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Inositol is considered a growth factor in yeast cells and it plays an important role in Candida as an essential precursor for phospholipomannan, a glycophosphatidylinositol (GPI)-anchored glycolipid on the cell surface of Candida which is involved in the pathogenicity of this opportunistic fungus and which binds to and stimulates human macrophages. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway, which controls many cell cycle events. Here, high-affinity myo-inositol uptake in Candida albicans has been characterized, with an apparent [K.sub.m] value of 240 [+ or -] 15 [mu]M, which appears to be active and energy-dependent as revealed by inhibition with azide and protonophores (FCCP, dinitrophenol). Candida myo-inositol transport was sodium-independent but proton-coupled with an apparent [K.sub.m] value of 11.04 [+ or -] 1.1 nM for [H.sup.+], equal pH 7.96 [+ or -] 0.05, suggesting that the C. albicans myo-inositoI-[H.sup.+] transporter is fully activated at physiological pH. C. albicans inositol transport was not affected by cytochalasin B, phloretin or phlorizin, an inhibitor of mammalian sodium-dependent inositol transport. Furthermore, myo-inositol transport showed high substrate specificity for inositol and was not significantly affected by hexose or pentose sugars as competitors, despite their structural similarity. Transport kinetics in the presence of eight different inositol isomers as competitors revealed that proton bonds between the C-2, C-3 and C-4 hydroxyl groups of myo-inositol and the transporter protein play a critical role for substrate recognition and binding. It is concluded that C. albicans myo-inositoI-[H.sup.+] transport differs kinetically and pharmacologically from the human sodium-dependent myo-inositol transport system and constitutes an attractive target for delivery of cytotoxic inositol analogues in this pathogenic fungus.

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities. During growth on 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) and 3-chlorobenzoate stoichiometric amounts of chloride were released. It also utilized all three isomeric dichlorobenzenes and 1,2,3-trichlorobenzene as the sole carbon and energy source. Furthermore, the bacterium grew well on a great number of n-alkanes ranging from n-heptane to n-triacontane and on the branched alkane 2,6,10,14-tetramethylpentadecane (pristane) and slowly on n-hexane and n-pentatriacontane. It was able to grow at temperatures from 5 to 30[degrees]C, with optimal growth at 20[degrees]C, and could tolerate 6% NaCl in mineral salts medium. Genes encoding the initial chlorobenzene dioxygenase were detected by using a primer pair that was designed against the [alpha]-subunit (TecA1) of the chlorobenzene dioxygenase of Ralstonia (formerly Burkholderia) sp. strain PS12. The amino acid sequence of the amplified part of the [alpha]-subunit of the chlorobenzene dioxygenase of Rhodococcus sp. strain MS11 showed > 99 % identity to the [alpha]-subunit of the chlorobenzene dioxygenase from Ralstonia sp. strain PS12 and the parts of both [alpha]-subunits responsible for substrate specificity were identical. The subsequent enzymes dihydrodiol dehydrogenase and chlorocatechol 1,2-dioxygenase were induced in cells grown on 1,2,4,5-TeCB. During cultivation on medium-chain-length n-alkanes ranging from n-decane to n-heptadecane, including 1-hexadecene, and on the branched alkane pristane, strain MS11 produced biosurfactants lowering the surface tension of the cultures from 72 to [less than or equal to]29 mN [m.sup.-1]. Glycolipids were extracted from the supernatant of a culture grown on n-hexadecane and characterized by [sup.1]H- and [sup.13]C-NMR-spectroscopy and mass spectrometry. The two major components consisted of [alpha],[alpha]-trehalose esterified at C-2 or C-4 with a succinic acid and at C-2' with a decanoic acid. They differed from one another in that one 2,3,4,2'-trehalosetetraester, found in higher concentration, was esterified at C-2, C-3 or C-4 with one octanoic and one decanoic acid and the other one, of lower concentration, with two octanoic acids. The results demonstrate that Rhodococcus sp. strain MS11 may be well suited for bioremediation of soils and sediments contaminated for a long time with di-, tri- and tetrachlorobenzenes as well as alkanes.

Pseudomonas aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase, rhlA is indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P. aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties. P. aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility.

Research has been conducted on the virulent strains of Mycobacterium tuberculosis with the characteristic cell wall molecules, sulfated trehalose glycolipids. Results indicate that aryl-sulfotransferase gene Rv1373 encodes glycolipid sulfotrasferase towards the eukaryotic glycolipid galactosylceramide and glucosylceramide.

A study which was aimed at characterizing new Thermus strains and confirming the presence of glycerol-linked and diol-linked glycolipids within these strains was conducted. The methods used were extraction and purification of polar lipids, hydrolysis and analysis of glycolipids, preparation of fatty acid methyl esters (FAMEs) and derivatization. Results showed that partial structures of intact diol-containing lipids were present in the bacteria and some Thermus strains contain a mixture of diol- and glycerol-based glycolipids which were not present in the Thermomicrobium roseum.

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1[Beta] (IL-1[Beta]) and tumor necrosis factor [Alpha] are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1[Beta] steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1[Beta] mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor [Alpha] and Staphylococcus induction of IL-1[Beta] gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1[Beta] mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the -310 to -57 nucleotide region of the IL-1[Beta] promoter was found to mediate LPG's inhibitory activity. The requirement for the -310 to -57 promoter gene sequence for LPG's effect is demonstrated by the abrogation of LPG's inhibitory activity by truncation or deletion of the -310 to -57 promoter gene sequence. Furthermore, the minimal IL-1[Beta] promoter (positions -310 to +15) mediated LPG's inhibitory activity with dose and kinetic profiles that were similar to LPG's suppression of steady-state IL-1[Beta] mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a 'gene silencer,' a function, to our knowledge, not previously described. LPG's inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

The mannan chains of Klyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal [Alpha]1 [right arrow] 2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetyiglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.

Mutation in the hglK gene of the cyanobacterium Anabaena sp. strain PCC 7120 results into vegetative cells which are more cylindrical and have thicker septa than those in the wild type in an environment without nitrogen, and heterocysts without the glycolipid layer outside the cell wall in a nitrogen-rich environment. Findings indicate that hglK encodes a protein needed for heterocyst glycolipid localization.