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Applied and Environmental Microbiology, December 2001, p. 5370-5376, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5370-5376.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Surface Expression of the Conserved C Repeat Region of Streptococcal M6 Protein within the Pip Bacteriophage Receptor of Lactococcus lactis

Bruce L. Geller,1,2,3,* Nadine Wade,1 Thomas D. Gilberts,1 Dennis E. Hruby,1,2 Ryan Johanson,1 and Ljubisa Topisirovic4

Department of Microbiology,1 The Center for Gene Research and Biotechnology,2 and The Western Dairy Center,3 Oregon State University, Corvallis, Oregon 97331-3804, and Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 283, 11001 Belgrade, Yugoslavia4

Received 19 March 2001/Accepted 9 August 2001

The C repeat region of the M6 protein (M6c) from Streptococcus pyogenes was expressed within the Pip bacteriophage receptor on the surface of Lactococcus lactis. M6c was also detected in the culture medium. The pip-emm6c allele was integrated into the chromosome and stably expressed without antibiotic selection. The level of cell-associated surface expression of PipM6c was 0.015% of total cellular protein. The amount of PipM6c on the cell surface was increased about 17-fold by expressing pip-emm6c from a high-copy-number plasmid. Replacing the native pip promoter with stronger promoters isolated previously from Lactobacillus acidophilus increased surface expression of PipM6c from the high-copy-number plasmid up to 27-fold. Concomitantly, the amount of PipM6c in the medium increased 113-fold. The amount of PipM6c did not vary greatly between exponential- and stationary-phase cultures. Western blots indicated that the full-length PipM6c protein and most of the numerous proteolytic products were found only on the cell surface, whereas only one proteolytic fragment was found in the culture medium.

* Corresponding author. Mailing address: Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1845. Fax: (541) 737-0496. E-mail: gellerb{at}orst.edu.

Applied and Environmental Microbiology, December 2001, p. 5370-5376, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5370-5376.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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