Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli.

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of the fatty acid synthetic pathway. Although ACC has often been proposed to be a major rate-controlling enzyme of this pathway, no direct tests of this proposal in vivo have been reported. We have tested this proposal in Escherichia coli.

In vivo enzymatic protein biotinylation.

Biotin is biologically active only when protein-bound and is covalently attached to a class of important metabolic enzymes, the biotin carboxylases and decarboxylases. Biotinylation is a relatively rare modification, with between one and five biotinylated protein species found in different organisms. We discuss the mechanism and structures involved in this extraordinarily specific protein modification and its exploitation in tagging recombinant proteins.

Optimal management strategy for use of compression US for deep venous thrombosis in symptomatic patients: a cost-effectiveness analysis.

Rationale And Objectives: The authors' purpose was to identify the optimal strategy for using compression ultrasonography (US) in patients suspected of having deep venous thrombosis (DVT).

Materials And Methods: The authors developed a decision-analytic model representing the natural history of DVT and the benefits and risks of anticoagulation therapy. They evaluated six initial imaging strategies: (a) unilateral examination of the common femoral and popliteal veins; (b) unilateral examination of the common femoral, popliteal, and femoral veins; (c) bilateral examination of the common femoral and popliteal veins; (d) bilateral examination of the common femoral, popliteal, and femoral veins; (e) complete unilateral examination of the symptomatic leg (including calf veins); and (f) complete bilateral examination of both legs.

Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli.

Holo-(acyl carrier protein) synthase (AcpS) post-translationally modifies apoacyl carrier protein (apoACP) via transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 gamma-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth.

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V.

The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity.

Biotin is a coenzyme essential to all life forms. The vitamin has biological activity only when covalently attached to certain key metabolic enzymes. Most organisms have only one enzyme for attachment of biotin to other proteins and the sequences of these proteins and their substrate proteins are strongly conserved throughout nature.

Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli.

Cyclopropane fatty acid (CFA) formation is a post-synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid.

Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal.

N-formyl-methionine termini are formed in the initiation reaction of bacterial protein synthesis and processed during elongation of the nascent polypeptide chain. We report that the formyl group must be removed before the methionine residue can be cleaved by methionine aminopeptidase. This has long been implicitly assumed, but that assumption was based on inconclusive data and was in apparent conflict with more recently published data.

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP.

Detecting hepatic lesions: the added utility of CT liver window settings.

Purpose: To prospectively evaluate the utility of adding computed tomographic (CT) liver windows to conventional soft-tissue windows for the detection of hepatic disease.

Materials And Methods: One of four radiologists experienced in abdominal imaging interpreted 1,175 consecutive abdominal CT scans from one institution. Hepatic images were first interpreted by using standard soft-tissue windows.

Acyl homoserine-lactone quorum-sensing signal generation.

Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme.

Molecular biology of biotin attachment to proteins.

Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification. Both biotin domains and biotin protein ligases are very strongly conserved throughout biology. This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins.

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA.

Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS).

Strains of Escherichia coli carrying mutations at the relA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3',5'-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification.

Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase.

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins.

FadR, transcriptional co-ordination of metabolic expediency.

FadR is an Escherichia coli transcriptional regulator that optimizes fatty acid metabolism in response to exogenously added fatty acids. Many bacteria grow well on long-chain fatty acids as sole carbon source, but at the expense of consuming a useful structural material. Exogenous fatty acids are readily incorporated into membrane phospholipids in place of the acyl chains synthesized by the organism, and phospholipids composed of any of a large variety of exogenously derived acyl chains make biologically functional membranes.

Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli.

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III.

Improved microsurgical anastomotic patency with low molecular weight heparin.

Blood flow to a free flap may be impaired by thrombotic occlusion at the anastomosis or by microemboli occluding microvessels. The purpose of this study was to test the hypothesis that unfractionated heparin (UFH) or low molecular weight fractions of heparin (LMWH) could improve both the patency of microvascular anastomoses and microcirculatory perfusion. Sixty-six rats underwent orthotopic elevation of 3- x 10-cm epigastric free flaps.

Percutaneous biopsy in diffuse renal disease: comparison of 18- and 14-gauge automated biopsy devices.

Purpose: To compare 18-gauge-needle automated biopsy guns to 14-gauge systems for diagnostic efficacy and safety in percutaneous renal biopsy.

Materials And Methods: One hundred sixty-one computed tomographic (CT) guided biopsies for diffuse renal disease were retrospectively reviewed. An automated biopsy gun with an 18-gauge needle was used in 74 procedures, and a 14-gauge needle was used in 87 cases.

Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster.

The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y. Zhang and J.

In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases.

Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in beta-oxidative fatty acid degradation.

A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase.

Bacillus subtilis grown at 37 degrees C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37 degrees C are transferred to 20 degrees C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B.