Bacterial Pathogenesis

For isolation of nucleic acids from Listeriu spp. complete lysis of the bacte-
ria is essential. In contrast to Gram-negative bacteria where lysis is easily
performed using NaOH/SDS, Gram-positive bacteria are particularly
resistant because of the more complex structure of their cell wall peptido-
glycan. Fliss et al. (1991) described the use of mutanolysin, an endo-N-
acetyl muramidase prepared from the culture supernatant of S treptornyces
globisporus strain 1829, as a potential agent in the lysis of Listeriu and other
Gram-positive bacteria. A modified lysis protocol is given in Table 7.20.
An alternative method for the recovery of nucleic acids of Listeria is the
use of bacteriophage-encoded endolysins specific for Lis teria species
(Loessner et al., 1995). Isolation of chromosomal DNA is described in
Table 7.22.
For isolation of plasmids start with 1 ml culture and follow the lysis
protocol described in Table 7.20. Subsequent steps in plasmid extraction
are carried out as for E. coli plasmids (Birnboim and Doly, 1973).
Cloning of DNA fragments in Listeriu requires special cloning vectors
that can be classified into conjugative and non-conjugative vectors. Non-
conjugative vectors are based upon the plasmids pWVOl and pE194
Table 7.20. Lysis of Listeria spp.
1. Inoculate Listeriu spp. into BHI (Sambrook et al., 1989) and incubate at
the appropriate temperature with or without shaking until the culture
has reached the optical density required.
2. Harvest lOml of the culture in centrifuge tubes and centrifuge at
3000xg at 4°C for 10 min. Carefully decant the supernatant.
3. Wash pellet in 5 ml SET buffer (see Table 7.21).
4. Resuspend cell pellet in 5 ml ice cold acetone and chill the cells on ice
for 10 min. Mix gently by inversion from time to time.
5. Centrifuge at 3000 x g at 4°C for 10 min and carefully decant the ace-
tone. Allow the pellet to dry to remove acetone completely.
6. Resuspend the pellet in 5 ml lysis buffer (see Table 7.21). Add 50 pl
proteinase K and 10 fl mutanolysin (see Table 7.21). Incubate the sus-
pension at 37°C for 15-20 min until it becomes clear. Use the lysate for
extraction of chromosomal DNA or isolation of plasmids.
Table 7.2 I. Solutions
0 SET buffer: 50 m~ NaC1,30 m~ Tris-HC1,5 m~ EDTA, pH 8.0. Store at
room temperature.
0 Lysis buffer: 50 m~ Tris-HC1, pH 6.5. Store at room temperature.
0 Proteinase K: 20 mg ml-' (Merck, Rahway, NJ, USA). Store at -20°C.
0 Mutanolysin: Resuspend mutanolysin (Sigma, Diesenhofen,
Germany) as described by the manufacturer. Aliquot at 100 U 20 pl"
and store at -20°C.
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