Vol. 55, No. 9 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1989, p. 2251-2256 0099-2240/89/092251-06$02.00/0 Copyright © 1989, American Society for Microbiology Cloning of a Gene Encoding a Major Secreted Polypeptide of Listeria monocytogenes and Its Potential Use as a Species-Specific Probe ROBERT K. FLAMM,'t DAVID J. HINRICHS,2 AND MICHAEL F. THOMASHOW3* Department of Microbiology, Washington State University, Pullman, Washington 991641; Research Service 151-S, Veterans Administration Medical Center, Portland, Oregon 972072; and Department of Crop and Soil Science and Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824-13253 Received 27 February 1989/Accepted 21 June 1989 A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridiza- tion conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed. Listeria monocytogenes-contaminated food and dairy products have been the sources of disease outbreaks leading to severe illness and death (4, 7). A rapid and sensitive method to screen for the presence of L. monocytogenes would aid in the prevention of such outbreaks. Currently, the primary method of identifying food products contam- inated with L. monocytogenes is in vitro culture. However, there are a number of problems associated with this method. It is both time consuming and cumbersome. In addition, although L. monocytogenes may be present, it may not grow or may grow only with the use of cold enrichment (1). Finally, culture identification is complicated by the fact that there are a number of different Listeria species. A DNA probe specific for L. monocytogenes could potentially over- come these problems. Such a probe, in conjunction with polymerase chain reaction technology (26), could provide a very sensitive assay for the detection of low numbers of bacteria and even nongrowing organisms. Secreted polypeptides, such as hemolysins, toxins, and siderophores, often play a role in survival of an organism and/or its ability to cause disease. In addition, secreted polypeptides often play a significant role in the elicitation of the host immune response. Thus, genes encoding major extracellular products could have important roles in the interaction of L. monocytogenes with host cells and could potentially provide a useful marker for identification of L. monocytogenes. Here, we report the cloning of a gene, msp, that encodes a major secreted polypeptide of L. monocytogenes. Hybrid- ization experiments indicated that msp is highly conserved among L. monocytogenes isolates and that under stringent hybridization conditions, homology is observed only with this species of Listeria. * Corresponding author. t Present address: Squibb Institute for Medical Research, Prince- ton, NJ 08540. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Media. Escherichia coli strains were grown in LB (18) broth, on LB agar, or on tryptic soy agar (Difco Laborato- ries, Detroit, Mich.) containing 5% (vol/vol) sheep blood (SBA). All Listeria species were grown in brain heart infusion (BHI) (Difco) broth or on SBA plates. DNA isolation and transformation. Procedures for isolating total DNA (6), lambda bacteriophage DNA (8), and plasmid DNA (2) were as previously described. E. coli was trans- formed by the CaC12 heat shock method (14). Restriction enzyme analysis. Samples of DNA were di- gested with restriction endonucleases as recommended by the supplier (New England BioLabs, Inc., Beverly, Mass.), and the DNA fragments were fractionated by agarose gel electrophoresis as previously described (6). Southern blot analysis. DNA digests were fractionated by agarose gel electrophoresis, and the DNA fragments were transferred to nitrocellulose filters by the method of South- ern (31). The filters were hybridized with DNA probes that had been labeled in vitro by the nick translation reaction (15). Hybridization conditions were as described previously (32). Blots were washed under stringent conditions with a buffer consisting of 0.3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M trisodium citrate) and 0.1% sodium dodecyl sulfate (SDS) at 68°C or under nonstringent conditions with a buffer composed of 3x SSC, 0.2% SDS, and 5 mM EDTA at 55°C. Molecular cloning procedures. An L. monocytogenes gene bank was constructed by using the lambda vector EMBL3A (8). Total DNA isolated from L. monocytogenes 10403 was partially digested with Sau3A, the restriction fragments were separated by agarose gel electrophoresis, and the fragments 15 to 23 kilobases (kb) in size were collected and ligated to BamHI-digested EMBL3A DNA by using T4 DNA ligase. The DNA mixture was then packaged in vitro into phage (11) and amplified in E. coli NM535. Subcloning of the L. 2251