Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer.

We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-beta-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Ideally 3H-R-BrP would be transported in plasma, taken up by tissues and activated by the enzyme acyl-CoA synthetase (ACS) like native FFA, but then 3H-labeled metabolites would be trapped. In vitro we found that 2-bromopalmitate and palmitate compete equivalently for the same ligand binding sites on albumin and intestinal fatty acid binding protein, and activation by ACS was stereoselective for the R-isomer. In vivo, oxidative and non-oxidative FFA metabolism was assessed in anesthetized Wistar rats by infusing, over 4 min, a mixture of 3H-R-BrP and [U-14C] palmitate (14C-palmitate). Indices of total FFA utilization (R*f) and incorporation into storage products (Rfs') were defined, based on tissue concentrations of 3H and 14C, respectively, 16 min after the start of tracer infusion. R*f, but not Rfs', was substantially increased in contracting (sciatic nerve stimulated) hindlimb muscles compared with contralateral non-contracting muscles. The contraction-induced increases in R*f were completely prevented by blockade of beta-oxidation with etomoxir. These results verify that 3H-R-BrP traces local total FFA utilization, including oxidative and non-oxidative metabolism. Separate estimates of the rates of loss of 3H activity indicated effective 3H metabolite retention in most tissues over a 16-min period, but appeared less effective in liver and heart. In conclusion, simultaneous use of 3H-R-BrP and [14C]palmitate tracers provides a new useful tool for in vivo studies of tissue-specific FFA transport, utilization and metabolic fate, especially in skeletal muscle and adipose tissue.

Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB.

Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.

Function of the htrB high temperature requirement gene of Escherichia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate.

By assaying lysates of Escherichia coli generated with the hybrid lambda bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), we identified two clones (lambda232 and lambda233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). The E. coli DNA inserts in lambda232 and lambda233 suggested that a known gene (htrB) required for rapid growth above 33 degrees C might encode the lauroyl transferase. Using the intermediate (Kdo)2-lipid IVA as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity. Cells harboring hybrid htrB+ plasmids overproduced transferase activity 100-200-fold. The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)2-lipid IVA. The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis.

Inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

The gene coding for 3-deoxy-manno-octulosonic acid transferase and the rfaQ gene are transcribed from divergently arranged promoters in Escherichia coli.

The gene kdtA in Escherichia coli codes for 3-deoxy-D-manno-octulosonic acid transferase, the enzyme responsible for attachment of the two 3-deoxy-D-manno-octulosonic acid residues that constitute the link between lipid A and the core oligosaccharide of the lipopolysaccharide. Cloning and subsequent sequencing of the region upstream of kdtA revealed an open reading frame identified as the first gene (rfaQ) in an rfa gene cluster. The kdtA and rfaQ transcripts were identified, and the 5' ends of the transcripts were mapped by primer extension. Two main, divergently arranged promoters were found. These promoters generated transcripts with 5' ends separated by 289 bases. That the two divergent transcripts from the identified promoters represent the kdtA and rfaQ transcripts was confirmed by fusing different parts of the intergenic region between the promoterless lacZ and phoA genes in promoter-screening plasmid pCB267.

A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing.

An autoradiographic assay applicable to colonies immobilized on filter paper was developed for obtaining temperature-sensitive mutants of Escherichia coli defective in the transfer of 3-deoxy-D-manno-octulosonic acid (KDO) from CMP-KDO to a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A, designated lipid IVA. Cell-free extracts from two mutants found in a population of 30,000 mutagen-treated cells showed normal KDO transferase activity when assayed at 30 degrees C, but almost no activity at 42 degrees C. The mutation was mapped by mating one of the mutants with different Hfr strains and analyzing genetic linkage of KDO transferase activity to selectable markers. The lesion was located to a position between 80 and 84 min on the E. coli chromosome. A plasmid from the Clarke and Carbon collection (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99), pLC17-24, known to contain genes from the rfa region (81 min), was shown to overexpress KDO transferase activity 4-5 times and to correct the mutation when the plasmid was conjugated into the mutant strains. The KDO transferase gene, designated kdtA, was subcloned from pLC17-24 into a multicopy vector. The resulting plasmid, pCL3, overproduced transferase activity approximately 100-fold. The kdtA gene was shown to code for a 43-kDa polypeptide, as judged by radiolabeling of minicells. Its DNA sequence was determined. The results demonstrate that overexpression of this single gene product greatly stimulates the incorporation of two stereochemically distinct KDO residues during lipopolysaccharide biosynthesis in extracts of E. coli.

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii.

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.

Transmembrane electrical potential affects the lipid composition of Acholeplasma laidlawii.

In membranes of Acholeplasma laidlawii, lipid composition is regulated as a function of several stimuli affecting the volume and length of the hydrocarbon chains and the hydrocarbon-water interfacial area. This regulation is vizualized as changes in the relative amounts of the major polar lipids monoglucosyl diglyceride and diglucosyl diglyceride. These lipids form reversed hexagonal and lamellar phases with water, respectively. However, mixtures of the two lipids, in the molar proportions found in the A. laidlawii membrane, form a lamellar phase. By adjustment of the glycolipid ratio as a response to environmental stimuli, a certain stability of the lamellar membrane is maintained. In growing cells with oleoyl membrane lipids, a transmembrane electrical potential of approximately -50 mV (inside negative), but no transmembrane pH difference, was found. Addition of the K+ ionophore valinomycin caused a rapid and dose-dependent hyperpolarization remaining for at least 7 h. Simultaneously, a rapid and lasting metabolic decrease in the ratio monoglucosyl diglyceride/diglucosyl diglyceride occurred. The increase in potential and the decrease in the lipid ratio were both reversed in a dose-dependent manner by extracellular KCl. Likewise, the lipophilic cation tetraphenylphosphonium caused a dose-dependent decrease in membrane potential and an increase in the monoglucosyl diglyceride/diglucosyl diglyceride ratio, respectively. The ionophores monensin and particularly nigericin had similar but less pronounced effects on the potential and lipid ratios as valinomycin. The uncoupler carbonyl cyanide m-chlorophenylhydrazone had no effect on cell growth, membrane potential, or lipid regulation at 10 microM. These dissimilar structures and the low concentrations used make a direct disturbance of drug molecules on lipid packing in membranes less likely.(ABSTRACT TRUNCATED AT 250 WORDS)