Lipoic acid is a covalently bound disulfide-containing cofactor required for function of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine cleavage enzyme complexes of Escherichia coli. Recently we described the isolation of the lplA locus, the first gene known to encode a lipoyl-protein ligase for the attachment of lipoyl groups to lipoate-dependent apoenzymes (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Biol. Chem. 269:16091-16100, 1994). Here, we report an unexpected redundancy between the functions of lplA and lipB, a gene previously identified as a putative lipoate biosynthetic locus. First, analysis of lplA null mutants revealed the existence of a second lipoyl ligase enzyme. We found that lplA null mutants displayed no growth defects unless combined with lipA (lipoate synthesis) or lipB mutations and that overexpression of wild-type LplA suppressed lipB null mutations. Assays of growth, transport, lipoyl-protein content, and apoprotein modification demonstrated that lplA encoded a ligase for the incorporation of exogenously supplied lipoate, whereas lipB was required for function of the second lipoyl ligase, which utilizes lipoyl groups generated via endogenous (lipA-mediated) biosynthesis. The lipB-dependent ligase was further shown to cause the accumulation of aberrantly modified octanoyl-proteins in lipoate-deficient cells. Lipoate uptake assays of strains that overproduced lipoate-accepting apoproteins also demonstrated coupling between transport and the subsequent ligation of lipoate to apoprotein by the LplA enzyme. Although mutations in two genes (fadD and fadL) involved in fatty acid failed to affect lipoate utilization, disruption of the smp gene severely decreased lipoate utilization. DNA sequencing of the previously identified slr1 selenolipoate resistance mutation (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) showed this mutation (now called lplA1) to be a G76S substitution in the LplA ligase. When compared with the wild-type allele, the cloned lplA1 allele conferred a threefold increase in the ability to discriminate against the selenium-containing analog. These results support a two-pathway/two-ligase model of lipoate metabolism in E. coli.

The enzyme, aspartate 1-decarboxylase (L-aspartate 1-carboxy-lyase; EC 4.1.1.15), that catalyzes the reaction aspartate-fl-alanine + C02 was found in extracts of Escherichia coli. panD mutants of E. coli are defective in 8-alanine biosynthesis and lack aspartate 1-decarboxylase. Therefore, the enzyme functions in the biosynthesis of the ,8-alanine moiety of pantothenate. The genetic lesion in these mutants is closely linked to the other pantothenate (pan) loci of E. coli K-12. D-Pantothenate, the condensation product of

No abstract

The inhibition of phospholipid synthesis engendered by starving glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli (plsB or gpsA) for G3P is incomplete; 5 to 10% of the normal rate of phospholipid synthesis remains, even after prolonged starvation. We report that G3P starvation of a strain having lesions in both the gpsA and plsB genes resulted in essentially complete (>98.5%) inhibition of phospholipid synthesis, indicating that all de novo glycerolipid synthesis in E. coli proceeds by acylation of G3P.

Unsaturated fatty acid auxotrophs of Escherichia coli are able to use only unsaturated fatty acids of the cis configuration as the required growth supplement. A mutation in thefat4 gene allows such auxotrophs to utilize unsaturated fatty acids with a trans double bond as well as fatty acids having a cis double bond. ThefatA gene was mapped to min 69 near argG, and the allele studied (fatl41) was found to be dominant over the wild-type gene. fatAl mutant strains grew at similar rates when supplemented with elaidate (trans-9-octadecenoate) or oleate (cis-9-octadecenoate). The fat' strain, however, lysed when supplemented with the trans fatty acid.Physiological characterization of the fatA mutant strain was undertaken. The mutation appeared not to be involved with long-chain fatty acid transport. Introduction of lesions in known fatty acid transport genes abolished trans fatty acid utilization in thefatA mutant strain. Also, growth characteristics of thefat' and the fatAl1 mutant strains on elaidate as the sole carbon source were identical, which indicated comparables rate of fatty acid accumulation. The mutation appeared to be involved with recognition of the trans configuration after uptake into the cell. The levels of trans fatty acid incorporation into the phospholipids of thefat' and thefatA strains differed considerably, with the mutant incorporating much higher levels. No significant accumulation of elaidate into nonphospholipid cellular components was observed. The fatA mutation did not appear to be involved with the cellular metabolic state, as cyclic AMP had no effect on the ability of the strains to utilize trans fatty acids.Escherichia coli unsaturated fatty acid auxotrophs have been very useful in elucidating the dependence of cell physiology on the physical state of the membrane phospholipids (for review, see reference 8). Such auxotrophs, which map to either of two genes, fabA (encoding ,B-hydroxydecanoyl thioester dehydrase) or fabB (encoding 3-ketoacylacyl carrier protein synthase), grow when supplemented with any of a surprisingly wide range of cis unsaturated fatty acids (9). These strains are also able to grow on unsaturated fatty acids having a double bond in the unnatural trans configuration but require a second mutational lesion for growth on trans fatty acids (14,17,28,29,31). Strains carrying this second lesion in addition to the original lesion in unsaturated fatty acid synthesis grow well when supplemented with either cis or trans unsaturated fatty acids but fail to grow, and ultimately lyse, when supplemented with saturated fatty acids or when left unsupplemented. These strains have been previously termed elaidate variants (13, 38, 39), since they grow when supplemented with elaidic acid (trans-9-octadecenoic acid), the trans isomer of oleic acid (cis-9-octadecenoic acid).Our interest in the elaidate variant mutational lesion stemmed from two aspects. First, much of the work on the interrelationships of cell physiology and the phospholipid order-disorder phase transition has used such st...