[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2014; 9(2):e86628. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by β-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.
Journal of bacteriology 11/2011; 194(3):553-60. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
Journal of bacteriology 07/2011; 193(18):4598-611. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2011; 6(12):e29335. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 08/2010; 76(15):4960-8. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.
Journal of bacteriology 04/2010; 192(7):2020-1. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of bacteriology 02/2010; 192(9):2359-72. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Biotechnology and Bioengineering 12/2009; 105(5):955-64. · 3.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: In this paper, we describe the context sensitivity problem encountered in partitioning a heterogeneous biological sequence into statistically homogeneous segments. After showing signatures of the problem in the bacterial genomes of Escherichia coli K-12 MG1655 and Pseudomonas syringae DC3000, when these are segmented using two entropic segmentation schemes, we clarify the contextual origins of these signatures through mean-field analyses of the segmentation schemes. Finally, we explain why we believe all sequence segmentation schems are plagued by the context sensitivity problem.
[show abstract][hide abstract] ABSTRACT: In this paper, we extend a previously developed recursive entropic segmentation scheme for applications to biological sequences. Instead of Bernoulli chains, we model the statistically stationary segments in a biological sequence as Markov chains, and define a generalized Jensen-Shannon divergence for distinguishing between two Markov chains. We then undertake a mean-field analysis, based on which we identify pitfalls associated with the recursive Jensen-Shannon segmentation scheme. Following this, we explain the need for segmentation optimization, and describe two local optimization schemes for improving the positions of domain walls discovered at each recursion stage. We also develop a new termination criterion for recursive Jensen-Shannon segmentation based on the strength of statistical fluctuations up to a minimum statistically reliable segment length, avoiding the need for unrealistic null and alternative segment models of the target sequence. Finally, we compare the extended scheme against the original scheme by recursively segmenting the Escherichia coli K-12 MG1655 genome.
[show abstract][hide abstract] ABSTRACT: 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.
Applied and environmental microbiology 04/2009; 75(9):2720-6. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The availability of complete genome sequences for three P. syringae pathovars (P. s. pv. tomato DC3000, P. s. pv. phaseolicola 1448A, and P. s. pv. syringae B728a) provides researchers with unprecedented opportunities to explore factors contributing to pathogenicity and virulence
in these pathogens. One method employed for genome sequence analysis involves identification of genes and regulatory elements
through association with conserved sequence motifs. Using this approach, ~50 and ~30 genes for T3SS-dependent Hop/Avr effector
proteins have been identified in Pst DC3000 and Psp 1448A, respectively, on the basis of their proximity to conserved “hrp
box” regulatory motifs and the presence of Hop-associated patterns at the N-terminus of the encoded proteins. These analyses
have also led to a comprehensive catalog of the hrpL regulons of all three pathovars. Similar pattern-based searches have led to comprehensive identification of other regulatory
elements in the Pst DC3000 genome, including binding sites for RpoD, RpoE, RpoN, RpoS, and Fur. Genome comparison represents
a second powerful tool for identification of the genes and regulatory elements involved in diverse stages of the plant–pathogen
association. For example, genome comparison of the three sequenced P. syringae pathovars sheds light on genes contributing to their different pathogenic strategies. More broadly, genes distinguishing
the plant-associated pseudomonads from pseudomonads occupying other niches can be identified as described in the study comparing
the Pst DC3000 genome with that of the saprophyte P. putida and the animal pathogen P. aeruginosa. Using this approach, 44 “LSRs” (lineage-specific regions) were identified in the Pst DC3000 genome. Enriched for genes encoding
known virulence factors, the LSRs also contain a large number of genes encoding uncharacterized “hypothetical” proteins with
potential roles in the bacteria–plant interaction. Comparisons among genomes of plant-associated pseudomonads and enterobacteriaceae
may also yield insight into the underlying, broadly conserved functions required for bacteria to associate with their plant
hosts. However, the effective use of genomic data for any analysis requires that genome annotation be accurate, up-to-date,
and enhanced as needed by improvements such as consistent nomenclature (as done for the effectors) or addition of gene ontology
(GO) terms. Furthermore, the genome annotation, results of large-scale genomic analyses, and relevant tools must be readily
accessible to the research community. To this end, the Pseudomonas–Plant Interaction (PPI) web site (http://pseudomonas-syringae.org)
was developed and continues to be maintained for the benefit of the P. syingae research community.