Effects on Baci//us subtilis of conditional expression of the accBC operon encoding subunits of acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis (original) (raw)
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Microbiology-sgm, 1998
A Bacillus subtilis strain was constructed in which the operon accBC, encoding the biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits of acetyl-CoA carboxylase (ACC), was placed under the control of the IPTGinducible promoter spac. A reduction in the levels of BCCP resulted in a decrease of de novo fatty acid synthesis and in the total content of membrane fatty acids. This strain was dependent upon the presence of IPTG for a normal growth phenotype. Growth was specifically restored by supplying exogenous long branched-chain fatty acids in the medium, indicating that the inducerdependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. The strain showed a strong decrease in sporulation frequency when it was induced to sporulate in an IPTG-free medium. Germination and outgrowth were both delayed in spores of the mutant obtained in the absence of IPTG. (1995). The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.
Archives of Microbiology, 2001
Acetyl-CoA carboxylase catalyses the synthesis of malonyl-CoA, the first intermediate in fatty acid (hence phospholipid) synthesis. The accBC operon from Bacillus subtilis codes for two subunits of acetyl-CoA carboxylase, biotin carboxyl-carrier and biotin carboxylase. In this work, the 5′-end of the accBC mRNA was determined by primer-extension, and transcriptional fusion analysis of the accBC promoter was carried out under a variety of growth conditions. A direct correlation between the levels of transcription of the accBC genes and the rate of cellular growth is reported. Consistent results were also obtained in nutritional upshift and downshift experiments.
Journal of Biological Chemistry, 2000
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 ratecontrolling enzyme of this pathway, no direct tests of this proposal in vivo have been reported. We have tested this proposal in Escherichia coli. The genes encoding the four subunits of E. coli ACC were cloned in a single plasmid under the control of a bacteriophage T7 promoter. Upon induction of gene expression, the four ACC subunits were overproduced in equimolar amounts. Overproduction of the proteins resulted in greatly increased ACC activity with a concomitant increase in the intracellular level of malonyl-CoA. The effects of ACC overexpression on the rate of fatty acid synthesis were examined in the presence of a thioesterase, which provided a metabolic sink for fatty acid overproduction. Under these conditions ACC overproduction resulted in a 6-fold increase in the rate of fatty acid synthesis.
Expression of Two Escherichia coli Acetyl-CoA Carboxylase Subunits Is Autoregulated
Journal of Biological Chemistry, 2003
Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid biosynthesis, the synthesis of malonyl-CoA from acetyl-CoA using ATP and bicarbonate. In Escherichia coli and most other bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Prior work from this laboratory showed that the in vivo expression of the accBC operon had a strikingly nonlinear response to gene copy number (Li, S.-J, and Cronan, J. E., Jr. (1993) J. Bacteriol. 175, 332-340) in that the presence of 50 or more copies of the accBC operon resulted in only a 2-3-fold increase in AccB and AccC. We now report that AccB functions to negatively regulate transcription of the accBC operon. Expression of a chimeric protein consisting of the N terminus of E. coli AccB and the C-terminal bioinylation domain of Bacillus subtilis AccB down-regulated transcription of the E. coli accBC operon. A truncated form of AccB consisting of the N-terminal 68 amino acids of E. coli AccB was sufficient to negatively regulate the accBC operon. In vivo bypass of acetyl-CoA carboxylase activity by expression of a malonyl-CoA synthase from Rhizobium trifolii allowed construction of strain deleted for the accA and accB genes. Unexpectedly, the ⌬accB mutation could not be resolved from the ⌬accA mutation. Transcription of the accBC operon in the ⌬accB ⌬accA strain continued well into stationary phase under growth conditions that normally result in greatly decreased transcription. These data support a model in which AccB acts as an autoregulator of accBC operon transcription.
Applied and Environmental Microbiology, 2001
Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded -ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the  and ␣ subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.
Journal of Bacteriology, 2000
A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the β-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of ...
The Biochemical journal, 1996
Acetyl-CoA carboxylase is a rate-limiting enzyme in the biogenesis of long-chain fatty acids. In the present study, the 5' end and flanking region of the acetyl-CoA carboxylase (ACC) gene was analysed in the chicken. A genomic clone was isolated containing the first three exons, the third one containing the ATG codon. Using nuclease-mapping experiments and primer-extension analyses, the transcription-initiation site was located 153 nucleotides upstream of the ATG codon. In contrast with rat ACC gene expression, reverse transcriptase PCR analysis performed on chicken liver mRNA did not reveal alternative splicing in the 5'-untranslated region of these messengers. The promoter region is very G+C rich, and contains no TATA or CAAT boxes. Analysis by transient transfection in a human hepatoma cell line (HepG2) demonstrates that the promoter activity requires the presence of symmetrical sequences located upstream of the GC boxes. Transcription of this gene is found to be controll...
Planta, 1996
The cerulenin-insensitive 13-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C4, C6, C8 and Clo) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 I~M cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS Ill-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC50 = 0.45 ~tM), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata