The ECF sigma factor, PSPTO_1043, in Pseudomonas syringae pv. tomato DC3000 is induced by oxidative stress and regulates genes involved in oxidative stress response.

The bacterial plant pathogen Pseudomonas syringae adapts to changes in the environment by modifying its gene expression profile. In many cases, the response is mediated by the activation of extracytoplasmic function (ECF) sigma factors that direct RNA polymerase to transcribe specific sets of genes. In this study we focus on PSPTO_1043, one of ten ECF sigma factors in P. syringae pv. tomato DC3000 (DC3000). PSPTO_1043, together with PSPTO_1042, encode an RpoERsp/ChrR-like sigma/anti-sigma factor pair. Although this gene pair is unique to the P. syringae group among the pseudomonads, homologous genes can be found in photosynthetic genera such as Rhodospirillum, Thalassospira, Phaeospirillum and Parvibaculum. Using ChIP-Seq, we detected 137 putative PSPTO_1043 binding sites and identified a likely promoter motif. We characterized 13 promoter candidates, six of which regulate genes that appear to be found only in P. syringae. PSPTO_1043 responds to the presence of singlet oxygen (1O2) and tert-butyl hydroperoxide (tBOOH) and several of the genes regulated by PSPTO_1043 appear to be involved in response to oxidative stress.

Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions.

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the "hrp promoter." Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.

Analysis of the small RNA spf in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000.

Bacteria contain small non-coding RNAs (ncRNAs) that are typically responsible for altering transcription, translation or mRNA stability. ncRNAs are important because they often regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA of Pseudomonas syringae DC3000, spot 42 (now referred to as spf), was investigated. A putative RpoE binding site was identified upstream of spf in strain DC3000. RpoE is shown to regulate the expression of spf. Also, deletion of spf results in increased sensitivity to hydrogen peroxide compared with the wild-type strain, suggesting that spf plays a role in susceptibility to oxidative stress. Furthermore, expression of alg8 is shown to be influenced by spf, suggesting that this ncRNA plays a role in alginate biosynthesis. Structural and comparative genomic analyses show this ncRNA is well conserved among the pseudomonads. The findings provide new information on the regulation and role of this ncRNA in P. syringae.

Genomic plasticity enables phenotypic variation of Pseudomonas syringae pv. tomato DC3000.

Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements in recombination events that facilitate genomic reorganization in P. syringae.

CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000.

Small non-coding RNAs (ncRNAs) are important components of many regulatory pathways in bacteria and play key roles in regulating factors important for virulence. Carbon catabolite repression control is modulated by small RNAs (crcZ or crcZ and crcY) in Pseudomonas aeruginosa and Pseudomonas putida. In this study, we demonstrate that expression of crcZ and crcX (formerly designated psr1 and psr2, respectively) is dependent upon RpoN together with the two-component system CbrAB, and is influenced by the carbon source present in the medium in the model plant pathogen Pseudomonas syringae pv tomato DC3000. The distribution of the members of the Crc ncRNA family was also determined by screening available genomic sequences of the Pseudomonads. Interestingly, variable numbers of the Crc family members exist in Pseudomonas genomes. The ncRNAs are comprised of three main subfamilies, named CrcZ, CrcX and CrcY. Most importantly the CrcX subfamily appears to be unique to all P. syringae strains sequenced to date.

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).

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.

Construction of an rsmX co-variance model and identification of five rsmX non-coding RNAs in Pseudomonas syringae pv. tomato DC3000.

Non-coding RNAs (ncRNAs) are important components of many regulatory pathways and have key roles in regulating diverse functions. In the Pseudomonads, the two-component system, GacA/S, directly regulates at least two well-characterized ncRNAs, RsmZ and RsmY, which act by sequestration of translation repressor proteins to control expression of various exoproducts. Pseudomonas fluorescens CHA0 possesses a third ncRNA, RsmX, which also participates in this regulatory pathway. In this study we confirmed expression of five rsmX ncRNAs in Pseudomonas syringae pv. tomato DC3000, and determined the distribution of the members of the rsmX ncRNA family by screening available genomic sequences of the Pseudomonads. Variable numbers of the rsmX family exist in Pseudomonas genomes, with up to five paralogs in Pseudomonas syringae strains. In Pseudomonas syringae pv. tomato DC3000, the rsmX genes are 112 to 120 nucleotides in size and are predicted by structural analysis to contain multiple exposed GGA motifs, which is consistent with structural features of the Rsm ncRNAs. We also found that these rsmX ncRNA genes share a conserved upstream region suggesting that their expression is dependent upon the global response regulator, GacA.

Recombineering using RecTE from Pseudomonas syringae.

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

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.

Oligonucleotide recombination: a hidden treasure.

In Swingle et al. we demonstrate that it is possible to use recombineering to direct a variety of changes in wild-type bacterial cells without the addition of phage-encoded proteins. This discovery is potentially applicable to biological engineering in a wide variety of bacterial species. Here we describe key features of oligo recombination as it is currently understood, and propose strategies for expanding the utility of oligo recombination for bioengineering.

Oligonucleotide recombination in Gram-negative bacteria.

This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single-stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage-encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here were designed with an eye towards learning to use oligo recombination in order to bootstrap identification and development of phage-encoded recombination systems for recombineering in a wide range of bacteria. The results show that oligo concentration and sequence have the greatest influence on recombination frequency, while oligo length was less important. Apart from the utility of oligo recombination, these findings also provide insights regarding the details of recombination mediated by phage-encoded functions. Establishing that oligos can recombine with bacterial genomes provides a link to similar observations of oligo recombination in archaea and eukaryotes suggesting the possibility that this process is evolutionary conserved.

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: the role of iron in cell growth and virulence factor induction.

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h(-1) with an initial cell mass of less than 0.6 OD(600). Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD(600) or higher, or at low dilution rates (<0.05 h(-1)) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states.Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron-supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron-limited chemostat cultures was observed compared to iron-supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology.

The effect of target vector selection on the invariance of classifier performance measures.

In this paper, the multiclass supervised training problem is considered when a discrete set of classes is assumed. Upon generating affine models for finite data sets, we have observed the invariance of certain measures of performance after a trained classifier has been presented with test data of unknown classification. Specifically, after constructing mappings between training vectors and their desired targets, the class membership and ranking of test data has been found to remain either invariant or close to invariant under a transformation of the set of target vectors. Therefore, we derive conditions explaining how this type of invariance can arise when the multiclass problem is phrased in the context of linear networks. A bioinformatics example is then presented in order to demonstrate various principles outlined in this work.

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.

Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs.

BACKGROUND: Identification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many sigmaB-dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic DeltasigB mutant, which does not express the alternative sigma factor sigmaB, a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase. RESULTS: Overall, 83% of all L. monocytogenes genes were transcribed in stationary phase cells; 42% of currently annotated L. monocytogenes genes showed medium to high transcript levels under these conditions. A total of 96 genes had significantly higher transcript levels in 10403S than in DeltasigB, indicating sigmaB-dependent transcription of these genes. RNA-Seq analyses indicate that a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs. Application of a dynamically trained Hidden Markov Model, in combination with RNA-Seq data, identified 65 putative sigmaB promoters upstream of 82 of the 96 sigmaB-dependent genes and upstream of the one sigmaB-dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5'- and 3'-UTR regions. CONCLUSIONS: The results from these studies provide powerful evidence that RNA-Seq data combined with appropriate bioinformatics tools allow quantitative characterization of prokaryotic transcriptomes, thus providing exciting new strategies for exploring transcriptional regulatory networks in bacteria.

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.

Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads.

Bacteria that survive under variable conditions possess an assortment of genetic regulators to meet these challenges. The group IV or extracytoplasmic function (ECF) sigma factors regulate gene expression in response to specific environmental signals by altering the promoter specificity of RNA polymerase. We have undertaken a study of PvdS, a group IV sigma factor encoded by Pseudomonas syringae pv. tomato DC3000 (DC3000), a plant pathogen that is likely to encounter variations in nutrient availability as well as plant host defences. The gene encoding PvdS was previously identified by sequence similarity to the Pseudomonas aeruginosa orthologue, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition, and is responsible for the characteristic fluorescence of the pseudomonads. We identified 15 promoters regulated by PvdS in DC3000 and characterized the promoter motif using computational analysis. Mutagenesis of conserved nucleotides within the motif interfered with promoter function and the degree of the effect was different depending on which region of the motif was mutated. Hidden Markov models constructed from alignments of sequence motifs extracted from DC3000 and PAO1 were used to query genomes of DC3000 and other fluorescent pseudomonads for similar motifs. We conclude that the role of PvdS as a regulator of pyoverdine synthesis is conserved among the fluorescent pseudomonads, but the promoters recognized by PvdS orthologues may differ subtly from species to species.

Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains.

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.

Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes.

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.