Bacterial persister cells constitute a small portion of a culture which is tolerant to killing by lethal doses of bactericidal antibiotics. These phenotypic variants are formed in numerous bacterial species, including those with clinical relevance like the opportunistic pathogen Pseudomonas aeruginosa. Although persisters are believed to contribute to difficulties in the treatment of many infectious diseases, the underlying mechanisms affecting persister formation are not well understood. Here we show that even though P. aeruginosa cultures have a significantly smaller fraction of multidrug-tolerant persister cells than cultures of Escherichia coli or Staphylococcus aureus, they can increase persister numbers in response to quorum-sensing-related signaling molecules. The phenazine pyocyanin (and the closely related molecule paraquat) and the acyl-homoserine lactone 3-OC12-HSL significantly increased the persister numbers in logarithmic P. aeruginosa PAO1 or PA14 cultures but not in E. coli or S. aureus cultures.Over the last 50 years, Pseudomonas aeruginosa has emerged as a major cause of nosocomial infections in immunocompromised patients, including those relying on mechanical ventilation and suffering from neutropenia or severe burns, and is perhaps most well known as the agent primarily responsible for the decline in lung function leading to death in cystic fibrosis (CF) patients (8,14,16,23,38). P. aeruginosa possesses a multiplicity of virulence factors that are elicited upon access to susceptible individuals, including various toxins, secretion systems, siderophores, surface appendages, endotoxin (lipopolysaccharide [LPS]), alginate, and phenazines. These can generate acute toxicity/injury, leading P. aeruginosa to be labeled as the "hyena" of the bacterial world (15). Treatment of infections by P. aeruginosa is hindered by its high level of intrinsic resistance to antibiotics due primarily to a combination of the impermeable outer membrane and a number of broad-spectrum efflux pumps (50, 51). P. aeruginosa is also thought to enter into a biofilm mode of growth in CF lung infections (36,48,63), contributing both to pathogenicity/colonization and resistance to therapeutic intervention.In P. aeruginosa, global regulation, mediated by at least 3 quorum-sensing (QS) systems, controls population behaviors and synthesis of the majority of these pathogenicity factors (59, 66). This bacterium possesses two N-acyl-homoserine lactone (HSL)-mediated quorum-sensing systems, las and rhl (26,43,46,47), and a Pseudomonas quinolone signal (PQS) system mediated by 2-heptyl-3-hydroxy-4-quinolone (49). In the HSL-mediated systems, the HSL synthases LasI and RhlI are responsible for the synthesis of the autoinducers N-(3-oxododecanoyl)-L-HSL (3-OC12-HSL) and N-butyryl-L-HSL (C4-HSL), respectively. Expressions of the lasI and rhl genes are regulated by the transcriptional activators LasR and RhlR in response to their cognate HSL signal molecules. Among the numerous cellular and secreted virulence factors in P. aeruginosa whose...

SummaryThe MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown. Here, the role of MtrAB was studied with C. glutamicum as model organism. In contrast to M. tuberculosis , it was possible to delete the mtrAB genes in C. glutamicum . The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol. In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays. Three genes showed a more than threefold increased RNA level in the mutant, i.e. mepA ( NCgl2411 ) encoding a putative secreted metalloprotease, ppmA ( NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function. Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA. The mRNA level of two genes was more than fivefold decreased in the mutant, i.e. betP and proP which encode transporters for the uptake of betaine and proline respectively. The microarray results were confirmed by primer extension and RNA dot blot experiments. In the latter, the transcript level of genes involved in osmoprotection was tested before and after an osmotic upshift. The mRNA level of betP , proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased. The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP , proP , lcoP , mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the D D D D mtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichiacoli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.

The two-component system MtrBA is involved in the osmostress response of Corynebacterium glutamicum. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress.The ability of bacteria to sense and respond to environmental changes is a prerequisite for survival in their natural habitats. The most frequent bacterial sensory and signal transduction systems are two-component systems. Prototypical two-component systems consist of a membrane-bound histidine protein kinase and a cytoplasmic response regulator generally acting as a transcription factor. The homodimeric histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates in trans in an ATP-dependent manner at a conserved histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue of the response regulator. In turn, this transfer affects the DNA-binding properties of the response regulator. Most histidine kinases also possess phosphatase activity that allows the dephosphorylation of the cognate response regulator. The balance between the autokinase and the phosphatase activity of the sensor kinase is thought to control the net phosphorylation of the response regulator (23,24).Corynebacterium glutamicum, a gram-positive soil bacterium, contains 13 two-component systems. Recently, a set of 12 strains was constructed in order to analyze the individual functions of these systems (15,17). One of these systems, the so-called MtrB-MtrA system, is highly conserved in sequence and genomic organization among actinobacteria (7,17). The response regulator MtrA seems to be essential in Mycobacterium tuberculosis (29), whereas a single ⌬mtrA deletion mutant as well as a ⌬mtrAB double deletion mutant of C. glutamicum can be constructed (2, 17). The function of this system is still rather ill-defined, and different target genes in corynebacteria and mycobacteria have been described. In mycobacteria, MtrB-MtrA seems to be involved in the regulation of the cell wall permeability (3) and in cell cycle progression, since dnaA is under its control (6). In C. glutamicum, a number of genes involved in osmoregulation and peptidoglycan metabolism show altered mRNA levels in the ⌬mtrAB mutant. RNA hybridization experiments revealed that the genes encoding three out of the altogether four osmoregulated compatible-solute carriers, namely, betP, proP, and lcoP, are under the control of MtrB-MtrA (17), suggesting that the MtrB-MtrA system is important for the osmotic stress response of C. glutamicum. MtrB seems to be able to detect the extent of osmotic stress, since the expression of the transporter genes was induced according to...

The MtrB-MtrA two component system of Corynebacterium glutamicum was recently shown to be in involved in the osmostress response as well as cell wall metabolism. To address the question of whether the histidine protein kinase MtrB is an osmosensor, the kinase was purified and reconstituted into liposomes in a functionally active form. The activity regulation was investigated by varying systematically physicochemical parameters, which are putative stimuli that could be used by the bacterial cell to detect osmotic conditions. Membrane shrinkage was ruled out as a stimulus for activation of MtrB. Instead, MtrB was shown to be activated upon the addition of various chemical compounds, like sugars, amino acids, and polyethylene glycols. Because of the different chemical nature of the solutes, it seems unlikely that they bind to a specific binding site. Instead, they are proposed to act via a change of the hydration state of the protein shifting MtrB into the active state. For MtrB activation it was essential that these solutes were added at the same side as the cytoplasmic domains of the kinase were located, indicating that hypertonicity is sensed by MtrB via cytoplasmatically located protein domains. This was confirmed by the analysis of two MtrB mutants in which either the large periplasmic loop or the HAMP domain was deleted. These mutants were regulated similar to wild type MtrB. Thus, we postulate that MtrB belongs to a class of histidine protein kinases that sense environmental changes at cytoplasmatic protein domains independently of the periplasmic loop and the cytoplasmic HAMP domain.