Journal of Biological Chemistry
Volume 286, Issue 41, 14 October 2011, Pages 35782-35794
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Gene Regulation
Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator: A CASE STUDY IN SHEWANELLA ONEIDENSIS*

https://doi.org/10.1074/jbc.M111.267963Get rights and content
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Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis ΔhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [13C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

Bacteria
Carbohydrate Metabolism
Computational Biology
Glycolysis
Repressor Protein
Transcription Regulation
HexR
Proteobacteria

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*

This work was supported in part by the United States Department of Energy, Office of Science, as part of the Genomic Science Program under Contract DE-AC05-76RLO with Pacific Northwest National Laboratory (Genomic Science Program Foundational Scientific Focus Area), Sanford-Burnham Medical Research Institute, Lawrence Berkeley National Laboratory Grant DE-SC0004999, National Science Foundation Grant DBI-0850546, Russian Foundation for Basic Research Grant 10-04-01768, and the Russian Academy of Sciences Program Molecular and Cellular Biology.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S4 and Figs. S1–S4.

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Supported by National Science Foundation of China Grants 30970035 and 31070033 and Knowledge Innovation Program of Chinese Academy of Sciences Grant KSCX2-EW-G-5.