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Appl Environ Microbiol, May 1998, p. 1812-1815, Vol. 64, No. 5
Department of Genetics, Howard Hughes Medical
Institute, University of Pennsylvania,1 and
Department of Bioscience and Biotechnology, Drexel
University,3 Philadelphia, Pennsylvania 19104, and
Microbial Food Safety Research Unit, Eastern Regional
Research Center, Agricultural Research Service, U.S. Department of
Agriculture, Wyndmoor, Pennsylvania 190382
Received 6 October 1997/Accepted 9 March 1998
Virulent serotypes of Yersinia enterocolitica carry a
plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The
plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Western blotting with a monoclonal antibody (3.2C)
that is specific for a 25-kDa released protein to show that 32°C is
the lowest temperature at which plasmid-encoded proteins are expressed
in quantity. The highest calcium concentration allowing full expression
of these proteins was 445 to 545 µM at 32°C. Calcium concentrations
of 745 µM and above at 37°C completely prevented the loss of pYV
during multiple subcultures, while at 32°C, calcium concentrations of
245 µM and greater were sufficient to stabilize the plasmid. Growth
of Y. enterocolitica at pH 5.5 was slower than at neutral
pH values, but it also resulted in greatly increased stability of pYV.
These studies showed that bacterial growth, retention of pYV, and
expression of plasmid-encoded proteins may be maximized at 32°C with
445 µM calcium and that pYV stability is enhanced by growth at low
pH. These observations suggest new approaches for isolation of
plasmid-bearing virulent strains of Y. enterocolitica from
samples contaminated with this organism and also may improve our
understanding of pYV retention in vivo.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Maximizing Plasmid Stability and Production of
Released Proteins in Yersinia enterocolitica
*
Corresponding author. Mailing address: Department of
Bioscience and Biotechnology, 32nd and Chestnut Streets, Drexel
University, Philadelphia, PA 19104. Phone: (215) 895-6906. Fax: (215)
895-1273. E-mail: mageewe{at}post.drexel.edu.
Appl Environ Microbiol, May 1998, p. 1812-1815, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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