ABSTRACT
The plasmid pSymA, in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, carries a 750-bp ORF (SMa1978) designated, hdhA, which encodes a novel dehalogenase that can detoxify haloacid compounds, showing a preference for haloacetic acids. Purified His-tagged HdhA demonstrated the apparent ability to dehalogenate chloroacetic acid and trifluoroacetic acid. In addition, upstream of hdhA, a gene encoding a lysR-type transcription regulator denoted, hdhR (SMa1979), has been identified to be a transcriptional repressor of hdhA expression. In an hdhR knockout mutant, hdhA promoter activity was markedly increased. Purified 32-kDa His-tagged HdhR repressed expression of hdhA by specifically binding to the promoter region of hdhA, as demonstrated by gel mobility shift assay and DNase I foot printing experiments. Moreover, the pesticide, pentachlorophenol, was also found to induce hdhA expression via HdhR. Site-directed mutants, in which the Cys residues at positions 160 and 192 in HdhR were changed to Ser, were constructed. C160S and C192S single mutants showed diminished HdhR-mediated repression of hdhA expression, while a C160S:C192S double mutant could no longer repress expression of hdhA.
Subject(s)
Hydrolases/genetics , Sinorhizobium meliloti/genetics , Transcription Factors/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , DNA Footprinting/methods , Gene Expression Regulation, Bacterial , Hydrolases/metabolism , Molecular Sequence Data , Mutation , Promoter Regions, Genetic/genetics , Protein Binding , Sequence Alignment , Sinorhizobium meliloti/metabolism , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The broad-spectrum organophosphate insecticide chlorpyrifos (CPF)-inducible locus, chpAB, was identified on the endogenous plasmid pSymB in Sinorhizobium meliloti. The S. meliloti chpA promoter was highly induced by CPF and was induced at much lower levels by diazinon and ethion. Transcription of chpA was dependent on chpR, a CadC family transcriptional regulator located upstream of, and divergently transcribed from, chpAB. ChpR was able to mediate the CPF-inducible expression of the S. melilotichpA promoter in Escherichia coli through direct interaction with the chpAB promoter. The chpR-chpA intergenic regions of several bacterial chpRAB operons were aligned and a putative ChpR-binding sequence was proposed. Both the ChpR transcription factor and chpA promoter constitute a good candidate system for genetic-based biosensor development.
Subject(s)
Bacterial Proteins/physiology , Chlorpyrifos/metabolism , Gene Expression Regulation, Bacterial , Sinorhizobium meliloti/physiology , Transcription Factors/physiology , Transcription, Genetic , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Cloning, Molecular , Diazinon/metabolism , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Multigene Family , Organothiophosphorus Compounds/metabolism , Plasmids , Promoter Regions, Genetic , Protein Binding , Sequence Alignment , Transcription Factors/geneticsABSTRACT
Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.
