ABSTRACT
The low-calcium response (lcr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37 degrees C in Ca(2+)-depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence-plasmid-encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature-regulated activator and by Ca(2+)-regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface-located when Yersinia is grown at 37 degrees C. yopN was found to be part of an operon that is induced during the low-calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild-type phenotype of the yopN mutant. Thus the surface-located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.
Subject(s)
Bacterial Proteins/metabolism , Calcium/metabolism , Membrane Proteins , Signal Transduction , Yersinia pseudotuberculosis/metabolism , Amino Acid Sequence , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Blotting, Northern , DNA Mutational Analysis , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Immunoblotting , Molecular Sequence Data , Plasmids/genetics , Repressor Proteins/physiology , Restriction Mapping , Temperature , Transcription Factors/physiology , Transcription, Genetic , Yersinia pseudotuberculosis/pathogenicityABSTRACT
Leukocytes in synovial fluid and peripheral blood samples from patients with Yersinia-triggered reactive arthritis were analyzed after DNA amplification using the polymerase chain reaction. The primers applied were specific for the virulence plasmid-coded 1crE genes of Yersinia enterocolitica O:3 and Yersinia pseudotuberculosis III. No Yersinia DNA was observed within the synovial fluid cells or peripheral blood cells by polymerase chain reaction techniques. However, Yersinia antigens were detected in the synovial fluid cells by immunofluorescence techniques. These results suggest that only parts of the causative agents, not the entire microbe, can enter the joint and initiate the inflammation that leads to a reactive arthritis.
Subject(s)
Arthritis, Infectious/microbiology , Yersinia Infections , Adolescent , Adult , Base Sequence , Blood Cells/chemistry , DNA, Bacterial/analysis , Female , Fluorescent Antibody Technique , Humans , Immunoblotting , Male , Middle Aged , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction , Synovial Fluid/chemistryABSTRACT
The low-calcium response (lcr) region of the virulence plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica has been associated with calcium-dependent growth of bacteria. Mutations in the previously identified lcrE locus within the lcr region lack the repressor control of production of the lcr specific proteins, Yersinia outer membrane proteins (Yops) and V and W antigens. We sequenced a 3.3-kilobase-pair BamHI-ClaI fragment of the lcrE locus of pYVO3, the virulence plasmid of Y. enterocolitica O:3. The sequence of lcrE locus revealed six tightly packed open reading frames (ORFs), one of which was identified as the structural gene, lcrE, of the 32.9-kilodalton outer membrane protein LcrE (formerly known as Yop4b or YopN). Detection of large (greater than 2.3-kilobase-pair) transcripts strongly supports the conclusion that the lcrE gene and ORF1 to -5 function as an operon. Transcription of the lcrE-containing operon and the adjacent lcrB locus was found to be divergent, and the corresponding transcripts overlapped about 1,200 nucleotides. This extremely long overlap of the 5' ends of the transcripts produced from face-to-face promoters is a new finding; the longest overlap thus far found has been a few hundred nucleotides. Temperature was found to play the major role in regulation of transcription of the lcrE-containing operon of pYVO3, whereas Ca2+ concentration seemed to affect it only moderately.
Subject(s)
Calcium/pharmacology , Genes, Bacterial , Operon , Yersinia enterocolitica/genetics , Amino Acid Sequence , Bacterial Proteins/analysis , Base Sequence , Blotting, Northern , Cloning, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
No homology was observed between Yersinia enterocolitica O:3 and HLA-B27 at DNA level when Yersinia enterocolitica chromosomal probes were hybridized with human HLA-B27 positive leukocyte DNA or in the hybridization of Yersinia DNA with HLA-B27 specific probe. Our results do not exclude the existence of molecular mimicry between Yersinia proteins and HLA-B27 antigen, since the crossreactive epitope might be a conformational determinant not detected with hybridization.
Subject(s)
DNA, Bacterial , DNA , HLA-B Antigens/genetics , Nucleic Acid Hybridization , Yersinia enterocolitica/genetics , Arthritis, Infectious/etiology , HLA-B27 Antigen , Humans , Reference Values , Sequence Homology, Nucleic Acid , Yersinia Infections/geneticsABSTRACT
Total RNA was purified from human fetal calvaria and articular cartilage. Messenger RNAs for type I and II collagens were identified by hybridization using cDNA clones for chicken pro alpha 1(I)-, pro alpha 2(I)- and pro alpha 1(II)collagen mRNAs and by analysis of cell-free translation products of these RNAs by polyacrylamide gel electrophoresis. The size of human pro alpha 1(II)collagen mRNA is approx. 5100 bases. Translatability of cartilage specific type II collagen mRNA was found to be concentration dependent: with increasing total RNA concentrations the relative translation of type II collagen mRNA was reduced with respect to type I mRNAs.
