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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.

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.

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.

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.

Gramene: a resource for comparative grass genomics.

Gramene (http://www.gramene.org) is a comparative genome mapping database for grasses and a community resource for rice. Rice, in addition to being an economically important crop, is also a model monocot for understanding other agronomically important grass genomes. Gramene replaces the existing AceDB database 'RiceGenes' with a relational database based on Oracle. Gramene provides curated and integrative information about maps, sequence, genes, genetic markers, mutants, QTLs, controlled vocabularies and publications. Its aims are to use the rice genetic, physical and sequence maps as fundamental organizing units, to provide a common denominator for moving from one crop grass to another and is to serve as a portal for interconnecting with other web-based crop grass resources. This paper describes the initial steps we have taken towards realizing these goals.

A graph-theoretic approach to comparing and integrating genetic, physical and sequence-based maps.

For many species, multiple maps are available, often constructed independently by different research groups using different sets of markers and different source material. Integration of these maps provides a higher density of markers and greater genome coverage than is possible using a single study. In this article, we describe a novel approach to comparing and integrating maps by using abstract graphs. A map is modeled as a directed graph in which nodes represent mapped markers and edges define the order of adjacent markers. Independently constructed graphs representing corresponding maps from different studies are merged on the basis of their common loci. Absence of a path between two nodes indicates that their order is undetermined. A cycle indicates inconsistency among the mapping studies with regard to the order of the loci involved. The integrated graph thus produced represents a complete picture of all of the mapping studies that comprise it, including all of the ambiguities and inconsistencies among them. The objective of this representation is to guide additional research aimed at interpreting these ambiguities and inconsistencies in locus order rather than presenting a "consensus order" that ignores these problems.

Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.).

A total of 2414 new di-, tri- and tetra-nucleotide non-redundant SSR primer pairs, representing 2240 unique marker loci, have been developed and experimentally validated for rice (Oryza sativa L.). Duplicate primer pairs are reported for 7% (174) of the loci. The majority (92%) of primer pairs were developed in regions flanking perfect repeats > or = 24 bp in length. Using electronic PCR (e-PCR) to align primer pairs against 3284 publicly sequenced rice BAC and PAC clones (representing about 83% of the total rice genome), 65% of the SSR markers hit a BAC or PAC clone containing at least one genetically mapped marker and could be mapped by proxy. Additional information based on genetic mapping and "nearest marker" information provided the basis for locating a total of 1825 (81%) of the newly designed markers along rice chromosomes. Fifty-six SSR markers (2.8%) hit BAC clones on two or more different chromosomes and appeared to be multiple copy. The largest proportion of SSRs in this data set correspond to poly(GA) motifs (36%), followed by poly(AT) (15%) and poly(CCG) (8%) motifs. AT-rich microsatellites had the longest average repeat tracts, while GC-rich motifs were the shortest. In combination with the pool of 500 previously mapped SSR markers, this release makes available a total of 2740 experimentally confirmed SSR markers for rice, or approximately one SSR every 157 kb.