FIG. 2.

Genetic map of GdhR-regulated loci and Northern blot analysis. (A) The position of the cDNA (closed rectangles) corresponding to either up-regulated (d) or down-regulated (a and f) genes in the GdhR-defective strain is indicated with respect to the available genetic map of MC58 (44), an ET-5 strain phylogenetically related to H44/76 (35). Open rectangles locate the NMB2147-, the zwf-, the NMB1965-, the NMB1964-, and the NMB1961-specific probes used in Northern blot (B) and slot blot (C) experiments. The RNA sequences of the palindromic elements mapping in the intercistronic NMB1963-NMB1962 region and at the 3′ end of NMB1958 are reported. (B) Northern blot analysis of GdhR-regulated transcripts in isogenic strains H44/76 and H44/76ΩgdhR. Top, H44/76 (lanes 1, 3, 5, and 7) and H44/76ΩgdhR (lanes 2, 4, 6, and 8) were grown to late logarithmic phase (OD₅₅₀ of 0.8) in complex GC medium. Total RNAs (10 μg) were extracted and analyzed by Northern blotting using the ³²P-labeled NMB2147-, zwf-, NMB1965-, and NMB1961-specific probes shown in panel A. Bars indicate GdhR-regulated transcripts whose approximate sizes were deduced on the basis of the relative migration of 23S and 16S rRNA (arrowheads). Bottom, Ethidium bromide staining of ribosomal RNAs is reported as a loading control. (C) Relative levels of NMB2147-, zwf-, NMB1965-, NMB1964,- and NMB1961-specific mRNAs in strains H44/76 and H44/76ΩgdhR determined by slot blot experiments. For NMB2147, NMB1965, NMB1964, and NMB1961, transcript levels in H44/76 are arbitrarily assumed to be equal to 100%. For zwf the transcript level in H44/76ΩgdhR is assumed to be equal to 100%. Values represent means from three independent experiments, each with triplicate samples. Bars indicate standard deviations.