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Temperature, among other environmental factors, influences the incidence and severity of many plant diseases. Likewise, numerous traits, including the expression of virulence factors, are regulated by temperature. Little is known about the underlying genetic determinants of thermoregulation in plant-pathogenic bacteria. Previously, we showed that the expression of both fliC (encoding flagellin) and syfA (encoding a nonribosomal polypeptide synthetase) was suppressed at high temperatures in Pseudomonas syringae. In this work, we used a high-throughput screen to identify mutations that conferred overexpression of syfA at elevated temperatures (28°C compared to 20°C). Two genes, Psyr_2474, encoding an acyl-coenzyme A (CoA) dehydrogenase, and Psyr_4843, encoding an ortholog of RppH, which in Escherichia coli mediates RNA turnover, contribute to thermoregulation of syfA. To assess the global role of rppH in thermoregulation in P. syringae, RNA sequencing was used to compare the transcriptomes of an rppH deletion mutant and the wild-type strain incubated at 20°C and 30°C. The disruption of rppH had a large effect on the temperature-dependent transcriptome of P. syringae, affecting the expression of 569 genes at either 20°C or 30°C but not at both temperatures. Intriguingly, RppH is involved in the thermoregulation of ribosome-associated proteins, as well as of RNase E, suggesting a prominent role of rppH on the proteome in addition to its effect on the transcriptome., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.

Using a sensitive assay, we observed low levels of an unknown surfactant produced by Pseudomonas syringae pv. syringae B728a that was not detected by traditional methods yet enabled swarming motility in a strain that exhibited deficient production of syringafactin, the main characterized surfactant produced by P. syringae. Random mutagenesis of the syringafactin-deficient strain revealed an acyltransferase with homology to rhlA from Pseudomonas aeruginosa that was required for production of this unidentified surfactant, subsequently characterized by mass spectrometry as 3-(3-hydroxyalkanoyloxy) alkanoic acid (HAA). Analysis of other mutants with altered surfactant production revealed that HAA is coordinately regulated with the late-stage flagellar gene encoding flagellin; mutations in genes involved in early flagellar assembly abolish or reduce HAA production, while mutations in flagellin or flagellin glycosylation genes increase its production. When colonizing a hydrated porous surface, the bacterium increases production of both flagellin and HAA. P. syringae was defective in porous-paper colonization without functional flagella and was slightly inhibited in this movement when it lacked surfactant production. Loss of HAA production in a syringafactin-deficient strain had no effect on swimming but abolished swarming motility. In contrast, a strain that lacked HAA but retained syringafactin production exhibited broad swarming tendrils, while a syringafactin-producing strain that overproduced HAA exhibited slender swarming tendrils. On the basis of further analysis of mutants altered in HAA production, we discuss its regulation in Pseudomonas syringae.

A genetically tractable model plant pathosystem, Pseudomonas syringae pv. tomato DC3000 on tomato and Arabidopsis thaliana hosts, was used to investigate the role of salicylic acid (SA) and iron acquisition via siderophores in bacterial virulence. Pathogen-induced SA accumulation mediates defense in these plants, and DC3000 contains the genes required for the synthesis of SA, the SA-incorporated siderophore yersiniabactin (Ybt), and the fluorescent siderophore pyoverdin (Pvd). We found that DC3000 synthesizes SA, Ybt, and Pvd under iron-limiting conditions in culture. Synthesis of SA and Ybt by DC3000 requires pchA, an isochorismate synthase gene in the Ybt genomic cluster, and exogenous SA can restore Ybt production by the pchA mutant. Ybt was also produced by DC3000 in planta, suggesting that Ybt plays a role in DC3000 pathogenesis. However, the pchA mutant did not exhibit any growth defect or altered virulence in plants. This lack of phenotype was not attributable to plant-produced SA restoring Ybt production, as the pchA mutant grew similarly to DC3000 in an Arabidopsis SA biosynthetic mutant, and in planta Ybt was not detected in pchA-infected wild-type plants. In culture, no growth defect was observed for the pchA mutant versus DC3000 for any condition tested. Instead, enhanced growth of the pchA mutant was observed under stringent iron limitation and additional stresses. This suggests that SA and Ybt production by DC3000 is costly and that Pvd is sufficient for iron acquisition. Further exploration of the comparative synthesis and utility of Ybt versus Pvd production by DC3000 found siderophore-dependent amplification of ybt gene expression to be absent, suggesting that Ybt may play a yet unknown role in DC3000 pathogenesis.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

Two methionine biosynthetic genes in Pseudomonas syringae pv. syringae, metX and metW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine O-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera. Both metX and metW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including metX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.

We have identified a recombinant plasmid, pCUV8, from a cosmid library of Pseudomonas syringae genomic DNA which contains a functional analog of the Escherichia coli recA gene. The plasmid was initially identified by its ability to restore UV resistance to E. coli HB101. Quantitative analysis demonstrated that it restored both recombination proficiency and UV resistance to an E. coli recA deletion mutant. By these criteria, pCUV8 appears to contain the P. syringae recA gene. Several pathogenic and epiphytic strains of P. syringae, but not E. coli, showed sequence homology to pCUV8 under normal stringency.