Fig. 3.

Expression of genes involved in raffinose uptake. Levels of gene expression were quantitated by qRT-PCR using the standard curve method and normalized to levels of cyclophilin D (SPD_1367 in the D39 genome). Genes examined were as follows: (A) rafK: raffinose ABC transporter ATP-binding protein; (B) aga: alpha-galactosidase; (C) rafG: raffinose ABC transporter permease; (D) rafR: potential raffinose transport transcriptional activator; (E) dldh: dihydrolipoamide dehydrogenase. Strains compared: D39, wild type; DLDH, dldh mutant; P2A1, control mutant that lacks expression of lplA; RafK, rafK deletion with Janus insertion; RafE, rafE interruption resulting in polar effects on the raf operon. Bars represent starting quantity (SQ) and are the combined data from duplicates of two or more experiments. Error bars represent standard deviations. All statistical tests were Student t tests in comparison to data for D39. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ND, not determined. (F) Expression of RafK protein in wild-type and DLDH-negative bacteria (C0832). Cell wall, membrane, and cytosolic fractions from the bacteria were separated by gel electrophoresis, and RafK was detected with anti-RafK antibodies by Western blotting. Lanes: 1, D39 cell wall fraction; 2, C0832 cell wall fraction; 3, D39 membrane fraction; 4, C0832 membrane fraction; 5, D39 cytosolic fraction; 6, C0832 cytosolic fraction.