title: Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora.
abstract: The type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by an RpoN-HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant-pathogenic bacteria. Complementation of the hrpL mutant with promoter deletion constructs of the hrpL gene and promoter activity analyses using various lengths of the hrpL promoter fused to a promoter-less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between -138 and -125 nucleotides (TGCAA-N4-TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrpL gene among plant enterobacterial pathogens. Results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and electrophoresis mobility shift assay coupled with site-directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E. amylovora was characterized by complementation of the hrpS mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E. amylovora.
authors.affiliation: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Jae Hoon
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: George W
authors.initials: GW
authors.lastname: Sundin
authors.affiliation: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Youfu
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D044084:Erwinia amylovora / Q000235:genetics*; D017343:Genes, Plant; D005816:Genetic Complementation Test; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D031989:Pyrus / Q000382:microbiology; D014158:Transcription, Genetic; D000069296:Type III Secretion Systems / Q000235:genetics*; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D000069296:Type III Secretion Systems
keywords: HrpL; RpoN; T3SS; fire blight; sigma factor; virulence