The FabR (YijC) Transcription Factor Regulates Unsaturated Fatty Acid Biosynthesis in Escherichia coli *

Unsaturated fatty acid biosynthesis is a vital facet of Escherichia coli physiology and requires the expression of two genes, fabA and fabB, in the type II fatty acid synthase system. This study links the FabR (YijC) transcription factor to the regulation of unsaturated fatty acid content through the regulation of fabB gene expression. TheyijC (fabR) gene was deleted by replacement with a selectable cassette, and the resulting strains (fabR::kan) possessed significantly elevated levels of unsaturated fatty acids, particularlycis-vaccenate, in their membrane phospholipids. The altered fatty acid composition was observed in thefabR::kan fabF1 double mutant pinpointing fabB as the condensing enzyme responsible for the increased cis-vaccenate production. ThefabR::kan strains had 4- to 8-fold higher levels of fabB and a 2- to 3-fold increase infabA transcripts as judged by Northern blotting, Affymetrix array analysis, and real-time PCR. FabR did not regulate the enzymes of fatty acid β-oxidation. The elevated level offabB mRNA was reflected by higher condensing enzyme activity in fabR::kan fabF1 double mutants. Thus, FabR functions as a repressor that potently controls the expression of the fabB gene, which in turn, modulates the physical properties of the membrane by altering the level of unsaturated fatty acid production.

[1]  R. Heath,et al.  The FadR·DNA Complex , 2001, The Journal of Biological Chemistry.

[2]  S. Subrahmanyam,et al.  FadR, transcriptional co‐ordination of metabolic expediency , 1998, Molecular microbiology.

[3]  C. Rock,et al.  Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis. , 1996, Biochimica et biophysica acta.

[4]  J. Cronan,et al.  A new mechanism of transcriptional regulation: Release of an activator triggered by small molecule binding , 1992, Cell.

[5]  P. Black,et al.  Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals. , 2000, The Journal of nutrition.

[6]  J. Cronan,et al.  Thermal regulation of membrane fluidity in Escherichia coli. Effects of overproduction of beta-ketoacyl-acyl carrier protein synthase I. , 1983, The Journal of biological chemistry.

[7]  C. DiRusso,et al.  Cloning and characterization of a gene (fadR) involved in regulation of fatty acid metabolism in Escherichia coli , 1985, Journal of bacteriology.

[8]  C. Cantor,et al.  Analysis of DNA Sequences by Hybridization , 2002 .

[9]  J. W. Campbell,et al.  Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. , 2001, Annual review of microbiology.

[10]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[11]  J. Cronan,et al.  Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli. , 1980, The Journal of biological chemistry.

[12]  J. W. Campbell,et al.  Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene , 2001, Journal of bacteriology.

[13]  J. Liu,et al.  Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes. , 2001, Nucleic acids research.

[14]  R. Heath,et al.  Roles of the FabA and FabZ β-Hydroxyacyl-Acyl Carrier Protein Dehydratases in Escherichia coli Fatty Acid Biosynthesis* , 1996, The Journal of Biological Chemistry.

[15]  C. DiRusso,et al.  Analysis of Acyl Coenzyme A Binding to the Transcription Factor FadR and Identification of Amino Acid Residues in the Carboxyl Terminus Required for Ligand Binding (*) , 1995, The Journal of Biological Chemistry.

[16]  D. V. van Aalten,et al.  Crystal structure of FadR, a fatty acid‐responsive transcription factor with a novel acyl coenzyme A‐binding fold , 2000, The EMBO journal.

[17]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[18]  E. Gelmann,et al.  Physical properties of membrane lipids: biological relevance and regulation. , 1975, Bacteriological reviews.

[19]  C. Raetz,et al.  An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. , 1994, The Journal of biological chemistry.

[20]  S. Kauppinen,et al.  β-ketoacyl-ACP synthase I of Escherichia coli: Nucleotide sequence of thefabB gene and identification of the cerulenin binding residue , 1988, Carlsberg research communications.

[21]  J. Cronan,et al.  Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli , 1983, Journal of bacteriology.

[22]  D. Laporte,et al.  Regulated expression of a repressor protein: FadR activates iclR , 1996, Journal of bacteriology.

[23]  D. V. van Aalten,et al.  The structural basis of acyl coenzyme A‐dependent regulation of the transcription factor FadR , 2001, The EMBO journal.

[24]  B. Booth.  Cell surface proteins of E. coli. , 1980, Biochemical and biophysical research communications.

[25]  W. F. Thompson,et al.  Rapid isolation of high molecular weight plant DNA. , 1980, Nucleic acids research.

[26]  C. Rock,et al.  Preparative enzymatic synthesis of acyl-acyl carrier protein. , 1981, Methods in enzymology.

[27]  C. DiRusso,et al.  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. , 1992, The Journal of biological chemistry.

[28]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[29]  P. Black,et al.  Molecular inroads into the regulation and metabolism of fatty acids, lessons from bacteria. , 1999, Progress in lipid research.

[30]  J. Cronan,et al.  Beta-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. , 1980, The Journal of biological chemistry.

[31]  H. Schulz,et al.  Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase , 1988, Journal of bacteriology.

[32]  J. Cronan,et al.  Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation. , 1991, Journal of molecular biology.

[33]  M. Polacco,et al.  β-Hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis , 1983 .

[34]  P. Højrup,et al.  Fatty Acyl-CoA Binding Domain of the Transcription Factor FadR , 1998, The Journal of Biological Chemistry.

[35]  J. Cronan.  In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor , 1997, Journal of bacteriology.

[36]  J. Cronan,et al.  Thermal regulation of membrane lipid fluidity in bacteria , 1983 .

[37]  K. Bloch.  15 β-Hydroxydecanoyl Thioester Dehydrase , 1971 .