Biology Reference
In-Depth Information
3. Methods
The methods described below outline (1) the construction of an isogenic
mutant defect in the deacetylation of PIA, (2) different purification and (3)
detection techniques, and (4) chemical analysis of PIA. Bacterial cultures for
each experiment are inoculated from pre-cultures grown overnight at a dilution
of 1:100 and incubated at 37 °C with shaking at 120 rpm for 16 h, unless
otherwise noted.
3.1. Generation of an icaB Mutant in Staphylococcus epidermidis
DNA manipulations were performed by standard procedures for recombinant
DNA to construct a temperature-sensitive plasmid for homologous recombi-
nation
(6)
and are not described here in detail. Briefly, PCR-amplified regions
flanking the
icaB
gene, which encodes a PIA-deacetylating enzyme, and an
erythromycin resistance cassette,
ermB
, were cloned into the plasmid pBT2,
leading to pBT
icaB
. Plasmid pBT
icaB
was transformed into the wild-type
S. epidermidis
strain 1457 by electroporation. Allelic replacement of the
icaB
gene by the erythromycin resistance cassette was performed as described
(4)
.
Successful deletion of
icaB
was verified by sequencing of the genomic DNA
of the putative
icaB
mutant and determination of
icaB
expression by TaqMan
analysis
(4)
. Thewild-type strain
S. epidermidis
1457and its isogenicmutant strain
S. epidermidis
1457
icaB
were used for further investigations (
see
Note 1
).
3.2. Purification of PIA
PIA of staphylococci can be isolated from the cell surface or from the
bacterial culture filtrate. The majority of partially deacetylated PIA is attached
to the bacterial cell wall, whereas completely acetylated PIA (from the
icaB
deletion mutant strain) is released into the culture filtrate
(4)
. In addition, small
amounts of deacetylated PIA released from the cell surface by mechanical shear
force might be detectable in the culture filtrates
(1)
.
3.2.1. Isolation of Crude PIA
Isolation of crude PIA is suitable to screen an extended range of samples
with methods that do not require highly pure PIA preparations.
3.2.1.1. Isolation of Crude Surface-Attached PIA
1. Harvest 1 mL of a 16-h staphylococcal culture by centrifugation at 10,000 ×
g
for 1 min at 4 °C.
2. Wash bacterial cell pellet with 1 mL of PBS buffer and repeat centrifugation step.
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