Fig 1.

Analysis of aconitase enzyme activity and expression levels in citB mutant strains. (A) Cell extracts were prepared from B. subtilis strains grown in DS medium and analyzed for aconitase specific activity. Data from a representative experiment of two are shown. (B) The concentration of citrate in the culture supernatant during growth in DS medium was determined. The means and standard deviations from two biological replicates are shown. (C) The expression level of a citB-lacZ fusion during growth in DS medium was determined. Samples were taken at the indicated time points, and β-galactosidase activity was measured. Data from a single experiment are shown; all strains were assayed in multiple experiments and gave similar results. (D) Cell extracts were prepared from strains grown in DS medium and analyzed by Western blotting using antibodies specific to Acn and CodY (a loading control). Bands were quantitated, and values are presented as a ratio of Acn to CodY, normalized to the ratio of the citB⁺ strain expression level at 2.5 h. Data from a representative experiment of two are shown; an immunoblot from a single time point (2.5 h) from that experiment is shown in the inset. The strains used were wild-type (KBP94), citB null (AWS174), citB2 (KBP118), and citB7 (KBP81) strains.