The Biofilm Exopolysaccharide Polysaccharide Intercellular Adhesin—A Molecular and Biochemical Approach - Bacterial Pathogenesis: Methods and Protocols - page 94

respective genes in other bacteria are similar to those of staphylococci but have
been given different names, such as pga in E. coli and Actinobacillus . PIA is a
positively charged homopolymer of -1,6-linked N -acetylglucosamine (NAG)
residues (3) . The positive charge of PIA is enzymatically introduced by the
IcaB protein through a mechanism of deacetylation (4) . IcaA and IcaD form a
NAG-transferase responsible for the oligomerization of monomeric NAG units.
IcaC is a putative transporter thought to be involved in the export of elongated
PIA strands through the bacteria cell membrane (5) . Our current knowledge
of the chemical structure of PIA, its biological function with regard to biofilm
formation and immune evasion, and the processes involved in the regulation
of PIA expression is based on intensive studies carried out predominantly in
staphylococci. Here we describe a range of techniques to study PIA with an
emphasis on molecular and biochemical techniques applied for staphylococci.
These techniques are likely transferable to investigate PIA-related structures in
other bacteria.

2. Materials
1. Tryptic soy broth (TSB) (Difco, Franklin Lakes, NJ), supplemented with 0.5%
glucose after sterilization by autoclaving.
2. Enzymes: DNase (0.5 mg/mL final concentration), RNase (0.5 mg/mL
final concentration), lysostaphin (0.5 mg/mL final concentration), lysozyme
(0.5 mg/mL final concentration), and proteinase K (4 mg/mL final concentration);
store at -20 °C.
3. Standard (or wash) buffer: phosphate-buffered saline (PBS, 10 m M sodium
phosphate, pH 7.4, 150 m M NaCl) or Tris-buffered saline (TBS, 150 m M NaCl,
10 m M Tris-HCl, pH 7.4), and 0.5 M EDTA, pH 8.0; prepare fresh as required.
4. Ultrafiltration devices: Amicon Ultrafree-MC-YM-10, and Amicon Centriprep-
YM-10 (Millipore Corporation, Bedford, MA).
5. Tangential flow filtration: regenerated cellulose Prep/Scale Spiral Wound TFF-6
cartridge, Prep/Scale Holder, and peristaltic pump (Millipore Corporation).
6. Miscellaneous materials: dialysis membranes ( 10 kDa cut off), skim milk,

∼

spectrophotometer, and nitrocellulose membranes.
7. Miscellaneous chemicals: 2,3,6-trinitrobenzenesulfonic acid (TNBS) and sodium
bicarbonate (NaHCO 3 ).
8. Chromatography equipment: high-performance liquid chromatography (HPLC)
and coupled electro-spray ionization mass spectrometer, Jordi PolarPac WAX
10,000A 300 × 7.8 mm column (Alltech, Deerfield, IL), and HiLoad

26/60 Superdex 200 gel filtration column (Amersham Pharmacia Biotech,

Piscataway, NJ).
9. Staphylococcus epidermidis strains 1457 and 1457 icaB .