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Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis
A Forsberg1, I Bölin, L Norlander
1Department of Microbiology, National Defence Research Institute, Umeå, Sweden.
Abstract:
A number of plasmid-associated proteins (YOPs) of Y. pseudotuberculosis are induced and expressed at high levels when the pathogen is grown at 37 degrees C in absence of Ca2+ ions. These proteins were recovered both from the outer membrane fraction and the culture supernatant. Two hours after a temperature-shift the YOPs were only found in the culture supernatant, amounting to about 5% of the total cell protein. After 4 h of incubation they were also detected in the outer membrane fraction. Separation by 2-D gel electrophoresis revealed that the YOPs could be separated into 6 different polypeptides; YOP2a (45 kDa), YOP2b (45 kDa), YOP3 (41-42 kDa), YOP4a (34 kDa), YOP4b (34 kDa) and YOP5 (26 kDa). The structural genes of all of these YOPs, except the YOP2a gene, were cloned to pBR322 and their respective genetic localization was established. It was found that the genes were not part of a common operon but scattered around plasmid plB1. Only the YOP4b protein was found to map within the Ca2+ region. The hybrid plasmid plB572 coded for a number of plasmid plB1 specific proteins, one of which showed a molecular weight of 38 kDa. This polypeptide could be precipitated by monospecific V-antiserum, showing that this protein is the V-antigen.
Insights
Plasmid-associated proteins (YOPs) in Y. pseudotuberculosis are temperature- and calcium-regulated, with six distinct polypeptides identified. Their genes are scattered on plasmid pIB1, not in a common operon.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Yersinia pseudotuberculosis secretes plasmid-associated proteins (YOPs) under specific environmental conditions.
- YOPs are crucial virulence factors, with their expression tightly regulated by temperature and ion availability.
Purpose of the Study:
- To characterize the YOPs of Y. pseudotuberculosis, including their expression, purification, and genetic localization.
- To investigate the relationship between YOPs and plasmid structure.
Main Methods:
- Bacterial culture under specific temperature and calcium conditions.
- Protein fractionation (outer membrane and supernatant).
- 2-D gel electrophoresis for polypeptide separation.
- Gene cloning into pBR322 and genetic mapping.
- Immunoprecipitation using V-antiserum.
Main Results:
- Six YOP polypeptides (YOP2a, YOP2b, YOP3, YOP4a, YOP4b, YOP5) were identified with distinct molecular weights.
- YOPs were detected in the culture supernatant and outer membrane fraction, with temporal changes after temperature shift.
- Genes for most YOPs were located on plasmid pIB1, scattered and not in an operon.
- YOP4b mapped to the Ca2+ regulated region.
- A 38 kDa protein from hybrid plasmid pIB572 was identified as V-antigen.
Conclusions:
- YOP expression and localization are dynamic and influenced by environmental cues.
- The genetic organization of YOPs on pIB1 is complex, with individual gene regulation.
- This study provides insights into the molecular basis of Yersinia virulence and pathogenesis.