Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.Cyanobacteria present a diverse group of Gram-negative bacteria capable of oxygenic photosynthesis (Margulis, 1975). Their two photosystems, as well as other genetic and morphological similarities, identified them as putative predecessors of chloroplasts of eukaryotic plants (Wallace, 1982; Pakrasi, 1995). The structural similarities of cyanobacteria and chloroplasts are reflected in part by equivalence of biochemical pathways and their components. For instance, cyanobacterial fatty acid and glycerolipid compositions closely resemble those of the inner envelope and thylakoid membranes of chloroplasts (Roughan et al., 1980; Heinz and Roughan, 1983). In cyanobacteria, as well as in chloroplasts, fatty acids are synthesized by a type II fatty acid synthase (FAS) complex utilizing a freely dissociable acyl carrier protein (ACP; Froehlich et al., 1990). The products of FAS are released as acyl ACPs and may serve directly as substrates for acyltransferases, incorporating the fatty acids into membrane lipids (Frentzen et al., 1983). The substrate specificity of the acyltransferases establishes in cyanobacteria as well as in plastids the typical prokaryotic fatty acid pattern characterized by C16 fatty acids esterified to the sn-2 position. The correspondence of metabolic pathways between cyanobacteria and chloroplasts is reflected by the shared presence of closely related enzymes that catalyze key reactions. Besides the many similarities, however, there are also clear discrepancies that in part account for the fact that cyanobacteria are unicellular organisms, whereas chloroplasts are embedded in the metabolism of a eukaryotic cell. In terms of lipid metabolism, such differences become obvious if one considers the fact that the plastidial FAS also supplies the extraplastidic compartment with fatty acids (Browse et al., 1986). Fatty acid export from the chloroplast necessitates the release of synthesized acyl chains from ACP to allow transport across both envelope membranes. The release is achieved by the action of acyl-ACP thioesterases that hydrolyze the acyl-ACP thioester to liberate the fatty acid (Voelker et al., 1997). In cyanobacteria such export would obviously result in an unfavorable loss of fatty acids, and consequently homologous proteins to acyl-ACP thioesterases cannot be found here. Whereas cyanobacteria seem to be unable to release fatty acids enzymatically from their activated state, all cyanobacterial genomes available to date encode an activity most likely responsible for the activation of free fatty acids. The respective sequences are annotated as acyl-CoA synthetases. Conserved motifs in the amino acid sequence identify these proteins as members of the well-established superfamily of AMP-binding proteins. This protein family comprises several hundred amino acid sequences spreading across all organisms analyzed so far. The family members are annotated in the PROSITE database under entry number PS00455. Although these predicted fatty acid-activating enzymes of cyanobacteria are annotated as acyl-CoA synthetases due to their sequence similarity to proteins with such enzymatic activity, there is a much higher degree of similarity to certain AMP-binding proteins of plant origin with less-well-established function. These plant proteins are predicted to reside in chloroplasts and one member of this subgroup from Arabidopsis (Arabidopsis thaliana) designated as AAE15 was recently described as acyl-ACP synthetase. The conclusions were based on the comparison of enzymatic activity between plant extracts of wild-type and knockout mutant lines (Koo et al., 2005). Whereas the biological role of this activity remained largely elusive, it was shown that the capacity of plant extracts to elongate supplied medium fatty acids depended on AAE15 activity. Since the elongation of medium chain fatty acids in the plastid depends on the FAS requiring acyl ACPs, it was concluded that the fatty acids must have been activated by ACP. The elongated fatty acids ultimately appeared in membrane lipids. Together these findings suggested that AAE15 is an acyl-ACP synthetase.Besides encoding a protein homologous to AAE15 from Arabidopsis, cyanobacteria are also able to utilize exogenous fatty acids like it was shown for isolated chloroplasts. It is well established that feeding different cyanobacteria with free fatty acids results in the incorporation of these fatty acids into membrane lipids. For this process the activation of the fatty acids is believed to be essential. This causal relationship was clearly shown at least for other unicellular organisms like Escherichia coli and yeast (Saccharomyces cerevisiae) where the deletion of acyl-CoA synthetase activity resulted in the inability to utilize exogenous fatty acids (Overath et al., 1969; Knoll et al., 1995). It is not easy to assess how regularly cyanobacterial cells are exposed to exogenous free fatty acids in nature but at least for marine strains this is most likely a rather artificial situation. Therefore, it can be speculated that the capacity to activate free fatty acids might be of different relevance in the lipid metabolism of cyanobacteria in vivo.In this article, we investigated the fatty acid metabolism of cyanobacteria. We isolated candidate genes potentially encoding enzymes involved in fatty acid activation from the strains Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Synechococcus elongatus PCC 7942 (hereafter Synechococcus) and performed heterologous expression in E. coli. The recombinant proteins were shown to possess acyl-ACP synthetase activity with broad substrate specificity. Knockout mutant strains deficient in acyl-ACP synthetase activity were characterized by secretion of endogenous free fatty acids into the culture medium. Combined with labeling experiments, the results suggest an essential role for acyl-ACP synthetase in fatty acid recycling in cyanobacteria.     

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.     

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.     

异染色质相关蛋白TRIM28调控锌指蛋白和原钙黏蛋白家族基因的转录

目的 TRIM28是一种异染色质相关蛋白,通过和SETDB1、HP1相互作用参与H3K9me3修饰的建立,本文旨在更深入地研究TRIM28的相关功能。方法 本文利用CRISPR/Cas9技术、染色质免疫共沉淀技术、免疫印迹技术和实时荧光定量PCR技术,建立HEK293F Trim28基因敲除细胞系,分析一系列实验数据结果。结果 Trim28主要抑制内源表达水平较低的基因转录,进一步分析发现Trim28调控锌指蛋白家族基因和原钙黏蛋白β家族基因的转录。在Trim28敲除细胞系中,锌指蛋白家族基因H3K27ac修饰、H3K4me1修饰和H3K4me3修饰都显著上升,H3K9me3修饰下降。原钙黏蛋白β家族基因的H3K4me3修饰显著上升,H3K9me3修饰下降。结论 这些结果提示TRIM28通过改变染色质的开放程度调控锌指蛋白和原钙黏蛋白β家族基因的转录,为更深入研究TRIM28的功能提供了新的思路。     

Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.Capsule polysaccharide (CPS) of Gram-positive bacteria, external to the cell wall, provides resistance to phagocytosis. CPS in Streptococcus pneumoniae is the most important virulence factor and the target of pneumococcal vaccines (2). Ninety individual CPS serotypes have been recognized so far by immunological and chemical techniques (9). Each has a structurally distinct CPS, composed of repeating oligosaccharide units joined by glycosidic linkages. The components of the repeat units are transferred from nucleoside diphosphate (NDP) derivatives. Among the 54 identified CPS structures, several sugars and related compounds have been found. Seven NDP-monosaccharide precursors (glucopyranose, N-acetylglucosamine, galactopyranose, N-acetylgalactosamine, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose, ribitol-phosphate, and phosphorylcholine) are available from housekeeping metabolic pathways, and the biosynthetic genes for 14 NDP-monosaccharide precursors were found in the pneumococcal cps loci. Among the 14 components, the pathways of five (NDP-d-mannitol, NDP-d-arabinitol, NDP-ribofuranose [Rib], CDP-glycerol [CDP-Gro], and NDP-2-glycerol) are putative and have not yet been identified (1, 4).Glycerol-2-phosphate is rarely present in bacteria and has been found in S. pneumoniae types 15A and 23F. The NDP-2-glycerol biosynthetic pathway has been proposed to include three enzymes: Gtp1, Gtp2, and Gtp3. Gtp3 has been proposed to be a glyceraldehyde-2-phosphotransferase and to be involved in the synthesis of glyceraldehyde-2-phosphate from glyceraldehyde. Gtp1, a putative dehydrogenase, has been proposed to be responsible for the conversion of glyceraldehyde-2-phosphate to glycerol-2-phosphate. The last step of the synthesis of CDP-2-glycerol is catalyzed by the putative glycerol-2-phosphate cytidyltransferase Gtp2 (14). The three genes, gtp1, gtp2, and gtp3, have also been found to be present in the cps loci of S. pneumoniae serotypes 15B, 15C, 15F, 23A, 23B, 28A, and 28F (4). However, the biosynthetic pathway for NDP-2-glycerol has never been identified by molecular and biochemical methods.In this study, we found that the enzymes were not reactive by the previously proposed CDP-2-glycerol biosynthetic pathway. Therefore, a new pathway was proposed, and the three enzymes, Gtp1, Gtp2, and Gtp3, were identified and confirmed biochemically as 1,3-dihydroxyacetone/glyceraldehyde reductase, glycerol-2-phosphate cytidylyltransferase, and glycerol-2-phosphotransferase, respectively. This is the first report on the characterization of the CDP-2-glycerol biosynthetic pathway.     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-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. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.     

Pollination-Induced Expression of Ethylene Biosynthetic Genes at Transcription Level in Orchid Flowers

The authors investigated pollination-induced ethylene production and expression patterns of genes encoding 1-aminocyclopropane-l-carboxylate (ACC) synthase and ACC oxidase in orchid flowers (Doritaenopsis hybrida Hort. ). Following pollination both ACC synthase and ACC oxidase mRNAs were detected in the different organs of flowers, and the patterns of both ACC synthase and ACC oxidase mRNA accumulation were similar, mRNA accumulation of ACC synthase mRNA was more organ-specific than that of ACC oxidase mRNA. However, ACC oxidase mRNAs were much more abundant than ACC synthase mRNAs in the flower organs.     

Ca2+ Binding/Permeation via Calcium Channel,CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein,CD36, and Fatty Acid Metabolism

Ca²⁺ permeation and/or binding to the skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) facilitates activation of Ca²⁺/calmodulin kinase type II (CaMKII) and Ca²⁺ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α₁ subunit of Ca_V1.1) gene that abolishes Ca²⁺ binding within the Ca_V1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that Ca_V1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Ca_v1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized Ca_V1.1-mediated pathway that regulates energy utilization in skeletal muscle.     

Distribution of Plasmid-Borne Stress Protein Genes in Streptococcus thermophilus and Other Lactic Acid Bacteria

The presence of heat stress protein genes (hsp) was tested by Southern hybridization analysis in total DNA extracts from species of the genus Streptococcus (47 strains), Lactobacillus (34 strains), Lactococcus (24 strains), and Leuconostoc (5 strains). The biotinylated hsp16.4 probe prepared from an ORF2 fragment of pER341 (2.8 kb) tested positively with restricted DNA extracts of seven Streptococcus thermophilus strains and a single strain of Lactococcus lactis subsp. cremoris. In all positive S. thermophilus strains, the hsp was located on plasmids ranging from ca. 2.8 kb to 11 kb in size, while hsp was present in a 7.5-kb plasmid in Lactococcus lactis subsp. cremoris. Southern blots with a rep probe showed that all hsp16.4 ⁺ plasmids in S. thermophilus strains also shared homology with the replication function (rep) of pER341, suggesting the common origin of these plasmids. Received: 18 July 1998 / Accepted: 19 August 1998     

Wnt-Lrp5 Signaling Regulates Fatty Acid Metabolism in the Osteoblast

The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism.     

Expression of Streptococcus pneumoniae Virulence-Related Genes in the Nasopharynx of Healthy Children

Colonization and persistence in the human nasopharynx are prerequisites for Streptococcus pneumoniae disease and carriage acquisition, which normally occurs during early childhood. Animal models and in vitro studies (i.e. cell adhesion and cell cytotoxicity assays) have revealed a number of colonization and virulence factors, as well as regulators, implicated in nasopharyngeal colonization and pathogenesis. Expression of genes encoding these factors has never been studied in the human nasopharynx. Therefore, this study analyzed expression of S. pneumoniae virulence-related genes in human nasopharyngeal samples. Our experiments first demonstrate that a density of ≥10⁴ CFU/ml of S. pneumoniae cells in the nasopharynx provides enough DNA and RNA to amplify the lytA gene by conventional PCR and to detect the lytA message, respectively. A panel of 21 primers that amplified S. pneumoniae sequences was designed, and their specificity for S. pneumoniae sequences was analyzed in silico and validated against 20 related strains inhabitants of the human upper respiratory tract. These primers were utilized in molecular reactions to find out that all samples contained the genes ply, pavA, lytC, lytA, comD, codY, and mgrA, whereas nanA, nanB, pspA, and rrgB were present in ∼91–98% of the samples. Gene expression studies of these 11 targets revealed that lytC, lytA, pavA and comD were the most highly expressed pneumococcal genes in the nasopharynx whereas the rest showed a moderate to low level of expression. This is the first study to evaluate expression of virulence- and, colonization-related genes in the nasopharynx of healthy children and establishes the foundation for future gene expression studies during human pneumococcal disease.