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.     

Cell individuality: the bistable gene expression of the type III secretion system in Dickeya dadantii 3937

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.     

丁香假单胞菌中的III型分泌系统及其调控机制研究进展

丁香假单胞菌(Pseudomonas syringae)是引起许多作物病害的一种革兰氏阴性病原细菌。该细菌入侵寄主植物细胞主要通过其III型分泌系统(type III secretion system,T3SS)将效应蛋白转入到寄主真核细胞内,抑制寄主免疫功能,以达到成功侵染和定殖的目的。III型分泌系统的主调控因子RhpR/S通过感受环境信号的变化直接调控hrpR/S及其他毒力相关通路。同时III型分泌系统基因的表达也受到其他调控因子的影响,包括σ因子HrpL、双组分系统GacA/S、Lon蛋白酶、第二信使分子和环境信号等。本文在简要介绍丁香假单胞菌III型分泌系统组成和功能的基础上,综述丁香假单胞菌III型分泌系统调控机制的最新研究进展,以期为深入探究病原菌的致病机制提供参考和思路。     

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.     

Commensal effect of pectate lyases secreted from Dickeya dadantii on proliferation of Escherichia coli O157:H7 EDL933 on lettuce leaves

The outbreaks caused by enterohemorrhagic Escherichia coli O157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms of Dickeya dadantii 3937, a broad-host-range phytopathogen, on the proliferation of the human pathogen E. coli O157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 with D. dadantii 3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-type D. dadantii 3937. Increased expression of pelD (encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS) in planta. These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases in D. dadantii 3937.     

Population behavior analysis of dspE and pelD regulation in Erwinia chrysanthemi 3937

Erwinia chrysanthemi 3937 (Ech3937) is a phytopathogenic bacterium with a wide host range. The pectinolytic enzymes secreted by the bacterium and the type III secretion system (T3SS) are essential for full virulence. We used the green fluorescent protein gene as a reporter to investigate the expression of dspE (a putative T3SS effector) and pelD (a major pectin-degrading enzyme) in populations of Ech3937 under different conditions. Gene expression was analyzed by measuring the fluorescence intensity of individual cells with a fluorescence-activated cell sorter. Ech3937 dspE was induced in minimal medium (MM) with only a portion of Ech3937 cells (43.03%) expressing dspE after 12 h of culture. The nutrient-rich King's medium B did not fully eliminate the expression of dspE; a small percentage of Ech3937 cells (5.55%) was able to express dspE after 12 h of culture in this medium. In all, 68.95% of Ech3937 cells expressed pelD after 12 h of culture in MM supplemented with polygalacturonic acid (PGA). However, 96.34% of Echl31 cells (an hrpL deletion mutant of Ech3937) expressed pelD after 12 h of culture in MM supplemented with PGA. In potato tubers, 6.32% of the bacterial cells expressed dspE 2 h after inoculation, whereas only 0.25% of the cells expressed pelD. However, after 24 h, the percentage of cells expressing pelD (68.48%) was approximately 3.5 times that of cells expressing dspE (19.39%). In contrast to potato tubers, similar proportion of Ech3937 cells expressing dspE (39.34%) and pelD (40.30%) were observed in Chinese cabbage 24 h after inoculation. From promoter activity and real-time quantitative results, the expression of pelD in Ech3937 was demonstrated to be downregulated by HrpL in MM supplemented with PGA.     

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.