Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative factors are the extracytoplasmic function (ECF) factors. In this paper, we demonstrate that the expression of the ECF factor V in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that V is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon V . Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for Vdependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.The majority of genes in actively growing bacteria are transcribed by RNA polymerase using the general "housekeeping" factor 70 . Bacteria often utilize alternative factors to regulate subsets of genes required for specific environmental conditions (18). The largest group of these alternative factors are the extracytoplasmic function (ECF) factors (18, 39). ECF factors represent the "third pillar" of bacterial signal transduction and are often involved in response to extracytoplasmic stress (18,39). ECF factors are members of the 70 family of factors and are characterized by the presence of only two regions of 70 , regions 2 and 4.2 (18). Bacillus subtilis encodes seven known ECF factors (18). ECF factors are often required for their own transcription; thus, the expression of an ECF factor promoter is often indicative of activity of the ECF factor (18, 39). The signals which induce the activity of several ECF factors are known. For instance, the expression of sigW is induced by antimicrobial peptides and pH change (6, 9, 14, 32, 45), while sigM expression is induced by inhibitors of cell wall biosynthesis, heat shock, paraquat, and ethanol stress (12,40). Like sigM, sigX is induced by in...

c Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV, which encodes V , an extracytoplasmic function factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that V is required for lysozyme resistance in C. difficile. Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon V and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon V . Expression of pdaV, which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile-associated disease. We conclude that the V signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.

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Clostridium difficile is an anaerobic, Gram-positive, spore-forming, opportunistic pathogen that is the most common cause of hospital-acquired infectious diarrhea. In numerous pathogens, stress response mechanisms are required for survival within the host. Extracytoplasmic function (ECF) factors are a major family of signal transduction systems, which sense and respond to extracellular stresses. We have identified three C. difficile ECF factors. These ECF factors, CsfT, CsfU, and CsfV, induce their own expressions and are negatively regulated by their cognate anti-factors, RsiT, RsiU, and RsiV, respectively. The levels of expression of these ECF factors increase following exposure to the antimicrobial peptides bacitracin and/or lysozyme. The expressions of many ECF factors are controlled by site 1 and site 2 proteases, which cleave antifactors. Using a retargeted group II intron, we generated a C. difficile mutation in prsW, a putative site 1 protease. The C. difficile prsW mutant exhibited decreased levels of expression of CsfT and CsfU but not of CsfV. When expressed in a heterologous host, C. difficile PrsW was able to induce the degradation of RsiT but not of RsiU. When the prsW mutant was tested in competition assays against its isogenic parent in the hamster model of C. difficile infection, we found that the prsW mutant was 30-fold less virulent than the wild type. The prsW mutant was also significantly more sensitive to bacitracin and lysozyme than the wild type in in vitro competition assays. Taken together, these data suggest that PrsW likely regulates the activation of the ECF factor CsfT in C. difficile and controls the resistance of C. difficile to antimicrobial peptides that are important for survival in the host.Clostridium difficile is an anaerobic, Gram-positive, sporeforming, opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea (3,29,32). C. difficile infections can range from mild diarrhea to life-threatening pseudomembranous colitis. People who develop C. difficile-associated disease are most commonly patients on antibiotics and/or who reside in hospitals or long-term care facilities. In recent years both the incidence and severity of C. difficile infections have increased in susceptible populations as well groups not traditionally considered to be at risk (33,38,39,43).In most patients, C. difficile infection is triggered by the disruption of the normal intestinal flora. C. difficile colonizes the intestinal tract of 12 to 20% of the adult population without causing disease (42). The disruption of the normal intestinal flora, most commonly due to treatment with antibiotics such as fluoroquinolones or clindamycin (13, 29), allows C. difficile to grow to high levels. How the normal flora blocks C. difficile growth is unclear. The normal flora may compete with C. difficile for nutrients and for binding sites in the intestinal tract or produce antimicrobial compounds that inhibit C. difficile growth (26, 45).The major known virulence determinants of C. diff...

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.