Figure 5.

Effect of BCAA on TpxD expression in bacteria challenged with H₂O₂. TpxD synthesis and expression levels were determined by Western blotting (A) and by RT-PCR (B) in D39 grown anaerobically (N) in CDM with (BCAA+) or without (BCAA-) BCAA. Bacteria were grown to OD₆₂₀ = 0.25 and then challenged with 1 mM H₂O₂ for 40 min. Control cultures were incubated without the addition of H₂O₂. Separated proteins were electroblotted onto 0.45 μm nitrocellulose membrane, and stained with Ponceau S to ensure equal loading, before incubation with antibodies. Values are the mean of duplicate determinations of at least two independent experiments, and were normalized to the expression level of D39 grown anaerobically in medium containing BCAA. Bars indicate standard deviation. Significant alterations in gene expression compared to bacteria grown without H₂O₂ were determined using Student's t-test.