Identification of the Actinobacillus pleuropneumoniae Leucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes

Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown thatthe limitation of branched-chain amino acids (BCAAs) is a cue thatinduces the expression of a subset of A. pleuropneumoniaegenes identified as specifically induced during infection of thenatural host animal by using an in vivo expression technology screen.Leucine-responsive regulatory protein (Lrp) is a global regulator andhas been shown in Escherichia coli to regulate many genes,including genes involved in BCAA biosynthesis. We hypothesized thatA. pleuropneumoniae contains a regulator similar to Lrp andthat this protein is involved in the regulation of a subset of genesimportant during infection and recently shown to have increasedexpression in the absence of BCAAs. We report the identification of anA. pleuropneumoniae serotype 1 gene encoding a protein withsimilarity to amino acid sequence and functional domains of otherreported Lrp proteins. We further show that purified A.pleuropneumoniae His₆-Lrp binds in vitro to the A.pleuropneumoniae promoter regions for ilvI, antisensecps1AB, lrp, and nqr. A genetically definedA. pleuropneumoniae lrp mutant was constructed using anallelic replacement and sucrose counterselection method. Analysis ofexpression from the ilvI and antisense cps1ABpromoters in wild-type, lrp mutant, and complementedlrp mutant strains indicated that Lrp is required forinduction of expression of ilvI under BCAAlimitation.