Ultrospec 2000 spectrophotometer

Hydrolysis of purified cell walls was measured using an Ultrospec 2000 spectrophotometer (Amersham Biosciences) and following the decrease in turbidity at 450 nm for 15 min at 37 °C in 25 mM Tris–HCl, pH 7.5, and 100 mM NaCl buffer. PG was resuspended at a concentration giving an absorbance at 450 nm of 0.6. Various dilutions of AtlA and its derivatives were tested to identify conditions in which the velocity of hydrolysis was proportional to enzyme concentration. Enzymatic activity was expressed as absorbance at 450 nm units per min per mmol of protein; the residual activity of individual mutants was expressed as the percentage of WT activity.
For zymogram analysis, crude extracts were separated by SDS-PAGE using gels containing 0.2% autoclaved M. lysodeikticus cells. After electrophoresis, the proteins were renatured by incubating the gel for 24 h in 25 mM Tris (pH 8.0) buffer containing 0.1% Triton at 37 °C. Lytic activities could be visualized as clear bands on the opaque SDS-PAGE.

The concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA), as an index of LPO, was measured in tissue homogenates (using a Ultrospec 2000 spectrophotometer, purchased from Amersham Pharmacia Biotech (Uppsala, Sweden)), as described elsewhere [11 (link)]. Protein was measured using the Bradford method [26 (link)].

Overnight cultures of C. jejuni strains were harvested from MH agar plates and suspended in MH broth to an OD₆₀₀ of 0.001 or 0.1. Cultures (20 mL) in 100-mL flasks were incubated at 42°C with shaking under microaerobic conditions. Samples were taken at 2- or 3-h intervals, and the OD₆₀₀ was measured in an Ultrospec 2000 spectrophotometer (Amersham Pharmacia Biotech, USA) or serially diluted in phosphate-buffered saline (PBS). The suspension was serially diluted and plated on MH agar to enumerate CFU. All experiments were repeated three times.