beta-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissociated (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region of the Escherichia coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymatic defect in fabH1 strains. The NruI-HindIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelectric point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the amino-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overproduction of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1992

Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12.

The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.

Amino Acid Sequence↗

PubMed1992

Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB operons of Escherichia coli K-12.

The glp regulon of Escherichia coli encodes the proteins required for utilization of sn-glycerol 3-phosphate and its precursors. Transcription of the divergently transcribed glpTQ and glpACB operons is initiated at sites separated by 132 base pairs (bp) of DNA. These operons are controlled negatively by glp repressor and positively by the cAMP-cAMP receptor protein (CRP) complex. The locations of the binding sites for the glp repressor and for cAMP.CRP in the control regions of these operons were determined by DNase I footprinting. Binding of the glp repressor protected the region -32 to -51 (OT) in the glpTQ promoter, which was also the binding site for cAMP.CRP. Four repressor binding sites (-41 to -60 (OA1), -9 to -28 (OA2), +12 to -8 (OA3), and +52 to +33 (OA4)) and two cAMP.CRP binding sites (+11 to -11 and -30 to -51) were found in the glpACB promoter region. Comparison of the sequences of the repressor binding sites found in the glpTQ-glpACB control region with those operators previously described in the glpD operon allowed formulation of a consensus operator sequence which was the palindrome 5'-WATGTTCGWTAWC-GAACATW-3' (W is A or T). The role of each operator was assessed by measuring repression in constructs where individual operators were altered by site-directed mutagenesis. Alteration of OT did not significantly decrease repression of either operon. Each of the glpACB operators contributed to repression of both operons. These results suggest involvement of glpACB operator(s) in control of glpTQ expression perhaps via formation of a repression loop. Evidence supporting this hypothesis was obtained by measuring the degree of repression of the glpTQ promoter in constructs containing 6- or 10-bp insertions between the glpTQ and glpACB operators. A 6-bp insertion located within OA2 or between OT and OA1 eliminated repression of the glpTQ promoter, whereas significant repression was maintained in the case of a 10-bp insertion within OA2.

Base Sequence↗

PubMed1992

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli.

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain. Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed1992

Physical locations of genes in the rne (ams)-rpmF-plsX-fab region of the Escherichia coli K-12 chromosome.

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Bacterial Proteins↗

PubMed1991

Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli.

The nucleotide sequences of the glpQ and ugpQ genes of Escherichia coli, which both encode glycerophosphoryl diester phosphodiesterases, were determined. The glpQ gene encodes a periplasmic enzyme of 333 amino acids, produced initially with a 25 residue long signal sequence, while ugpQ codes for a cytoplasmic protein of 247 amino acids. Despite differences in size and cellular location, significant similarity in the primary structures of the two enzymes was found suggesting a common evolutionary origin. The 3' end of the ugpQ gene overlaps an open reading frame that is transcribed in the opposite direction. This open reading frame encodes a polypeptide with an unusual composition, i.e., 46 of the 146 amino acids are Gln or Asn. This polypeptide and the UgpQ protein were identified in an in vitro transcription/translation system as proteins with apparent molecular weights of 19.5 and 27 kDa, respectively.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12.

Aerobic sn-glycerol 3-phosphate dehydrogenase, encoded by the glpD gene of Escherichia coli, is a cytoplasmic membrane-associated respiratory enzyme. The nucleotide sequence of glpD was determined. An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence. The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a glpD-lacZ translational fusion. The predicted molecular weight, 56,750, corresponds well with the reported value of 58,000 for purified sn-glycerol 3-phosphate dehydrogenase. The flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins from E. coli. Repetitive extragenic palindromic sequences were identified downstream of the glpD coding region. The site for transcription termination was located between 87 and 216 bp downstream of the translation stop codon.

Aerobiosis↗

PubMed1988

Purification and characterization of glpQ-encoded glycerophosphodiester phosphodiesterase from Escherichia coli K-12.

Periplasmic glycerophosphodiester phosphodiesterase (EC 3.1.4.2) of Escherichia coli was purified seven-fold to near homogeneity from the cold osmotic shock fraction of a strain harboring a multicopy plasmid carrying the glpQ gene. The enzyme had a minimum subunit molecular weight of 40,000 as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme was 70,000 as assessed by gel filtration chromatography and 75,000 as assessed by nondenaturing gradient polyacrylamide gel electrophoresis, indicating that the native state of the enzyme is dimeric. The enzyme hydrolyzed the deacylation products of all glycerophospholipids tested including glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoinositol, and glycerophosphoserine. The enzyme did not release glycerol or sn-glycerol 3-phosphate from phosphatidyl-DL-glycerol or lysophosphatidyl-DL-glycerol present in Triton X-100 micelles. The enzyme functioned optimally at pH 7.8. The enzyme was totally inactivated by dilution into 1 mM ethylenediaminetetraacetate or ethylene glycol bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Activity was restored by the addition of Ca2+ or Cd2+, and was partially restored by the addition of Mn2+ or Cu2+. Co2+, Mg2+, Zn2+, and Fe2+ did not restore activity. The presence of calcium ions decreased the Km of the enzyme for the substrate, glycerophosphoglycerol, and increased the Vmax.

Cadmium↗

PubMed1988

Structures of the promoter and operator of the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12.

The nucleotide sequence of a 690-base-pair DNA segment containing the control region for the glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase was determined. An ATG translation initiation codon with an adjacent ribosome-binding site was found which preceded an open reading frame continuing 61 codons to the end of the DNA that was sequenced. The start site for transcription, identified by using primer extension analysis, was located 42 base pairs upstream from the proposed Met start codon. The transcription start site was preceded by a region containing typical -10 and -35 sequences found in bacterial promoters. A binding site for the cyclic AMP-cyclic AMP receptor protein complex (identified by comparison with the consensus-binding sequence and verified by using DNase I footprinting) was located just upstream from the -35 sequence, centered at position -63. The interaction site for the glp repressor was identified by using DNase I footprinting. It consisted of a 49-base-pair region which started at the -10 sequence and continued to position +38. This region contained two directly repeated sequences, each possessing hyphenated dyad symmetry, which suggests that the operator is tandemly repeated. The presence of two adjacent operators may explain why expression of the glpD gene is the most sensitive to repressor when compared with expression of the other operons that are members of the glp regulon.

Aerobiosis↗

PubMed1987

Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12.

The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.

Escherichia coli↗

PubMed1987

Cloning and characterization of the aerobic sn-glycerol-3-phosphate dehydrogenase structural gene glpD of Escherichia coli K-12.

The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a lambda glpD transducing phage. The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes. The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment. Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene. The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments. The glpD gene product, a polypeptide of Mr 55,000, was detected in a maxicell system. Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates. The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site. Determination of the apparent Mr of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region. The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded beta-galactosidase activity. The glpD3 mutation was mapped within the glpD gene, providing additional evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.

Aerobiosis↗

PubMed1987

Divergent transcription of the sn-glycerol-3-phosphate active transport (glpT) and anaerobic sn-glycerol-3-phosphate dehydrogenase (glpA glpC glpB) genes of Escherichia coli K-12.

The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12. The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work. Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens. Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA. Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines. A fine-structure deletion map of the glpT gene was thus constructed. All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT. This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently. Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions. A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase. These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E. coli.

Alleles↗

PubMed1987

Reducing the handling of p.r.n. doses in a unit dose drug distribution system.

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Medication Systems↗

PubMed1986

Reconstitution of sugar phosphate transport systems of Escherichia coli.

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.

Anions↗