Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes.

The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.     

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.     

SlyA regulates type III secretion system (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937

The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.     

The Response regulator HrpY of Dickeya dadantii 3937 regulates virulence genes not linked to the hrp cluster

HrpX/Y is a putative two-component system (TCS) encoded within the type III secretion system (T3SS) gene cluster of Dickeya dadantii. A linear regulatory cascade initiated by HrpX/Y that leads to activation of the downstream T3SS genes via HrpS and HrpL was described previously. Therefore, in D. dadantii, HrpX/Y plays an important role in regulation of genes involved in bacteria-plant interactions and bacterial aggregation via the T3SS. HrpX/Y is the only TCS shared among the plant-pathogenic enterobacteria that is not also present in animal-associated enterobacteria. To date, the genes known to be regulated by HrpY are restricted to the hrp and hrc genes and no signal has been identified that triggers HrpY-dependent gene expression. We demonstrated that HrpY interacts with the hrpS promoter in vitro. We then used a transposon-based system to isolate previously unidentified HrpY-dependent genes, including genes previously shown to affect virulence, including kdgM and acsC. HrpY is a dual regulator, positively regulating at least 10 genes in addition to those in the hrp gene cluster and negatively regulating at least 5 genes. The regulatory effect on one gene depended on the culture medium used. Of the 16 HrpY-regulated genes identified in this screen, 14 are not present in Pectobacterium atrosepticum, the nearest relative of D. dadantii with a sequenced genome. None of the newly identified HrpY-regulated genes were required for bacterial aggregation; thus, neither acyl-homoserine lactone-mediated quorum sensing nor the Rcs signal transduction system which regulates colanic acid, a molecule that plays an important role in biofilm formation in other enterobacteria, are required for D. dadantii aggregation.     

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome

Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins.     

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.     

The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins.

A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.