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Transcriptional effects of CRP* expression in Escherichia coli.

Khankal R, Chin JW, Ghosh D, Cirino PC - J Biol Eng (2009)

Bottom Line: Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP.Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase.Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA. khankal@engr.psu.edu

ABSTRACT

Background: Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.

Results: The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.

Conclusion: While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

No MeSH data available.


Related in: MedlinePlus

Genome-wide transcriptional effects of glucose in strain W3110 expressing wild-type CRP, presented as expression ratios for individual genes showing significant differential expression in the presence and absence of glucose (WT G/WT). a) 629 genes show significant changes in expression level in response to the presence of glucose in strain W3110. b) The changes in expression levels of the same genes shown in (a) in CRP* strain PC05, in response to glucose. Gene names and expression levels are given in supplementary Table S2 (see Additional file 2).
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Figure 1: Genome-wide transcriptional effects of glucose in strain W3110 expressing wild-type CRP, presented as expression ratios for individual genes showing significant differential expression in the presence and absence of glucose (WT G/WT). a) 629 genes show significant changes in expression level in response to the presence of glucose in strain W3110. b) The changes in expression levels of the same genes shown in (a) in CRP* strain PC05, in response to glucose. Gene names and expression levels are given in supplementary Table S2 (see Additional file 2).

Mentions: Table 1 summarizes the genome-wide effects of CRP* expression under the conditions tested, while Table 2 lists the average signal values and expression ratios for specific genes mentioned in this paper. Supplementary Table S1 (see Additional file 1) contains signal values for the complete probe set data for the E. coli K-12 genome. Transcriptome analysis of strain W3110 reveals that 629 genes show significant changes in expression level in response to the presence of glucose (comparison between WT G and WT in Figure 1a). 375 of these genes are upregulated by glucose, as depicted in Figure 1a. The complete list of expression levels of the genes that are differentially expressed between WT G and WT is provided in Supplementary Table S2 (see Additional file 2). Catabolic genes, membrane-related components, and sugar transporters (especially non-glucose PTS related enzymes) represent a large portion of genes repressed by glucose. In a study of CRP-dependent gene expression, Gosset and coworkers reported transcriptome analysis of CRP-dependent genes in another E. coli K-12 strain BW25113 [30]. In Table 3 we compare their result to results from our study for common conditions tested (i.e., WT G/WT). While their study did not examine CRP*, this comparison provides an indication of the consistency of glucose-responsive gene expression among different but similar strains. Our comparison focuses on genes involved in central metabolism and shows that the genes which are subject to glucose repression in BW25113 (such as aceA (isocitrate lyase monomer), aldA (aldehyde dehydrogenase A), sdhA (succinate dehydrogenase) and sucA (oxoglutarate dehydrogenase)) are also downregulated in the presence of glucose for W3110. However, not all the genes which are upregulated in the presence of glucose in BW25113 are upregulated in W3110 under the same conditions (examples are aceE (pyruvate dehydrogenase E1 component), guaB (IMP dehydrogenase), rpsQ (30S ribosomal subunit protein S17)). This is likely to be due to differences between these two strains [26,31-33] as well as the differences in experimental methods.


Transcriptional effects of CRP* expression in Escherichia coli.

Khankal R, Chin JW, Ghosh D, Cirino PC - J Biol Eng (2009)

Genome-wide transcriptional effects of glucose in strain W3110 expressing wild-type CRP, presented as expression ratios for individual genes showing significant differential expression in the presence and absence of glucose (WT G/WT). a) 629 genes show significant changes in expression level in response to the presence of glucose in strain W3110. b) The changes in expression levels of the same genes shown in (a) in CRP* strain PC05, in response to glucose. Gene names and expression levels are given in supplementary Table S2 (see Additional file 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743635&req=5

Figure 1: Genome-wide transcriptional effects of glucose in strain W3110 expressing wild-type CRP, presented as expression ratios for individual genes showing significant differential expression in the presence and absence of glucose (WT G/WT). a) 629 genes show significant changes in expression level in response to the presence of glucose in strain W3110. b) The changes in expression levels of the same genes shown in (a) in CRP* strain PC05, in response to glucose. Gene names and expression levels are given in supplementary Table S2 (see Additional file 2).
Mentions: Table 1 summarizes the genome-wide effects of CRP* expression under the conditions tested, while Table 2 lists the average signal values and expression ratios for specific genes mentioned in this paper. Supplementary Table S1 (see Additional file 1) contains signal values for the complete probe set data for the E. coli K-12 genome. Transcriptome analysis of strain W3110 reveals that 629 genes show significant changes in expression level in response to the presence of glucose (comparison between WT G and WT in Figure 1a). 375 of these genes are upregulated by glucose, as depicted in Figure 1a. The complete list of expression levels of the genes that are differentially expressed between WT G and WT is provided in Supplementary Table S2 (see Additional file 2). Catabolic genes, membrane-related components, and sugar transporters (especially non-glucose PTS related enzymes) represent a large portion of genes repressed by glucose. In a study of CRP-dependent gene expression, Gosset and coworkers reported transcriptome analysis of CRP-dependent genes in another E. coli K-12 strain BW25113 [30]. In Table 3 we compare their result to results from our study for common conditions tested (i.e., WT G/WT). While their study did not examine CRP*, this comparison provides an indication of the consistency of glucose-responsive gene expression among different but similar strains. Our comparison focuses on genes involved in central metabolism and shows that the genes which are subject to glucose repression in BW25113 (such as aceA (isocitrate lyase monomer), aldA (aldehyde dehydrogenase A), sdhA (succinate dehydrogenase) and sucA (oxoglutarate dehydrogenase)) are also downregulated in the presence of glucose for W3110. However, not all the genes which are upregulated in the presence of glucose in BW25113 are upregulated in W3110 under the same conditions (examples are aceE (pyruvate dehydrogenase E1 component), guaB (IMP dehydrogenase), rpsQ (30S ribosomal subunit protein S17)). This is likely to be due to differences between these two strains [26,31-33] as well as the differences in experimental methods.

Bottom Line: Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP.Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase.Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA. khankal@engr.psu.edu

ABSTRACT

Background: Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.

Results: The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.

Conclusion: While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.

No MeSH data available.


Related in: MedlinePlus