Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

Changes in hepatic branched-chain α-keto acid dehydrogenase activity in response to isoleucine imbalance in growing chickens

Three experiments were carried out to determine the influence of isoleucine imbalance on hepatic branched-chain α-keto acid dehydrogenase (BCKAD) activity in growing chickens. An isoleucine imbalance was induced by adding a 5% imbalancing amino acid mixture to a basal diet that contained adequate concentrations of all indispensable amino acids except isoleucine, which was marginally adequate (0.64–0.76% of the diet). The imbalancing mixture caused depressions (P < 0.05) in feed intake and growth rate. The depression in feed intake appeared to occur prior to the depression in growth rate. The isoleucine concentration in plasma decreased (P < 0.05), but not consistently, among experiments in response to the imbalancing mixture of amino acids. Basal and total activities of hepatic BCKAD were increased (P < 0.05) 21% and 28%, respectively, within 24 hours in one experiment and were elevated (P < 0.05) 19% and 14%, respectively, at the end of the 13 days of a second experiment. The moisture, protein, and fat contents of whole body and liver were not affected by the imbalancing mixture of amino acids. It appears likely that broiler chicks did not adapt to the imbalanced diets because the depressed feed intake and growth rate and alterations in plasma isoleucine and hepatic BCKAD activity persisted through 13 days of experiment. The isoleucine requirement, expressed as percent of diet, was increased by the imbalancing mixture of amino acids, and the efficiency of isoleucine utilization for growth (grams of weight gain per milligram of isoleucine intake) was decreased in two of three experiments. These results suggest that BCKAD may have a play in the increased isoleucine requirement of broiler chicks under conditions of isoleucine imbalance.     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

Molecular basis of maple syrup urine disease: novel mutations at the E1 alpha locus that impair E1(alpha 2 beta 2) assembly or decrease steady-state E1 alpha mRNA levels of branched-chain alpha-keto acid dehydrogenase complex.

We report the occurrence of three novel mutations in the E1 alpha (BCKDHA) locus of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1 alpha gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1 alpha subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1 alpha mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1 alpha subunit impairs its proper assembly with the normal E1 beta. Unassembled as well as misassembled E1 alpha and E1 beta subunits are degraded in the cell.     

Identification of a new nuclear gene (CEM1) encoding a protein homologous to a β-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with β-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains. Implying that the CBM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it Is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741‐1743 (highly homologous to a Pseudomonas aeruginosa acyl‐CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886‐MSMEG_1741‐1743 strictly required oleic acid for growth. The ΔMSMEG_1886‐ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742‐43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.     

Novel SNPs of butyrophilin (<Emphasis Type="Italic">BTN1A1</Emphasis>) and milk fat globule epidermal growth factor (EGF) 8 (<Emphasis Type="Italic">MFG</Emphasis>-<Emphasis Type="Italic">E8</Emphasis>) are associated with milk traits in dairy goat

Butyrophilin (BTN1A1) and milk fat globule epidermal growth factor (EGF) 8 (MFG-E8) genes are both milk fat globule membrane proteins. BTN1A1 plays a key role in the secretion of milk lipid and production which has effects on performance traits, while the MFG-E8 is vital for the development of the mammary gland and phagocytic clearance of apoptotic cells. Therefore, BTN1A1 and MFG-E8 gene are candidate genes for quantitative traits in mammalian animals with respect to milk performance traits. The objective of this study is to investigate variations in goat BTN1A1 and MFG-E8 gene and analyze their associations with growth trait and milk performance. In this study, the goat BTN1A1 gene showed a novel single-nucleotide polymorphism (SNP): XM_001494179:g.8659C>T, resulting in a missense mutation: CTT (Leu)>TTT (Phe) at position 377 aa of the BTN1A1 (526 aa); the goat MFG-E8 gene showed four novel SNPs: NC_007319: g.843delA, 6417delC, 14892T>C and 14996A>C, only the 14892T>C result in a synonymous mutation. The associations between genotypes and production traits were analyzed. Significant statistical results implied that HinfI locus of BTN1A1 gene is associated with milk fat yield (P = 0.004), total solid (P = 0.002), solid-non fat (P = 0.018) and first milk yield (P = 0.030). The DA and EcoRV loci of MFG-E8 gene are associated with milk fat yield (DA locus: P = 0.000; EcoRV locus: P = 0.033) and total solid (DA locus: P = 0.002; EcoRV locus: P = 0.015) in the Xinong Saanen dairy goat.     

Role of Aminotransferase IlvE in Production of Branched-Chain Fatty Acids by Lactococcus lactis subsp. lactis

The objective of this study was to determine the role of a lactococcal branched-chain amino acid aminotransferase gene, ilvE, in the production of branched-chain fatty acids. Lactococcus lactis subsp. lactis LM0230 and an ilvE deletion mutant, JLS450, produced branched-chain fatty acids from amino and α-keto acids at levels above α-keto acid spontaneous degradation and the fatty acids' flavor thresholds. The deletion mutant produced the same amounts of branched-chain fatty acids from precursor amino acids as did the parent. This was not the case, however, for the production of branched-chain fatty acids from the corresponding precursor α-keto acids. The deletion mutant produced a set of fatty acids different from that produced by the parent. We concluded from these observations that ilvE plays a role in the specific type of fatty acids produced but has little influence on the total amount of fatty acids produced by lactococci.     

Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations.

We have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain alpha-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue (-26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus.     

Aptitude of cheese bacteria for volatile S-methyl thioester synthesis I. Effect of substrates and pH on their formation by Brevibacterium linens GC171

The aptitude of resting cells of Brevibacterium linens G171 to synthesize S-methyl thioesters was studied in presence of methanethiol and nine short-chain fatty acids individually or as a mixture. Esterification of acetic, propionic and methyl branched-chain acids occurred with methanethiol alone and was enhanced by fatty acid addition. Addition of n-chain, 3-hydroxybutyric and 2-hydroxyvaleric acids allowed synthesis of n-chain thioesters up to thiocaproate. The kinetics of production and the effect of concentrations of both substrates and of cells were tested. The optimum pH for synthesis varied according to the kind of thioesters produced. Results suggested that thioesters were derived mainly from acyl-CoA from different metabolic breakdowns, such as the degradation of fatty acids or some amino acids, and that several acyltransferases could be involved. Received: 1 August 1996 / Received revision: 10 October 1996 / Accepted: 18 October 1996     

A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins.

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described. The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.     

Fatty acids ofPseudomonas aeruginosa grown on media affecting polymyxin susceptibility

The readily extractable lipids ofPseudomonas aeruginosa 015 were significantly altered by growth on branched-chain amino acids and their acyl derivatives as sole carbon source. These cultural conditions, especially growth on the acyl derivatives, resulted in decreased amounts of unsaturated fatty acids and concomitant increases in cyclopropyl acids. The shift in the ratio of unsaturated to cyclopropyl acids was primarily due to the carbon source and did not correlate to the physiological or temporal age of the culture. Growth on carbon sources catabolized to propionyl-CoA affected the synthesis of odd-chain fatty acids. Growth on substrates metabolized to isobranched carbon skeletons resulted in the synthesis of branched-chain fatty acids. The carbon sources caused minor changes in the cellular percentage and phosphate content of the readily extractable lipids.     

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH)

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (~70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a ΔfabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.     

Variation of Branched-Chain Fatty Acids Marks the Normal Physiological Range for Growth in Listeria monocytogenes

The fatty acid composition of Listeria monocytogenes Scott A was determined by close-interval sampling over the entire biokinetic temperature range. There was a high degree of variation in the percentage of branched-chain fatty acids at any given temperature. The percentage of branched C₁₇ components increased with growth temperature in a linear manner. However, the percentages of iso-C_15:0 (i15:0) and anteiso-C_15:0 (a15:0) were well described by third-order and second-order polynomial curves, respectively. There were specific temperature regions where the proportion of branched-chain fatty acids deviated significantly from the trend established over the entire growth range. In the region from 12 to 13°C there were significant deviations in the percentages of both i15:0 and a15:0 together with a suggested deviation in a17:0, resulting in a significant change in the total branched-chain fatty acids. In the 31 to 33°C region the percentage of total branched-chain components exhibited a significant deviation. The observed perturbations in fatty acid composition occurred near the estimated boundaries of the normal physiological range for growth.