A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein

Yongchang Zhang, Rongsui Gao, Huiyan Ye, Qingjing Wang, Youjun Feng

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Protein Cell ›› 2014, Vol. 5 ›› Issue (12) : 928-939. DOI: 10.1007/s13238-014-0107-3
RESEARCH ARTICLE

A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein

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Abstract

Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single geneticallyrecessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

Keywords

FadR / fatty acid metabolism / crosstalk

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Yongchang Zhang, Rongsui Gao, Huiyan Ye, Qingjing Wang, Youjun Feng. A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein. Protein Cell, 2014, 5(12): 928‒939 https://doi.org/10.1007/s13238-014-0107-3

References

[1]
Campbell JW, Cronan JEJr (2001) Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene. J Bacteriol 183: 5982−5990
CrossRef Google scholar
[2]
Clark D (1981) Regulation of fatty acid degradation in Escherichia coli: analysis by operon fusion. J Bacteriol 148: 521−526
[3]
Clark D, Cronan J (2005) Two-carbon compounds and fatty acids as carbon sources. In: Bock A, Curtiss III R, Karper J, Karp P, Neidhardt F, Nystrom T, Slauch J, Squiress C, Ussery D (eds) EcoSal−Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, DC
[4]
Clark DP, DeMendoza D, Polacco ML, Cronan JE Jr (1983) Betahydroxydecanoyl thio ester dehydrase does not catalyze a ratelimiting step in Escherichia coli unsaturated fatty acid synthesis. Biochemistry 22: 5897−5902
CrossRef Google scholar
[5]
Cronan JE Jr (1997) In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor. J Bacteriol 179: 1819−1823
[6]
Cronan JE Jr, Subrahmanyam S (1998) FadR, transcriptional coordination of metabolic expediency. Mol Microbiol 29: 937−943
CrossRef Google scholar
[7]
DiRusso CC, Heimert TL, Metzger AK (1992) Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A. J Biol Chem 267: 8685−8691
[8]
DiRusso CC, Metzger AK, Heimert TL (1993) Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR. Mol Microbiol 7: 311−322
CrossRef Google scholar
[9]
DiRusso CC, Tsvetnitsky V, Hojrup P, Knudsen J (1998) Fatty acyl-CoA binding domain of the transcription factor FadR. Characterization by deletion, affinity labeling, and isothermal titration calorimetry. J Biol Chem 273: 33652−33659
CrossRef Google scholar
[10]
Farewell A, Kvint K, Nystrom T (1998) Negative regulation by RpoS: a case of sigma factor competition. Mol Microbiol 29: 1039−1051
CrossRef Google scholar
[11]
Feng Y, Cronan JE (2009a) Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. J Biol Chem 284: 29526−29535
CrossRef Google scholar
[12]
Feng Y, Cronan JE (2009b) A new member of the Escherichia coli fad regulon: transcriptional regulation of fadM (ybaW). J Bacteriol 191: 6320−6328
CrossRef Google scholar
[13]
Feng Y, Cronan JE (2010) Overlapping repressor binding sites result in additive regulation of Escherichia coli FadH by FadR and ArcA. J Bacteriol 192: 4289−4299
CrossRef Google scholar
[14]
Feng Y, Cronan JE (2011a) Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters. Mol Microbiol 80: 195−218
CrossRef Google scholar
[15]
Feng Y, Cronan JE (2011b) The Vibrio cholerae fatty acid regulatory protein, FadR, represses transcription of plsB, the gene encoding the first enzyme of membrane phospholipid biosynthesis. Mol Microbiol 81: 1020−1033
CrossRef Google scholar
[16]
Feng Y, Cronan JE (2012) Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PloS one 7: e46275
CrossRef Google scholar
[17]
Feng Y, Li M, Zhang H, Zheng B, Han H, Wang C, Yan J, Tang J, Gao GF (2008) Functional definition and global regulation of Zur, a zinc uptake regulator in a Streptococcus suis serotype 2 strain causing streptococcal toxic shock syndrome. J Bacteriol 190: 7567−7578
CrossRef Google scholar
[18]
Feng Y, Napier BA, Manandhar M, Henke SK, Weiss DS, Cronan JE (2014) A Francisella virulence factor catalyses an essential reaction of biotin synthesis. Mol Microbiol 91: 300−314
CrossRef Google scholar
[19]
Gui L, Sunnarborg A, LaPorte DC (1996) Regulated expression of a repressor protein: FadR activates iclR. J Bacteriol 178: 4704−4709
[20]
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177: 4121−4130
[21]
Haldimann A, Wanner BL (2001) Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structurefunction studies of bacteria. J Bacteriol 183: 6384−6393
CrossRef Google scholar
[22]
He XY, Yang SY, Schulz H (1997) Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli. Eur J Biochem 248: 516−520
CrossRef Google scholar
[23]
Henry MF, Cronan JE Jr (1992) A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding. Cell 70: 671−679
CrossRef Google scholar
[24]
Hughes KT, Simons RW, Nunn WD (1988) Regulation of fatty acid degradation in Escherichia coli: fadR superrepressor mutants are unable to utilize fatty acids as the sole carbon source. J Bacteriol 170: 1666−1671
[25]
Iram SH, Cronan JE (2005) Unexpected functional diversity among FadR fatty acid transcriptional regulatory proteins. J Biol Chem 280: 32148−32156
CrossRef Google scholar
[26]
Iram SH, Cronan JE (2006) The beta-oxidation systems of Escherichia coli and Salmonella enterica are not functionally equivalent. J Bacteriol 188: 599−608
CrossRef Google scholar
[27]
Maloy SR, Nunn WD (1981) Role of gene fadR in Escherichia coli acetate metabolism. J Bacteriol 148: 83−90
[28]
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
[29]
Miller JH (1992) A short course in bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor
[30]
Nunn WD, Giffin K, Clark D, Cronan JE Jr (1983) Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli. J Bacteriol 154: 554−560
[31]
van Aalten DM, DiRusso CC, Knudsen J, Wierenga RK (2000) Crystal structure of FadR, a fatty acid-responsive transcription factor with a novel acyl coenzyme A-binding fold. Embo J 19: 5167−5177
CrossRef Google scholar
[32]
van Aalten DM, DiRusso CC, Knudsen J (2001) The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR. Embo J 20: 2041−2050
CrossRef Google scholar
[33]
Xu Y, Heath RJ, Li Z, Rock CO, White SW (2001) The FadR.DNA complex. transcriptional control of fatty acid metabolism in Escherichia coli. J Biol Chem 276: 17373−17379
CrossRef Google scholar

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