title: Identification of the Actinobacillus pleuropneumoniae leucine-responsive regulatory protein and its involvement in the regulation of in vivo-induced genes.
abstract: Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset of A. pleuropneumoniae genes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown in Escherichia coli to regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized that A. pleuropneumoniae contains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of an A. pleuropneumoniae serotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purified A. pleuropneumoniae His6-Lrp binds in vitro to the A. pleuropneumoniae promoter regions for ilvI, antisense cps1AB, lrp, and nqr. A genetically defined A. pleuropneumoniae lrp mutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from the ilvI and antisense cps1AB promoters in wild-type, lrp mutant, and complemented lrp mutant strains indicated that Lrp is required for induction of expression of ilvI under BCAA limitation.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Trevor K
authors.initials: TK
authors.lastname: Wagner
authors.forename: Martha H
authors.initials: MH
authors.lastname: Mulks
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics*; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D051756:Leucine-Responsive Regulatory Protein / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
chemical_list: D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D051756:Leucine-Responsive Regulatory Protein; C080946:IlvI protein, Bacteria