ABSTRACT
In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Fluorescent Dyes/metabolism , Polymerase Chain Reaction/methods , Taq Polymerase/metabolism , Yersinia enterocolitica/isolation & purification , Animals , Feces/microbiology , Humans , Meat Products/microbiology , Sensitivity and Specificity , Swine , Yersinia Infections/microbiology , Yersinia enterocolitica/geneticsABSTRACT
Several LysR-type transcriptional regulators have been shown to require the carboxy-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase to activate their target genes. We show here that GcvA, a LysR-type protein, also uses the alphaCTD to activate the Escherichia coli gcvTHP operon. Amino acid residues in the alphaCTD important for GcvA-dependent activation, however, have no effect on GcvA-mediated repression of the operon.
Subject(s)
DNA-Directed RNA Polymerases/physiology , Escherichia coli Proteins , Gene Expression Regulation , Hydroxymethyl and Formyl Transferases/metabolism , Transcription Factors , Aminomethyltransferase , Bacterial Proteins/physiology , DNA-Directed RNA Polymerases/genetics , Escherichia coli/enzymology , Lac Operon/genetics , Mutation , Repressor Proteins/physiologyABSTRACT
The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.
