneChips (Affymetrix) was performed at the University of Tulsa Microarray Facility per Affymetrix protocols. Data analysis was performed using Affymetrix Microarray Suite software; only those genes differentially regulated at least threefold were considered. In general, 70% to 75% of the ϳ5,500 open reading frames (ORFs) in P. aeruginosa (19) were detectable on the GeneChips.The steady...

The ability of invading pathogens to proliferate within host tissues requires the capacity to resist the killing effects of a wide variety of host defense molecules. sap mutants of the facultative intracellular parasite Salmonella typhimurium exhibit hypersensitivity to antimicrobial peptides, cannot survive within macrophages in vitro and are attenuated for mouse virulence in vivo. We conducted a molecular genetic analysis of the sapG locus and showed that it encodes a product that is 99% identical to the NAD+ binding protein TrkA, a component of a low‐affinity K+ uptake system in Escherichia coli. SapG exhibits similarity with other E. coli proteins implicated in K+ transport including KefC, a glutathione‐regulated efflux protein, and Kch, a putative transporter similar to eukaryotic K+ channel proteins, sapG mutants were killed by the antimicrobial peptide protamine in the presence of both high and low K+, indicating that protamine hypersensitivity is not due to K+ starvation. Strains with mutations in sapG and either sapJ or the sapABCDF operon were as susceptible as sapG single mutants, suggesting that the proteins encoded by these loci participate in the same resistance pathway. SapG may modulate the activities of SapABCDF and SapJ to mediate the transport of peptides and potassium.

~~Protamine is a polycationic peptide found in the nuclei of sperm of different animal species. While it has long been known to have antimicrobial properties, its mode of action has remained elusive. We have investigated the mechanism of action of protamine and established that this peptide exerts its antibacterial effect without causing cell lysis or permeabilization of the cytoplasmic membrane. Respiring cells were more susceptible than nonrespiring cells, and loss of viability could be prevented by incubation at low pH or the addition of respiratory poisons. This indicates that protamine activity is influenced by the electrical membrane potential (by) : increased killing occurs at higher Ay values. Protamine caused inhibition of proline uptake, rapid efflux of proline from preloaded cells, and a reduction in the cellular ATP content. Furthermore, protamine-treated cells first lost the ability to accumulate leucine and then could not carry out protein synthesis. Cumulatively, our data indicate that protamine disrupts energy transduction and nutrient uptake functions, and suggest that the cytoplasmic membrane is the target of protamine action.

Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.