Molecular Characterization of Pantoea stewartii subsp. stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter

Abstract

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a 70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.The hrp/hrc genes of gram-negative, phytopathogenic bacteria encode type III secretion systems that deliver effector proteins into host cells. These proteins may elicit cell death or suppress host defenses. They are required for infection and colonization of plants by all biotrophic pathogens of the genera Pseudomonas, Ralstonia, Xanthomonas, Erwinia, and Pantoea (3). They also play a role in rapid establishment of necrotrophic, soft-rot bacteria in the genus Erwinia, where enzymatic tissue disintegration is delayed by hrp mutations (33). Pantoea stewartii subsp. stewartii (synonym, Erwinia stewartii) causes vascular wilting and leaf blight of sweet corn and maize. This pathogen carries a group I hrp gene cluster (1,7,8,11), similar to those in Pseudomonas, Erwinia, and other Pantoea species. The group I hrp gene clusters are regulated by the HrpS enhancer-binding protein and the HrpL extracytoplasmic function alternative sigma factor (12, 25, 36-39). P. stewartii hrp genes are also controlled by the HrpX/HrpY two-component regulatory system, which is also present in Pantoea agglomerans pv. gypsophilae, Erwinia amylovora, Erwinia carotovora subsp. carotovora, and Erwinia chrysanthemi (19,25,27,35,39). In erwinias and pantoeas, the hrpL-hrpXY-hrpS genes have the same map order, and the corresponding proteins share greater than 80% similarity at the amino acid level (25). However, in P. stewartii, a large remnant of an insertion sequence (IS) element i...

Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis

“…A DNase footprinting assay was performed to map the physical binding region of MAP2827 on the putative promoter sequence as described by Merighi et al (2006) and Zianni et al (2006). Cattle MAP mbtB or bfrA promoters carrying the predicted iron box sequence were cloned into pSM128 (see below).…”

Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

“…The reaction was stopped by the addition of 125 mM EDTA and cleaned using the QIAquick gel extraction kit (Qiagen). A 10-l sample of DNA eluted in dH 2 O was processed at VBI on the 3730 DNA analyzer (Life Technologies), with a G5 dye set, running an altered default genotyping module with increased injection time and injection voltage (8). The electropherograms of the probes generated from digestion in the presence of BSA and in the presence of HMGE were overlapped using Peak Scanner v1.0 (Life Technologies) to reveal bases that are protected from cleavage by DNase I.…”

“…Once in the xylem, P. stewartii forms a biofilm and overproduces an exopolysaccharide, stewartan, that obstructs the xylem and likely causes the vascular wilting associated with Stewart's wilt by blocking water transport (6,7). P. stewartii also possesses a hrp and wts gene cluster encoding a Hrp type III secretion system and effector proteins (8). Affected seedlings wither (wilt phase), and affected mature plants develop necrotic lesions (leaf blight phase), leading to losses in the crop yield (9).…”

Formation of an inverted repeat junction in the transposition of insertion sequence ISLC3 isolated from Lactobacillus casei

“…The quantity of RNA was determined by spectrophotometry (A 260 and A 280 ). Analysis of the 59 ends of the OrfA mRNA transcripts was performed by primer extension using 6-FAM-labelled primers (59-TTGAATTCTT-TGTCGTAACGA-39; 110-190 bp of ISLC3) (Merighi et al, 2006). A total of 100 pmol primer was annealed to 20 mg total RNA.…”