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. 2001 Feb 1;29(3):683-92.
doi: 10.1093/nar/29.3.683.

Combined Transcriptome and Proteome Analysis as a Powerful Approach to Study Genes Under Glucose Repression in Bacillus Subtilis

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Free PMC article

Combined Transcriptome and Proteome Analysis as a Powerful Approach to Study Genes Under Glucose Repression in Bacillus Subtilis

K Yoshida et al. Nucleic Acids Res. .
Free PMC article

Abstract

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Figures

Figure 1
Figure 1
Glucose-repressive gene products on 2D gels induced in B.subtilis cells during growth in DSM. Total proteins from strain 1A250 (wild-type) (A) and strain 1A147 (ccpA1) (B) grown in DSM to an A600 of 1.0 with and without 10 mM glucose (Glc) were subjected to 2D gel electrophoresis as described in the text. Identification of the proteins in 11 glucose-sensitive spots, which are indicated by red arrowheads and numbers, is shown in Table 1. The 2D gel patterns (B) for strain 1A147 cells indicated that out of the 11 spots, four were still glucose-sensitive (indicated by red arrowheads), whereas the others (green arrowheads) had become glucose-insensitive due to the ccpA1 mutation.
Figure 1
Figure 1
Glucose-repressive gene products on 2D gels induced in B.subtilis cells during growth in DSM. Total proteins from strain 1A250 (wild-type) (A) and strain 1A147 (ccpA1) (B) grown in DSM to an A600 of 1.0 with and without 10 mM glucose (Glc) were subjected to 2D gel electrophoresis as described in the text. Identification of the proteins in 11 glucose-sensitive spots, which are indicated by red arrowheads and numbers, is shown in Table 1. The 2D gel patterns (B) for strain 1A147 cells indicated that out of the 11 spots, four were still glucose-sensitive (indicated by red arrowheads), whereas the others (green arrowheads) had become glucose-insensitive due to the ccpA1 mutation.
Figure 2
Figure 2
Logarithmic-scale plots of normalized spot intensities (arbitrary units). Spot intensities were normalized after subtracting their backgrounds, i.e. the average spot intensities of 12 mammal gene spots. After substituting the intensities of less than 10 with that of 10, which was less than the standard deviations of their backgrounds, the intensities were plotted logarithmically. The correlation coefficient is indicated by r. (A) RNA of strain 1A250 (wild-type) grown with glucose was used for both Cy3 and Cy5 cDNA-labeling. After hybridization of microarrays with the Cy3 and Cy5 probes, the respective spot intensities for 4100 genes, calculated as above, were plotted. The backgrounds for Cy3 and Cy5 spot intensities were 244.6 ± 42.7 and 278.2 ± 74.4, respectively. (B) RNA of strain 1A250 (wild-type) grown with and without glucose (Glc) was used for Cy3- and Cy5-labeling, respectively. The backgrounds for Cy3 and Cy5 spot intensities were 189.3 ± 30.4 and 249.5 ± 46.7, respectively. (C) RNA of strain 147 (ccpA1) grown with and without glucose was used for Cy3- and Cy5-labeling, respectively. The backgrounds for Cy3 and Cy5 spot intensities were 186.3 ± 21.5 and 206.7 ± 31.4, respectively.
Figure 3
Figure 3
Pseudocolor imaging of glucose-repressive genes. Cy5[–glucose (Glc)] and Cy3 (+ glucose) spot intensities are denoted in red and green, respectively. The red and green images were overlaid, producing pseudocolors for all the spots. Red and green spots correspond to glucose-repressive and -inducible genes, respectively. The 12 iol genes are indicated by white arrowheads. (A) RNA of strain 1A250 (wild-type) grown with and without glucose was used for Cy3- and Cy5-labeling of their cDNAs, respectively (the same analysis as in Fig. 2B). (B) RNA of strain 1A147 (ccpA1) grown with and without glucose was used for Cy3- and Cy5-labeling of their cDNAs, respectively (the same analysis as in Fig. 2C).

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