Genomic insights into a Pseudomonas amygdali isolate from Hibiscus rosa-sinensis.

The taxonomy of Pseudomonas has been extensively studied, yet the determination of species is currently difficult because of recent taxonomic changes and the lack of complete genomic sequence data. We isolated a bacterium causing a leaf spot disease on hibiscus (Hibiscus rosa-sinensis). Whole genome sequencing revealed similarity to Pseudomonas amygdali pv. tabaci and pv. lachrymans. The genome of this isolate (referred to as P. amygdali 35-1) shared 4987 genes with P. amygdali pv. hibisci, but possessed 204 unique genes and contained gene clusters encoding putative secondary metabolites and copper resistance determinants. We predicted this isolate's type III secretion effector (T3SE) repertoire and identified 64 putative T3SEs, some of which are present in other P. amygdali pv. hibisci strains. Assays showed that the isolate was resistant to copper at a concentration of 1.6 mM. This study provides an improved understanding of the genomic relatedness and diversity of the P. amygdali species.

Complete Genome Sequence Resource for Xanthomonas hortorum Isolated from Greek Oregano.

In spring 2019, necrotic leaf spots were detected on Greek oregano (Origanum vulgare var. hirtum) plants in a commercial greenhouse operation. An isolate was recovered from the diseased plants. Partial 16S ribosomal RNA sequencing and multilocus sequence analysis revealed that the isolate was a Xanthomonas sp. but proved insufficient to identify the species with certainty. Therefore, whole-genome sequencing using both Nanopore and Illumina technologies was performed. Here, we report the complete and annotated genome sequence of Xanthomonas hortorum strain 108, which was originally isolated from Greek oregano in Long Island, NY, U.S.A.

Complete Genome Sequence Resources for the Onion Pathogen, Pantoea ananatis OC5a.

Here, we report on the genomic sequence and annotation for Pantoea ananatis OC5a, a strain that was isolated from an onion bulb grown in New York and that is pathogenic to onion, causing center rot of onion. OC5a is the first P. ananatis strain pathogenic to onion from New York to be completely assembled and sequenced. Having been assembled using long PacBio reads and high-fidelity Illumina reads, this genome is closed, complete, and of high quality.

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.

Genome-Wide Identification of Genes Important for Growth of Dickeya dadantii and Dickeya dianthicola in Potato (Solanum tuberosum) Tubers.

Dickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Though all three strains are pathogenic on potato, D. dadantii 3937 is a well-characterized model while D. dianthicola strains ME23 and 67-19 are recent isolates. Strain ME23 specifically was identified as a representative strain from a 2014 outbreak on potato. This study generated comparable gene fitness measurements across ecologically relevant conditions for both model and non-model strains. Tubers from the potato cultivars "Atlantic," "Dark Red Norland," and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.

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).

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.

Transcriptomic Profiling Suggests That Promysalin Alters the Metabolic Flux, Motility, and Iron Regulation in Pseudomonas putida KT2440.

Promysalin, a secondary metabolite produced by P. putida RW10S1, is a narrow-spectrum antibiotic that targets P. aeruginosa over other Pseudomonas spp. P. putida KT2440, a nonproducing strain, displays increased swarming motility and decreased pyoverdine production in the presence of exogenous promysalin. Herein, proteomic and transcriptomic experiments were used to provide insight about how promysalin elicits responses in PPKT2440 and rationalize its species selectivity. RNA-sequencing results suggest that promysalin affects PPKT2440 by (1) increasing swarming in a flagella-independent manner; (2) causing cells to behave as if they were experiencing an iron-deficient environment, and (3) shifting metabolism away from glucose conversion to pyruvate via the Entner-Doudoroff pathway. These findings highlight nature's ability to develop small molecules with specific targets, resulting in exquisite selectivity.

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.

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.

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.