P. E. Kolattukudy

Biochemistry and molecular genetics of cell‐wall lipid biosynthesis in mycobacteria

Tuberculosis and other mycobacterial infections are the most serious infectious diseases in terms of human fatalities. The high content of unique cell‐wall lipids helps these organisms to resist antimicrobial drugs and host defences. The biosynthesis of these lipids is discussed briefly. The recent advances in recombinant DNA technology have begun to help to elucidate the nature of some of the enzymes involved in this process and the genes that encode them. Gene disruption and other molecular genetic technologies are beginning to provide new approaches to test for the biological functions of these gene products and may lead to identification of new antimycobacterial drug targets.

Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation

Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas‐like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface‐exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead‐like growth morphology without affecting the overall growth rate.

Purification and characterization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG

Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.

Developmental changes in the expression of S-acyl fatty acid synthase thioesterase gene and lipid composition in the uropygial gland of mallard ducks (Anas platyrhynchos)☆

Developmental changes in the composition of the uropygial gland secretory lipids of the postembryonic mallard ducks (Anas platyrhynchos) were determined. During the first 3 weeks after hatching, the composition of the secretory lipids remained constant; the lipids consisted of long-chain wax esters composed of a complex mixture of n-, monomethyl, and dimethyl fatty acids esterified to n-C16 and n-C18 fatty alcohols. Afterward, as the ducks began to acquire adult feathers, short-chain wax esters composed of 2- and 4-monomethyl fatty acids began to appear with 2-methylhexanoyl and 4-methylhexanoyl as the major acyl components; esters of short-chain monomethyl fatty acids (less than or equal to C12) constituted 90% of the lipids when the ducks were 2 months old and had acquired adult plumage. The appearance of the short-chain acids in the acyl portion of the wax esters was accompanied by the appearance of S-acyl fatty acid synthase thioesterase, which can hydrolytically release short-chain acids from fatty acid synthase in the gland. Northern blot analysis showed that the gland-specific thioesterase gene transcripts began to appear in the gland only 3 weeks after hatching. The appearance of the transcripts and immunologically detectable thioesterase protein reached maximum levels 2 months after hatching, with the acquisition of the adult plumage. Thus, the developmental changes in lipid composition correlated with the changes in the level of expression of the thioesterase gene. Expression of other gland-specific genes has been previously found to begin just prior to hatching. The gland-specific thioesterase is the first case of delayed expression of a gland-specific gene.

Cuticular Lipids in Plant-microbe Interactions

Cuticle constitutes the boundary between higher plants and their environment. Therefore, this layer might be expected to play an important role in the interaction of the plant with environmental factors. The plant cuticle is composed almost entirely of lipids and the role of some of these lipids in the interaction between plants and microbes has become clear in the recent years. In this brief review, we shall confine our discussion to two specific examples of such interactions: a detrimental one with pathogenic fungi and a beneficial one with phyllospheric bacteria which might provide fixed nitrogen in return for the use of some of the cuticular components as the carbon source. In this context, we will deal only with the role of the insoluble 1ipid-derived polymer, cutin, but not the role of soluble waxes that are always constituents of the cuticle.

THE MALATE DEHYDROGENASE GENE FROM BOTRYOCOCCUS BRAUNII (CHLOROPHYTA, CHLOROPHYCEAE): CLONING, SEQUENCE ANALYSIS, AND EXPRESSION IN ESCHERICHIA COLI

The gene for malate dehydrogenase from Botryococcus braunii Kützing (Chlorophyceae) was isolated. The gene codes for a protein of 316 amino acids with a calculated molecular weight of 32.9 kDa. In the N‐terminal region, a pyridine nucleotide‐binding domain was observed. The G+C content of the gene is 65%. Six copies of the (A/G)GGCGG hexanucleotide, which forms the core binding site for the cellular transcription factor Sp1, were observed in the promoter region. Southern blot analysis showed that malate dehydrogenase is encoded by a single gene in B. braunii. Escherichia coli cells in which the B. braunii gene was expressed showed 3.6 times more activity in the conversion of oxaloacetate to malate than the noninduced ones, confirming the identity of the gene. Botryococcus braunii malate dehydrogenase has been subjected to phylogenetic analyses by the neighbor‐joining and parsimony procedures.

Cutinase and Pectinase in Host-Pathogen and Plant-Bacterial Interaction

The aerial parts of plants are covered by the cuticle which forms the boundary layer at which microbes come into contact with the plant. The cuticle is composed of an insoluble structural polymer, cutin, which is embedded in a complex mixture of soluble lipids collectively called wax (1,2). Cutin is composed of interesterified hydroxy and hydroxyepoxy fatty acids primarily derived from palmitic, oleic, and linoleic acids (Fig. 1). The component fatty acids are held together by ester bonds between the primary as well as secondary hydroxy groups and the carboxyls. The cutin polymer constitutes the major physical barrier to penetration of fungi into the aerial parts of plants (3). The cutin barrier is attached to a pectinaceous polymeric layer which may also serve as a barrier to penetration by pathogens. In this paper, we will briefly review progress recently made in our understanding of the role of fungal cutinases and pectinases in the interaction between pathogenic fungi and their host plants. We also describe a recent discovery that a phyllospheric bacterium which cohabits with an apparently nitrogen-fixing bacterium generates an extra-cellular cutinase and thus can provide a carbon source while receiving fixed N2 from the cohabiting partner.