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C Ratledge

Publications and source records attributed to C Ratledge.

At least 55 records · Page 3Linked to original sources
PubMed1990

Pathways to acetyl-CoA formation in Candida albicans.

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP:citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.

ATP Citrate (pro-S)-Lyase↗
PubMed1990

Aspartate metabolism in Mycobacterium avium grown in host tissue and axenically and in Mycobacterium leprae.

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M. leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. avium grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.

Animals↗
PubMed1990

Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.

Acetyltransferases↗
PubMed1990

Evidence for two fatty alcohol oxidases in the biosurfactant-producing yeast Candida (Torulopsis) bombicola.

Fatty alcohol oxidase activities in Candida (Torulopsis) bombicola ATCC 22214, which produces large amounts of glycolipids consisting of omega- and (omega-1)-hydroxyfatty acids and a sugar moiety, occurred only in the microsomal fraction whether the cells had grown on n-alkanes, carbohydrates or a mixture of the two. High activities occurred in glucose-grown cells. Aliphatic alcohols from octanol to hexadecanol were oxidized with two maxima in activities for decanol and for tetradecanol. Differences in their pH optima and in temperature stability suggest two separate enzymes are present. Long chain diols, but not omega-hydroxyfatty acids, were also oxidized.

Alcohol Oxidoreductases↗
PubMed1989

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum.

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.

Bacterial Proteins↗
PubMed1989

Metabolism of aspartate in Mycobacterium smegmatis.

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.

Aspartate Kinase↗
PubMed1988

Enzyme activities in oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids.

The activities of ATP: citrate lyase (ACL; EC 4. 1. 3. 8), carnitine acetyltransferase (CAT; EC 2. 3. 1. 7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1. 1. 1. 42), isocitrate lyase (ICL; EC 4. 1. 3. 1) and malic enzyme (malate dehydrogenase; EC 1. 1. 1. 40) were measured in four oleaginous yeasts, Candida curvata D, Trichosporon cutaneum and two strains of Rhodosporidium toruloides, grown either to accumulate lipid, or to utilize their own lipid reserves or an exogenous supply of lipid. During lipid utilization, activities of ACL and malic enzyme diminished to low levels; CAT and ICL increased considerably in activity and ICDH activity was slightly increased but catalase (EC 1. 11. 1. 6) diminished in activity in both strains of R. toruloides. In all cases, yeasts utilizing exogenous lipid showed greater changes in enzyme activities than cells utilizing their endogenous reserves. Electron micrographs of Candida curvata D showed proliferation of peroxisomes in starved cells utilizing their own lipid reserve though peroxisomes were more in evidence when the yeast had been grown on exogenous lipid. In Lipomyces starkeyi, which shows only minimal utilization of its stored lipid and furthermore cannot grow on exogenous lipid, only the occasional peroxisome was seen when cells were starved of carbon.

Candida↗
PubMed1988

Mycobactins and exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and other related species.

Following iron-deficient growth, mycobactins and exochelins were isolated from 11 strains of Mycobacterium tuberculosis (including type strains of the virulent H37Rv and avirulent H37Ra organisms) as well as from M. bovis (one strain), M. bovis BCG (two strains), M. africanum (eight strains) and M. xenopi (one strain) but not from M. microti (one strain). The mycobactins from the tuberculosis group (M. tuberculosis, M. bovis and M. africanum) were identical and could each be resolved into four compounds by thin-layer chromatography (TLC) and into several fractions by high-performance liquid chromatography, supporting previous claims that this group is a single taxon. Exochelins were all chloroform-soluble and showed no species specificity; no single exochelin was recognized by TLC that had not been previously seen in M. avium or a related species.

Chromatography, High Pressure Liquid↗
PubMed1988

Use of carbon sources for lipid biosynthesis in Mycobacterium leprae: a comparison with other pathogenic mycobacteria.

Carbon from glycerol and palmitate, but not significantly from five other carbon sources tested, was incorporated into lipids by suspensions of non-growing Mycobacterium leprae organisms. However, of the five other substrates three-citrate, glucose and pyruvate-were taken up. Nongrowing Mycobacterium microti and Mycobacterium avium incorporated carbon into lipids from most simple carbon sources tested unless they were obtained from growth media including palmitate or from experimentally infected animals, when incorporation of carbon into lipids from carbon sources except palmitate occurred up to 20 times more slowly. Thus, utilization of simple carbon appeared to be repressible while utilization of the one fatty acid tested, palmitate, appeared constitutive. In M. leprae, carbon from glycerol was incorporated into the glycerol moiety of acylglycerols but not into the fatty acid moieties or into free fatty acids. M. microti and M. avium incorporated carbon from simple carbon sources into fatty acids, even (though very slowly) when these organisms were obtained from host tissue. Isocitrate lyase, malate synthase and acetate kinase were detected in M. leprae. However acetyl-CoA synthetase was not detectable and phosphoacetylase was deficient; thus, M. leprae may be incapable of making acetyl-CoA from acetate. Phosphotransacetylase was readily detected in both host-grown M. avium and M. microti.

Acetate Kinase↗
PubMed1987

Exochelin-mediated iron acquisition by the leprosy bacillus, Mycobacterium leprae.

Exochelins, water-soluble siderophores of mycobacteria, were isolated and partially purified from culture filtrates of iron-deficiently grown cultures of Mycobacterium neoaurum NCTC 10439 and an armadillo-derived Mycobacterium (ADM 8563). Two biologically active fractions mediating iron uptake were isolated from each bacterium which not only were able to transport iron into the producing organism but also into suspensions of Mycobacterium leprae isolated from armadillo liver. The rate of exochelin-mediated iron uptake into M. leprae was about 1.5% of the rate observed into the producing organisms. The process of iron uptake appears to be by facilitated diffusion as it was not inhibited by HgCl2, NaN3, KCN, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. Since no uptake of iron occurred into iron-sufficient ADM cells, this may indicate that M. leprae, as recovered from an animal tissue, had been growing iron-deficiently in order for iron uptake to have been demonstrated in vitro.

Chromatography, Gel↗
PubMed1987

Effect of homoserine on growth of Mycobacterium smegmatis: inhibition of glutamate transport by homoserine.

Homoserine strongly inhibited growth of Mycobacterium smegmatis in medium containing glutamate as the sole source of nitrogen but was without effect when asparagine, alanine or glutamine was the sole nitrogen source. It was readily taken up by glutamate-grown cells, reaching an intracellular concentration of over 20 mM after 4 h incubation. The primary site of action of homoserine was deduced to be the non-competitive inhibition of glutamate transport.

Biological Transport↗
PubMed1987

Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake.

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.

Antibodies, Bacterial↗
PubMed1986

Participation of iron on the growth inhibition of pathogenic strains of mycobacterium avium and M. paratuberculosis in serum.

Serum, which is low in freely-available iron, was bacteriostatic for M. avium and M. paratuberculosis irrespective of the ability of these organisms to produce the iron-chelators mycobactin and exochelin for iron acquisition. Bacteriostasis was overcome when the serum was supplemented with exochelin or mycobactin, but only if the mycobacteria had previously adapted to growth in a low iron environment. The results imply that for studies on mycobacterial pathogenicity the test cultures should be grown under low iron conditions before being used. The role of the exochelins, mycobactin and iron in the development of in vivo growth is discussed.

Animals↗
PubMed1986

Metal analogues of mycobactin and exochelin fail to act as effective antimycobacterial agents.

Mycobactins and exochelins from mycobacteria formed complexes with many heavy metals in the absence of iron. Although many of these complexes affected the growth of M. tuberculosis and M. avium in laboratory culture medium and in serum the complexes were not stable enough in the presence of iron, which displaced the metal ion, to be of value as specific antimycobacterial agents.

Culture Media↗
PubMed1986

Mycobactin and the competition for iron between Mycobacterium neoaurum and M. vaccae.

Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0.02-0.05 microgram Fe ml-1) and iron-sufficiency (2-4 micrograms Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6-8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells. Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.

Iron↗