Construction of the temperature-sensitive vectors pLUCH80 and pLUCH88 for delivery of Tn917::NotI/SmaI and use of these vectors to derive a circular map of Listeria monocytogenes Scott A, a serotype 4b isolate.
A physical map of Listeria monocytogenes Scott A was generated by the pulsed-field technique of contour-clamped-homogeneous-electric-field (CHEF) electrophoresis. The circular genome of this serotype 4b strain contains 12 AscI fragments (38 to 790 kb), 5 NotI fragments (55 to 1,400 kb), 3 SrfI fragments (110, 1,110, and 2,000 kb), and 2 SfiI fragments (1,320 and 1,920 kb). Summation of individually sized fragments derived by digestion of Scott A genomic DNA with each of these four enzymes provided an average estimated genome length of 3,210 +/- 60 kb. Efforts to assemble the macrorestriction map benefited greatly from the construction and use of pLUCH80 and pLUCH88, temperature-sensitive vectors for delivering transposon Tn917::NotI/SmaI to the chromosome of Scott A. As another component of this study, the positions of four known virulence genes (inlA, mpl, hly, and prf) and three L. monocytogenes-specific sequences (lisM44, lisM51, and lisM52) were localized on the physical map of Scott A by hybridization. Probes prepared from lisM44, lisM51, and the four virulence genes hybridized within a cluster on a 150-kb fragment of the Scott A genome that overlaps part of the NotI-B and AscI-D fragments. The lisM52 probe hybridized with the AscI-F2 (120-kb) fragment of Scott A, which is separated from the NotI-B-AscI-D region by about 300 kb. These results established the first physical and genetic map of a serotype 4b strain of L. monocytogenes and provided further insight on this important food-borne pathogen at the genome level.
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Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706, USA.
Luchansky
J B
JB
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005822:Genetic Vectors; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D015183:Restriction Mapping; D012703:Serotyping; D013696:Temperature
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes