The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

To determine the contribution of sigma B ( B ) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ⌬sigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ⌬sigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ⌬sigB strain. These results suggest the existence in L. monocytogenes of both a B -dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase.B contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The ⌬sigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ⌬sigB strain lost viability more rapidly than the parent strain.B contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that B plays a role in protecting L. monocytogenes against environmental adversities.

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, B , which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ⌬sigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ⌬sigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ⌬sigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is B independent. B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functionalB reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms.B does not appear to contribute to pH i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH i buffering by the GAD system under the conditions examined in this study. In summary, a functional B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

The stress-responsive, alternative sigma factor sigmaB has been described in members of three Gram-positive genera, Bacillus, Listeria, and Staphylococcus. In these bacteria, sigmaB appears to play an important role in facilitating rapid adaptation to and survival in stressful environments. sigmaB activity is regulated through a complex system of phosphatases and kinases encoded by rsb (regulator of sigma B) genes. We describe the sigB operon structure for the facultative intracellular pathogen Listeria monocytogenes and apply this sequence as well as other previously described sigB operon sequences to probe the evolution and functional conservation of the sigmaB stress response system among different Gram-positive bacteria. While sigmaB as well as two Rsbs (RsbS and RsbT) are highly conserved (73%, 84%, and 79% average amino acid [aa] identities, respectively), the predicted aa sequences of the other Rsb proteins showed less conservation (62-71% aa identities). Furthermore, the sigB operon structure varies among bacterial species. Bacterial species differ in the numbers and identities of rsb genes encoded in their genomes. We thus conclude that the sigmaB stress-response system as represented by the sigB operon has diverged in both its overall components as well as in the sequences of its individual proteins, even among closely related bacterial species. Differential evolution of this stress response system among various genera may represent a strategy that enables bacteria to adapt cellular response and survival systems to a variety of stress conditions.