Fig 5.

(A) Representative RNA hybridization experiments with RNA isolated from C. glutamicum WT, C. glutamicum ΔramA, and C. glutamicum ΔsugR cultivated in minimal medium with 2% (wt/vol) of the indicated carbon sources. The RNA levels of musK, musE, musFGI, and 16S were monitored with DIG-labeled antisense RNA probes. The results of one representative experiment from a series of three independent experiments is shown. (B) Expression of a musF′-′cat fusion in C. glutamicum WT(pET2-PmusF), C. glutamicum ΔramA(pET2-PmusF), and C. glutamicum ΔsugR(pET2-PmusF). As controls, CAT activity was analyzed in C. glutamicum WT(pET2-PmusF-TS), which lacks the musF transcriptional start site, and C. glutamicum WT(pET2), which carries the promoterless cat gene. The reporter gene activity was determined in cell extracts from cultivations in CgC minimal medium with 1% (wt/vol) of each of the indicated carbon sources. The values represent averages and standard deviations from at least three independent experiments. (C) Representative EMSA using hexahistidyl-tagged RamA protein (0 to 7.5 μg) with the 380-bp musF-Pr fragment as a probe (20 ng) and the 211-bp ramBp3b fragment as a negative control (10 ng).