Ca2+-Induced Two-Component System CvsSR Regulates the Type III Secretion System and the Extracytoplasmic Function Sigma Factor AlgU in Pseudomonas syringae pv. tomato DC3000.

Two-component systems (TCSs) of bacteria regulate many different aspects of the bacterial life cycle, including pathogenesis. Most TCSs remain uncharacterized, with no information about the signal(s) or regulatory targets and/or role in bacterial pathogenesis. Here, we characterized a TCS in the plant-pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 composed of the histidine kinase CvsS and the response regulator CvsR. CvsSR is necessary for virulence of P. syringae pv. tomato DC3000, since ΔcvsS and ΔcvsR strains produced fewer symptoms than the wild type (WT) and demonstrated reduced growth on multiple hosts. We discovered that expression of cvsSR is induced by Ca2+ concentrations found in leaf apoplastic fluid. Thus, Ca2+ can be added to the list of signals that promote pathogenesis of P. syringae pv. tomato DC3000 during host colonization. Through chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and global transcriptome analysis (RNA-seq), we discerned the CvsR regulon. CvsR directly activated expression of the type III secretion system regulators, hrpR and hrpS, that regulate P. syringae pv. tomato DC3000 virulence in a type III secretion system-dependent manner. CvsR also indirectly repressed transcription of the extracytoplasmic sigma factor algU and production of alginate. Phenotypic analysis determined that CvsSR inversely regulated biofilm formation, swarming motility, and cellulose production in a Ca2+-dependent manner. Overall, our results show that CvsSR is a key regulatory hub critical for interaction with host plants.IMPORTANCE Pathogenic bacteria must be able to react and respond to the surrounding environment, make use of available resources, and avert or counter host immune responses. Often, these abilities rely on two-component systems (TCSs) composed of interacting proteins that modulate gene expression. We identified a TCS in the plant-pathogenic bacterium Pseudomonas syringae that responds to the presence of calcium, which is an important signal during the plant defense response. We showed that when P. syringae is grown in the presence of calcium, this TCS regulates expression of factors contributing to disease. Overall, our results provide a better understanding of how bacterial pathogens respond to plant signals and control systems necessary for eliciting disease.

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 family member, is needed to produce L-allo-isoleucine, a precursor for the phytotoxin coronatine.

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 µg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

Characterization of the Fur regulon in Pseudomonas syringae pv. tomato DC3000.

The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).

Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

Transcriptome analysis of Pseudomonas syringae identifies new genes, noncoding RNAs, and antisense activity.

To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.

Occurrence of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Product Verification Testing Samples from U.S. Department of Agriculture-Regulated Producing Establishments, 2005 through 2017.

ABSTRACT: Ready-to-eat (RTE) meat and poultry product samples collected between 2005 and 2017 from RTE-producing establishments for the U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) ALLRTE/RTEPROD_RAND (random) and RTE001/RTEPROD_RISK (risk-based) sampling projects were tested for Listeria monocytogenes (Lm). Data for 45,897 ALLRTE/RTEPROD_RAND samples collected from 3,607 distinct establishments and 112,347 RTE001/RTEPROD_RISK samples collected from 3,283 distinct establishments were analyzed for the presence of Lm. These data were also analyzed based upon the percentages of establishments with positive samples, annual production volume, sanitation control alternatives, geographic location, and season or month of sample collection. Results revealed low occurrence of Lm-positive samples from the random and risk-based sampling projects, with 152 (0.33%) positive samples for ALLRTE/RTEPROD_RAND and 403 (0.36%) positive samples for RTE001/RTEPROD_RISK. The percentage of positive samples significantly decreased over time, from about 0.7% in 2005 and 2006 to about 0.2% in 2017 (P < 0.05). From 2005 to 2017, 3.9% of establishments sampled under the ALLRTE/RTEPROD_RAND sampling project had at least one Lm-positive sample. Similarly, 10.0% of establishments sampled under the RTE001/RTEPROD_RISK sampling project had at least one positive sample. Samples positive for Lm were found in all geographic regions in all months. Thus, in 13 years of RTE product sampling in FSIS-regulated establishments (2005 through 2017), <0.4% of samples were positive for Lm in both risk-based and random sampling projects. The low prevalence of Lm in these products suggests that the combination of FSIS policies and industry practices may be effective for controlling Lm contamination. Information obtained from these sampling projects is relevant to the ongoing prevention of foodborne Lm illnesses from RTE meat and poultry products.

Effect of iron concentration on the growth rate of Pseudomonas syringae and the expression of virulence factors in hrp-inducing minimal medium.

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp-inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 microM Fe(3+). The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.

A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.

The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant resistance (R)-gene sentinels, which may convert effector virulence activities into a monolithic defense response. On the premise that some effectors target universal eukaryotic processes and that yeast (Saccharomyces cerevisiae) lacks R genes, the DC3000 effector repertoire was expressed in yeast. Of 27 effectors tested, HopAD1, HopAO1, HopD1, HopN1, and HopU1 were found to inhibit growth when expressed from a galactose-inducible GAL1 promoter, and HopAA1-1 and HopAM1 were found to cause cell death. Catalytic site mutations affecting the tyrosine phosphatase activity of HopAO1 and the cysteine protease activity of HopN1 prevented these effectors from inhibiting yeast growth. Expression of HopAA1-1, HopAM1, HopAD1, and HopAO1 impaired respiration in yeast, as indicated by tests with ethanol glycerol selective media. HopAA1-1 colocalized with porin to yeast mitochondria and was shown to cause cell death in yeast and plants in a domain-dependent manner. These results support the use of yeast for the study of plant-pathogen effector repertoires.

Global transcriptional responses of Pseudomonas syringae DC3000 to changes in iron bioavailability in vitro.

BACKGROUND: Pseudomonas syringae pv tomato DC3000 (DC3000) is a Gram-negative model plant pathogen that is found in a wide variety of environments. To survive in these diverse conditions it must sense and respond to various environmental cues. One micronutrient required for most forms of life is iron. Bioavailable iron has been shown to be an important global regulator for many bacteria where it not only regulates a wide variety of genes involved in general cell physiology but also virulence determinants. In this study we used microarrays to study differential gene regulation in DC3000 in response to changes in levels of cell-associated iron. RESULTS: DC3000 cultures were grown under highly controlled conditions and analyzed after the addition of iron citrate or sodium citrate to the media. In the cultures supplemented with iron, we found that cell-associated iron increased rapidly while culture densities were not significantly different over 4 hours when compared to cultures with sodium citrate added. Microarray analysis of samples taken from before and after the addition of either sodium citrate or iron citrate identified 386 differentially regulated genes with high statistical confidence. Differentially regulated genes were clustered based on expression patterns observed between comparison of samples taken at different time points and with different supplements. This analysis grouped genes associated with the same regulatory motifs and/or had similar putative or known function. CONCLUSION: This study shows iron is rapidly taken up from the medium by iron-depleted DC3000 cultures and that bioavailable iron is a global cue for the expression of iron transport, storage, and known virulence factors in DC3000. Furthermore approximately 34% of the differentially regulated genes are associated with one of four regulatory motifs for Fur, PvdS, HrpL, or RpoD.

Occurrence of Salmonella in Ready-to-Eat Meat and Poultry Product Samples from U.S. Department of Agriculture-Regulated Producing Establishments. II. Salmonella in Ready-to-Eat Pork Barbecue Products, from 2005 to 2012.

Ready-to-eat (RTE) meat and poultry product samples from the random ALLRTE and risk-based RTE001 sampling projects of the Food Safety and Inspection Service (FSIS) were tested for both Listeria monocytogenes and Salmonella. In the course of analyzing Salmonella data for calendar years 2005 to 2012, it was observed that 8 (17.0%) of 47 positive samples were from pork barbecue. The eight Salmonella-positive samples, from seven establishments in a single state, were from 1,085 pork barbecue samples tested nationwide (0.74% positive) and from 296 samples tested from that one state (2.7% positive). The seven establishments represented 30.4% of 23 federal establishments in that state that had pork barbecue samples tested for Salmonella. A follow-up sample from intensified verification testing at one of the seven establishments also was positive for Salmonella. Upon further examination, contamination appeared to be influenced by regional differences in production methods. Notably, the style of pork barbecue that tested positive for Salmonella used a vinegar- and pepper-based sauce in which the ingredients were mixed without cooking. All the establishments with Salmonella-positive samples followed the practice of first cooking the pork and then adding the barbecue sauce ingredients (vinegar, pepper, other spices, etc.) after cooking (postlethality exposure). In addition to the sauce ingredients, other possible sources of contamination included employee hygiene and food handling practices and cross-contamination from other Salmonella-contaminated products and from commonly used equipment. Based on these findings, the FSIS issued guidelines recommending changes in production methods that would minimize or eliminate pork barbecue as a potential source of foodborne Salmonella infections.

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A.

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Genome-wide identification of transcriptional start sites in the plant pathogen Pseudomonas syringae pv. tomato str. DC3000.

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5'-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5'-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5'RACE. As expected, many 5'-ends were positioned a short distance upstream of annotated genes. We also captured 5'-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5'-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels.

An inhibitor of gram-negative bacterial virulence protein secretion.

Bacterial virulence mechanisms are attractive targets for antibiotic development because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen of small molecule libraries and identified one compound, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and type II secretion-dependent functions, suggesting that its target could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.

Identification of a twin-arginine translocation system in Pseudomonas syringae pv. tomato DC3000 and its contribution to pathogenicity and fitness.

The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.

HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions.

Pseudomonas syringae pv. tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana. The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense. Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells. Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS. A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves. In non-host N. tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000. Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N. benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P. syringae pv. tabaci 11528, respectively. HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity. These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions.