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C Ratledge

Publications and source records attributed to C Ratledge.

At least 73 records · Page 4Linked to original sources
PubMed1986

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin.

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.

Chromatography, Ion Exchange↗
PubMed1986

Distribution and application of mycobactins for the characterization of species within the genus Rhodococcus.

Representatives of 11 species of Rhodococcus were examined for their ability to synthesize mycobactin, a lipid-soluble siderophore, following iron-limited growth on solidified glycerol/asparagine medium. Rhodococcus bronchialis, R. terrae and R. rubropertinctus formed mycobactins, whereas the remaining species (R. coprophilus, R. equi, R. erythropolis, R. rhodnii, R. rhodochrous, R. ruber, R. maris and R. luteus) failed to synthesize these compounds even under conditions of strictly iron-limited growth. The mycobactins from R. terrae and R. rubropertinctus showed close similarity by thin-layer chromatography and high-performance liquid chromatography and could be easily distinguished from that of R. bronchialis.

Actinomycetales↗
PubMed1985

Equivalence of mycobactins from Mycobacterium senegalense, Mycobacterium farcinogenes and Mycobacterium fortuitum.

Mycobactins were isolated from five strains designated Mycobacterium farcinogenes and a similar number designated Mycobacterium senegalense following growth under conditions of iron-limitation. These lipid-soluble iron-chelating compounds were characterized by a combination of thin-layer and high-performance liquid chromatography. The mycobactins from both the slow-growing M. farcinogenes and the rapidly-growing M. senegalense strains proved impossible to differentiate both from each other and from those produced by strains of Mycobacterium fortuitum, indicating a close relationship between all three species. However, Nocardia farcinica, previously implicated with the bovine farcy strains, produced a different mycobactin which was easily distinguished by thin-layer chromatography alone.

Chromatography, High Pressure Liquid↗
PubMed1985

Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.

Chemical Phenomena↗
PubMed1984

Mycobactins as chemotaxonomic characters for some rapidly growing mycobacteria.

Thirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, 'M. diernhoferi', M. duvalii, M. flavescens, M. fortuitum, M. gadium, 'M. gallinarum', M. neoaurum, M. parafortuitum, 'M. peregrinum', M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and 'M. kanazawa' failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and 'M. peregrinum') formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, 'M. diernhoferi', M. vaccae and M. parafortuitum). The mycobactin from 'M. gallinarum' was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.

Chromatography, High Pressure Liquid↗
PubMed1983

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by adenine nucleotides.

The regulation of mitochondrial citrate metabolism has been investigated in oleaginous and non-oleaginous yeasts to ascertain its importance in controlling the rate of citrate efflux from mitochondria. The following observations were made: 1. Citrate efflux from mitochondria of the oleaginous yeast Candida curvata D, in the presence of L-malate and pyruvate, was stimulated by adding ATP and reduced by AMP. In the non-oleaginous yeast, Candida utilis 359, there was very little stimulation of citrate efflux by ATP but it was reduced by AMP. These effects appeared to be generalized as similar results were obtained in an examination of eight further yeasts (seven oleaginous and one non-oleaginous). 2. The effects of ATP and AMP were not observed in mitochondria whose metabolism had been inhibited by antimycin A and rotenone indicating the direct regulation of the citrate translocase was not involved. 3. In C. curvata D, ATP increased the total mitochondrial citrate content and reduced that of 2-oxoglutarate whereas AMP had the reverse effect. In C. utilis 359, AMP had a similar effect but that of ATP was much smaller. 4. To explain these observations the mitochondrial NAD+-dependent isocitrate dehydrogenase was studied in a number of yeasts. The enzyme from oleaginous yeasts had a requirement for AMP for activity and was inhibited by ATP. In non-oleaginous yeasts the enzyme was active in the absence of AMP and increased in activity as the isocitrate concentration increased. 5. The enzyme in C. curvata D was constantly more sensitive to increasing energy charge than that of the non-oleaginous yeast. These results indicate that the supply of citrate (and hence acetyl-CoA) to the cytosol is controlled by the activity of the intramitochondrial NAD+-dependent isocitrate dehydrogenase which in turn is regulated by adenine nucleotides. The sensitivity of this enzyme to the ATP/AMP ratio during lipogenesis is therefore an important control in the accumulation of lipid by yeasts.

Adenosine Monophosphate↗
PubMed1983

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by long-chain fatty acyl-CoA esters.

1. Citrate efflux from a wide range of yeast mitochondria was inhibited by long-chain fatty-acyl-CoA esters. 2. Fatty-acyl-CoA esters with chain lengths of between 14 and 18 carbons were the most potent inhibitors of citrate efflux which was unaffected by the fatty acids themselves. 3. 50% inhibition of citrate transport was observed using palmitoyl-CoA and oleoyl-CoA at approx. 4-5 microM when the tricarboxylate carrier was saturated with L-malate as counter-anion. 4. The inhibition with palmitoyl-CoA and oleoyl-CoA was competitive with L-malate. 5. The possibility that the fatty-acyl-CoA esters were exerting their effect by acting as detergent was eliminated because of the low concentrations used and appropriate comparisons being made with non-specific detergents. Although detergents inhibited citrate efflux they also released citrate by causing membrane damage. 6. The effect of fatty-acyl-CoA esters on citrate efflux could be decreased by using higher mitochondrial protein levels and by adding bovine serum albumin. 7. The possibility is discussed that this inhibition represents a genuine feedback inhibition which could regulate the amount of lipid being synthesized by an oleaginous yeast.

Acyl Coenzyme A↗
PubMed1983

A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture.

The oleaginous yeast, Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates. Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogen-limited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D = 0.05/hr) in a chemostat. Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D = 0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized. The relative proportions of the major fatty acids (16:0, 18:0, 18:1, 18:2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions. However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed. Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18:0) 15% and the lowest linoleic acid (18:2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18:1) 51% and decreased palmitic acid (16:0) 25%.

Candida↗
PubMed1983

Biochemical activities during lipid accumulation in Candida curvata.

Intracellular and extracellular concentrations of citrate and the specific activities of ten different enzymes in Candida curvata D were examined in relation to lipid biosynthesis in batch and continuous culture. Citrate was found to accumulate prior to lipid production and declined markedly as lipid accumulated in batch culture. The cells excreted citrate as the culture became nitrogen-limiting after 30 hr of growth, but little more was expelled after 40 hr when lipid accumulation was more marked. In continuous culture, only low levels of citrate were detected at the lower dilution rates and citrate was completely absent from both the cells and medium above a dilution rate of 0.1/hr. The activity of malic enzyme, malate dehydrogenase and ATP:citrate lyase increased in batch culture on lipid accumulated and, in continuous culture, both malic enzyme and ATP:citrate lyase varied in parallel with the specific rate of lipid synthesis which increased with increasing dilution rate. Activity of malate dehydrogenase, citrate synthase and glucose-6-phosphate dehydrogenase decreased with increasing dilution rate. The regulatory significance of these enzymes in lipid accumulation by C. curvata is discussed.

Ammonia↗
PubMed1983

Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium.

Fifty-three strains of M. avium and related species all produced one or more exochelins, the extracellular iron-binding compounds of the mycobacteria, when grown iron deficiently. Only those strains which could grow without the addition of mycobactin (i.e., mycobactin independent) produced mycobactin, the intracellular iron-binding compound of the mycobacteria. Exochelins varied from 20 to 2,000 micrograms per g of cell dry weight; mycobactins were between 1 and 10 mg per g of cell dry weight. M. paratuberculosis (13 strains) and 13 strains of M. avium, both species dependent upon mycobactin for growth, failed to produce spectrophotometrically detectable amounts of mycobactin (less than 0.2 microgram per g of cell dry weight), although mycobactin could be recognized in one strain of M. avium grown with an additional supply of salicylate and examined by a radiolabeling technique. On repeated subculture three of the mycobactin-dependent strains of M. avium, but none of those of M. paratuberculosis, lost their mycobactin dependence and on reexamination were found to produce their own mycobactin at 0.3 mg per g of cell dry weight. It is concluded that mycobactin biosynthesis is probably strongly repressed in the mycobactin-dependent strains rather than being a genetic deletion. The exochelins, when examined by high-pressure thin-layer chromatography were revealed as being multiples of similar compounds, with up to 20 individual iron-binding compounds being recognizable with some strains. It is argued that the exochelins represent the single most important means of iron acquisition in mycobacteria growing in vitro and in vivo, and their elaboration by the fastidious M. paratuberculosis and related species explains how these organisms are able to grow in vivo in the absence of an external supply of mycobactin.

Chromatography, Thin Layer↗
PubMed1983

Exochelin-mediated iron uptake into Mycobacterium leprae.

Iron chelated to the exochelins from Mycobacterium neoaurum was taken up by a suspension of M. leprae, prepared from the liver of an infected armadillo, over 15 hr. No uptake occurred when the iron was chelated with exochelins from M. bovis BCG or M. smegmatis or to a single exochelin from M. vaccae. Uptake appeared to be by facilitated diffusion since it was not inhibited by either HgCl2, NaN3, or 2,4-dinitrophenol. This was similar to the mode of uptake of ferriexochelin into M. neoaurum itself.

2,4-Dinitrophenol↗
PubMed1982

Iron transport in Mycobacterium smegmatis: the location of mycobactin by electron microscopy.

Mycobactin, the lipid-soluble iron-binding compound of the mycobacteria, has been located using electron microscopy of whole cells stained with vanadate. It forms a discrete, discontinuous region close to, or even included in, the cytoplasmic membrane of Mycobacterium smegmatis. It does not occur throughout the whole thickness of the wall but is some distance within the envelope. Possible models for the accommodation of mycobactin are discussed. It is concluded from these observations that mycobactin may be acting either as a store of iron, or as an ionophore, or possibly fulfilling both roles.

Cell Membrane↗
PubMed1982

Iron transport in Mycobacterium smegmatis: Uptake of iron from ferric citrate.

In mycobacterial growth medium 40 to 400 microM citrate was required to solubilize 2 microM 55Fe. This solubilized 55Fe was taken up into both iron-deficient and iron sufficient washed cell suspensions of Mycobacterium smegmatis and Mycobacterium bovis BCG. Although the 55Fe was taken up into the cell, the citrate was not. The uptake system with M. smegmatis was not inhibited by electron transport inhibitors, uncouplers of oxidative phosphorylation, or thiol reagents and was saturable with iron at approximately 35 microM. The system was independent of the iron transport systems already known to exist in M. smegmatis: i.e., the two exochelin routes of assimilation as well as the mycobactin-salicylate system. It was not induced by the presence of 400 microM citrate in the growth medium, nor did the presence of citrate in the medium affect the production of either exochelin or mycobactin.

Biological Transport↗
PubMed1980

Specificity of exochelins for iron transport in three species of mycobacteria.

Iron chelated to the water-soluble extracellular iron-binding compound (exochelin) from Mycobacterium smegmatis was taken up by that organism by an energy-coupled transport system which was not found in M. bovis BCG or M. intracellulare. The uptake of iron from the chloroform-soluble exochelins produced by the latter two species was by an inhibitor-insenstive system which was also found in M. smegmatis.

Biological Transport, Active↗
PubMed1979

A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms.

The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor. The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.

ATP Citrate (pro-S)-Lyase↗
PubMed1978

The separation of the mycobactins from Mycobacterium smegmatis by using high-pressure liquid chromatography.

The family of mycobactins from Mycobacterium smegmatis were resolved into seven fractions by high-pressure liquid chromatography. This separation was by virtue of the differences in length and character of the long acyl substituents as shown by g.l.c. of the methyl esters of the isolated fatty acids from the fractions. As t.l.c. could also resolve the individual mycobactin fractions, it too must rely on the same differences to effect separation. As the lengths of the acyl chains were modulated by the growth conditions, a specific range of acyl groups may not be needed for mycobactin to function. This technique provides a simple means of rapidly characterizing crude mycobactins from all mycobacteria.

Chromatography, High Pressure Liquid↗