Bacterial Pathogenesis

performed using a fragment encompassing the 3’ end of the gene.
Integration directed by this fragment will not disrupt the gene in ques-
tion, but places a copy of the plasmid between the promoter and 3’
sequences of the gene.
Chromosome
% Slngbcmssover
Figure 7.10. Disruption of a chromosomal gene by plasmid integration. The inte-
grating plasmid cames a fragment of DNA internal to the coding sequence. A
single crossover event results in integration of the plasmid and disruption of the
gene. The plasmid is flanked by a direct duplication of the chromosomal DNA
fragment it carried.
++++++ ISOLATION OF GENOMIC DNA FROM
STAPHYLOCOCCI
Chromosomal DNA is more difficult to isolate from staphylococci than
from E. coli. S. epidemidis is particularly difficult. The method detailed in
Table 7.34 is modified from Pattee and Neveln (1975) and Lindberg et al.
(1972). Modifications to the published procedures include the use of
recombinant lysostaphin Ambicin L (obtained from Applied
Microbiology, New York or Alpin and Barrett, Trowbridge, Wilts) and the
recommendation for additional phenol extraction steps (the need for the
latter varies from one strain to another).
Recently we have used a Bacterial Genomic Kit (catalogue number
AG85171 from AGTC, Gaithersburg, MD) for pullfying genomic DNA
from S. uureus with the modification of adding lysostaphin to 200 pg ml-’
to the spheroplast buffer. This procedure is very quick and obviates the
need for phenol extraction.
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