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.

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.

c Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. There are three Yersinia species pathogenic to humans. Y. pestis is the causative agent of bubonic and pneumonic plague and is transmitted through a flea vector or through airborne transmission from one mammalian host to another (1). In contrast, the enteropathogenic Yersinia species Y. enterocolitica and Y. pseudotuberculosis grow in the environment but can be transmitted to mammalian hosts through ingestion of contaminated food or water (1). Y. enterocolitica and Y. pseudotuberculosis cause typically self-limiting mesenteric lymphadenitis or gastroenteritis in otherwise healthy individuals but can cause a serious blood-borne infection in people with iron overload disorders such as hereditary hemochromatosis (2). In addition, sequelae following enteropathogenic Yersinia infection, such as erythema nodosum and reactive arthritis, have also been reported (3-5).Human-pathogenic Yersinia species share a virulence plasmid, called pCD1 in Y. pestis and pYV in enteropathogenic yersiniae, encoding the Ysc type III secretion system (T3SS) essential for causing disease (6). These 70-kb plasmids carry dozens of T3SS structural genes and encode five or six T3SS effector proteins called Yops and their dedicated chaperones, as well as genes encoding proteins involved in regulating expression and function of the T3SS. One of these regulatory proteins, LcrF, serves as the Yersinia T3SS master regulator, controlling transcription of a large number of plasmid-borne genes. Several environmental cues influence expression of LcrF itself, possibly enabling Yersinia to control T3SS expression during transitions from one niche to another. Recent reviews have highlighted important advances in our understanding of T3SS structure and modulation of the innate immune response by T3SS effector proteins (7). In this review, we focus on factors controlling LcrF expression, the target genes LcrF regulates, and how LcrF activity influences Yersinia pathogenesis. LcrF HISTORY, STRUCTURE, AND FUNCTIONIt has long been appreciated that human-pathogenic Yersinia carrying T3SS genes requires millimolar concentrations of calcium to grow at 37°C, and this phenomenon was termed the low-calcium response, or Lcr (8, 9). The absence of calcium, in combination with a shift to 37°C, triggers secretion of T3SS effector proteins, mimicking t...