- Volume 128, Issue 6, 1982
Volume 128, Issue 6, 1982
- Biochemistry
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The Biodegradation of Diethanolamine and Triethanolamine by a Yellow Gram-negative Rod
More LessA yellow Gram-negative rod-shaped organism that could grow with ethanolamine, diethanolamine or triethanolamine as the sole source of carbon and energy was isolated from a laboratory-scale activated-sludge plant. Studies with whole cells and cell-free extracts have enabled the inducible pathways for the degradation of these compounds to be elucidated. Triethanolamine is converted, via triethanolamine N-oxide, into diethanolamine and glycolaldehyde; diethanolamine in turn is converted into ethanolamine and glycolaldehyde. Ethanolamine is converted into acetyl units via ethanolamine O-phosphate and acetaldehyde. In di- and triethanolamine-grown cells the specific activities of glyoxylate carboligase and tartronic-semialdehyde reductase are comparatively high whilst that of isocitrate lyase is low; this situation is reversed in ethanolamine-grown cells. Triethanolamine-N-oxide dehydroxyethylase appears to be a product-induced enzyme and an inducible transport system may be involved in the uptake of diethanolamine.
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Properties and Regulation of Valine-sensitive Acetolactate Synthase from Mitochondria of Euglena gracilis
More LessAcetolactate synthase from mitochondria of Euglena gracilis was markedly activated by ATP and feedback-inhibited by valine and, to a lesser extent, by other branched-chain amino acids. 2-Oxobutyrate, a substrate converted to acetohydroxybutyrate, had a much higher affinity for this enzyme than pyruvate and markedly inhibited the formation of acetolactate from pyruvate. The inhibition of growth of E. gracilis by threonine and the relief of this inhibition by valine are explained by these characteristic properties of E. gracilis acetolactate synthase, the key enzyme in the biosynthesis of branched-chain amino acids.
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Regulation of Alkaline Phosphatase Synthesis in Candida utilis
More LessSynthesis of alkaline phosphatase in Candida utilis, unlike that of the two molecular forms of acid phosphatase, is not controlled by inorganic phosphate in the culture medium. The nature of the carbon source seems to play an important role in the regulation of this enzyme. Growth on a fermentable carbon source such as glucose, fructose or mannose leads to a constitutive synthesis of the enzyme which stops as soon as the sugar is exhausted, even though the yeast continues to grow on the products of the sugar fermentation. On transfer of cells from a glucose-containing to an ethanol-containing medium alkaline phosphatase synthesis stops immediately, whereas synthesis of the enzyme starts immediately on transfer of cells from an ethanol-containing to a glucose-containing medium. Transfer to growth on a different carbon source has no effect on the synthesis of the two forms of acid phosphatase. Thus, it seems that synthesis of acid and alkaline phosphatase in Candida utilis is regulated by completely different mechanisms.
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Alkaline Extracellular Protease Produced by Saccharomycopsis lipolytica CX161–1B
More LessSaccharomycopsis lipolytica CX161-1B, a strain suitable for genetic studies, when grown at near neutral pH produced a single alkaline extracellular protease, lower levels of acid extracellular protease(s) and no neutral extracellular protease. The alkaline protease was purified to homogeneity (as determined by polyacrylamide gel electrophoresis) by ultrafiltration, gel filtration and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated by gel filtration to be 27000–30000, and the isoelectric point was pH5·7. The purified enzyme had an alkaline pH optimum (pH 9–10). It was completely inhibited by phenylmethylsulphonyl fluoride, reversibly inhibited by EDTA, partially inhibited by o-phenanthroline, and not inhibited by dithiothreitol, N-ethylmaleimide or 4-hydroxymercuribenzoic acid, indicating that it is a serine protease. The content of sulphur amino acids was determined, and the purified protease contained no more than 1.8% carbohydrate as determined by the phenol–sulphuric acid method. The N-terminal amino acid sequence (25 residues) was determined; the N-terminal amino acid was alanine.
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The Characteristics of Peptide Uptake in Streptococcus faecalis: Studies on the Transport of Natural Peptides and Antibacterial Phosphonopeptides
More LessTransport of natural peptides and antibacterial phosphonopeptide analogues was studied in Streptococcus faecalis ATCC 9790. Competition studies, and the isolation of peptide-transport deficient mutants, indicate the presence of two peptide permeases. One is a high-rate system used by dipeptides, and to a lesser extent tripeptides; the other is a low-rate oligopeptide system. Following uptake, peptides are cleaved and their amino acid residues may undergo rapid exodus. Different strains of S. faecalis differ in their rates of peptide transport.
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- Development And Structure
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Electron Microscopic Studies of Chlorella ellipsoidea Protoplast Formation
More LessThe intermediate stages of protoplast formation of Chlorella ellipsoidea were studied by transmission and scanning electron microscopy. Within the first few hours of treatment with a polysaccharide-degrading enzyme mixture, the microfibrillar inner component of the cell wall was almost completely digested and the remaining outermost component was released irregularly from the plasma membrane. As incubation proceeded, a crevice which occurred at a fold of the outer wavy layer gradually extended through the layer; then, the surface of the plasma membrane was exposed from the crevice. The gradually degrading outer layer peeled off and an almost spherical protoplast with fine furrows on its surface was released.
Degradation of the outermost thin layer of cell wall, probably composed of pectin, limits the rate of protoplast formation in C. ellipsoidea.
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- Genetics And Molecular Biology
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In vitro-constructed RP4-prime Plasmids Mediate Orientated Mobilization of the Proteus morganii Chromosome
More LessRP4-prime plasmids containing inserts of Proteus morganii strain 2815 chromosomal DNA (0·5–13 megadaltons in size) were constructed in vitro. When introduced into the latter organism, the hybrid plasmids promoted transfer of chromosomal markers in an orientated manner, with recombinant frequenciEs From 10−4 to 10−6 per donor. Transconjugants expressed the tetracycline-resistant phenotype of the plasmids. Plasmid-guided trajectories overlapped and a circular map of chromosome markers was constructed. Correspondence between size of inserted chromosomal DNA in hybrid plasmids and numbers of markers transferred suggested that homology played a role in chromosome mobilization by these plasmids.
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Genetic Block of Outer Plaque Morphogenesis at the Second Meiotic Division in an hfd1-1 Mutant of Saccharomyces cerevisiae
More LessAn hfdl-1 mutant of Saccharomyces cerevisiae, SOS4, characterized by predominant production of two-spored asci at 29 °C, undergoes normal meiotic nuclear divisions and produces four haploid nuclei, but only two non-sister nuclei among them are incorporated into mature ascospores. Spindle pole bodies and prospore wall membranes at the second meiotic division at 29 °C were observed in this mutant by electron microscopy. The spindle pole body at one pole of each spindle had a normal outer plaque which was larger than the inner plaque. At the other pole, the outer plaque was entirely absent and a normal prospore wall membrane was not detected. It was concluded that at 29 °C the hfdl-1 mutation blocks the morphogenesis of outer plaques and prospore wall membranes at the two non-sister poles in the second meiotic division, and consequently only non-sister nuclei in the resulting meiotic cell are incorporated into ascospores.
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Genetic Analysis and Phenotypic Characterization of Effects on the Cytoskeleton of Coumarin-sensitivity Mutations in Dictyostelium discoideum
More LessFive coumarin-sensitivity mutations were assigned to loci on three linkage groups: couB352 to group I: couC356, couE353 and couF354 to group II; and couD355 to group III. Coumarin inhibited cell division, affected cell shape and induced EDTA-sensitive cellcell adhesion in vegetative cells at lower concentrations in coumarin-sensitive mutants (1·0 to 2·5 mm) than in wild-type strains (3·0 mm). One mutant, HU609 (couB352), lysed in the presence of coumarin (1·6mm). Coumarin, thiabendazole, and cambendazole induced multiple tips in pseudoplasmodia. Coumarin, unlike thiabendazole and cambendazole, did not induce metaphase arrest or haploidization of diploids. The couA351 and couF354 coumarin-sensitivity mutations are linked to growth temperature-sensitivity mutations, tsgK21 and tsgT360 respectively, which may be pleiotropic effects of the coumarin-sensitivity mutations. Exposure of vegetative cells of strains carrying either the couA351 or the couF354 mutation to the restrictive temperature (28C) induced cell rounding and cellcell adhesion similar to that seen on treatment with coumarin. Wild-type strains, other coumarin-sensitive strains and other temperature-sensitive strains did not exhibit this phenotype at 28C. Furthermore, temperature-sensitivity and coumarin-sensitivity co-reverted in temperature-resistant derivatives selected from strains carrying the couA351/tsgK21 mutation.
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Norsolorinic Acid Mutant of Aspergillus flavus
More LessA mutant of Aspergillus flavus accumulating norsolorinic acid and approximately 50% less aflatoxin than the parent strain was recovered after treatment of conidia with N-methyl-N′-nitro-N-nitrosoguanidine. The gene nor controlling this mutant phenotype was recessive and linked to afl-1 and leu on linkage group VII. Diploids homozygous for nor were similar to haploids in norsolorinic acid accumulation. The analysis of recombinant diploids and haploids showed that afl-1 and nor were both distal to leu on the same chromosome arm.
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- Immunology
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Immunochemistry of the Cell-Surface Carbohydrate Antigens of Clostridium difficile
More LessTwo carbohydrate cell-surface antigens were extracted from Clostridium difficile. One was extracted from pure cell walls by NaOH and contained glucose, mannose, galactosamine and phosphate in the approximate molar proportions of 2:0.65:1:0.63. The other antigen was extracted with phenol from the disrupted contents of whole cells and purified by chromatography on Sepharose 6B and an immunoabsorbent column; it contained glucose, glucosamine, phosphate and fatty acid in the approximate molar proportions of 2:1:1.6:0.04. Both antigens showed partial immunological identity and both cross-reacted with Clostridium sordellii antiserum. They are analogues of the wall and membrane teichoic acids of other Gram-positive bacteria.
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- Pathogenicity And Medical Microbiology
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Correlations between Structure and Antimycobacterial Activity in a Series of 2-Acetylpyridine Thiosemicarbazones
More LessThe antimycobacterial activity of a new series of 2-acetylpyridine thiosemicarbazones was determined in vitro using Mycobacterium smegmatis ATCC 607. The resulting log minimal inhibitory concentration (μmol l−1) values were plotted against the partition coefficient (log P) values for each compound, and fell on a parabolic distribution curve having a log P opt of 3.0. Compounds having partition coefficients outside the range 2.0 to 4.0 were inactive against M. smegmatis. When similar assays were carried out using M. tuberculosis, M. kansasii, M. marinum, M. simiae, M. avium and M. intracellulare, a similar series of parabolic activity curves were obtained having log P opt values around 4.0. The significance of this shift in the log P opt value obtained using the slow-growing pathogenic mycobacteria compared to that observed with the rapid-growing M. smegmatis is discussed in relation to the structures of the variable substituents of these new 2-acetylpyridine thiosemicarbazone compounds.
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Purification of Chlamydia trachomatis Lymphogranuloma Venereum Elementary Bodies and their Interaction with HeLa Cells
More LessA procedure has been developed to yield infectious elementary bodies of the lymphogranuloma venereum strains LGV 434 and 404 of Chlamydia trachomatis, labelled during intracellular growth in HeLa 229 cells. The final preparation, obtained after velocity sedimentation of a polycarbonate membrane-filtered sample through a sucrose gradient, is free of host proteins and, more importantly, of chlamydial reticulate bodies. Using such purified preparations, it was found that the association of LGV 434 elementary bodies with HeLa 229 cultures was unaffected by the pretreatment of the host cells with a variety of lectins or with neuraminidases from Clostridium perfringens and Vibrio cholerae. The association was inhibited by dextran sulphate and by mild trypsin treatment of HeLa cultures. Treatment of purified elementary bodies with trypsin, chymotrypsin, neuraminidases and a variety of carbohydrates and lectins did not produce any change in the rate of association with HeLa cultures. Heat-inactivated elementary bodies were significantly less able to associate with the host cells.
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- Physiology And Growth
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Purification and Characterization of Two Phage PBSX-induced Lytic Enzymes of Bacillus subtilis 168: An N-Acetylmuramoyl-l-alanine Amidase and an N-Acetylmuramidase
More LessAfter heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-l-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.
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Positive Chemotaxis of Rhizobium leguminosarum and other Bacteria towards Root Exudates from Legumes and other Plants
More LessRhizobium leguminosarum showed positive chemotaxis towards root exudates of its host the edible pea (Pisum sativum L.). Only the fraction of the exudate containing substances with molecular weights less than 1000 showed significant chemotactic activity. Cationic, neutral and anionic fractions were all attractive, the cationic being the most potent and the anionic the least. A range of amino acids, sugars and carboxylic acids were present in the exudate, and many were shown to be attractants. Other Rhizobium species and Escherichia coli were also attracted by pea exudate, and R. leguminosarum and the other bacteria were attracted by exudates from roots of a range of plants including non-legumes. It was concluded that although positive chemotaxis probably facilitates infection of legumes by Rhizobium, it has little or no role in host-symbiont specificity.
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The Effect of Cations on Zoospores of the Fungus Phytophthora cinnamomi
More LessSwimming zoospores of the fungus Phytophthora cinnamomi exposed to a range of ions were much more sensitive to cations than anions. One cation, Ca2+, induced zoospores to encyst and subsequently germinate, but most cations induced encystment only and were toxic at higher concentrations. In general, the sensitivity of zoospores to a cation increased with its charge density. At 0·3 μm, La3+ reduced the viability of a zoospore population to 50%, while Fe3+ (20 μm) and Mn2+ (50 μm) induced encystment with only a slight decrease in viability. The other divalent and monovalent cations tested (Mg2+, Ca2+, Ba2+, Li+, Na+, K+, Cs+, NH4 +) were effective in inducing encystment or reducing viability at higher concentrations. Each ion showed a distinctive concentration–response curve. Only F−and CH3COO−among the anions tested (Cl−, NO3 −, F−, H2PO4 2-, SO4 2−, CH3COO−) had any effect on zoospores, and at 20 mm (pH 6·0) they reduced viability.
The cysts (cystospores) of the fungus were generally less sensitive to cations than were swimming zoospores, and only Cs+and K+(50 mm) reduced viability to the same extent in each population. Both zoospores and cysts of this fungus had a broad tolerance to pH and temperature, but cysts were more resistant to low temperatures than were motile zoospores.
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Arogenate (Pretyrosine) Pathway of Tyrosine and Phenylalanine Biosynthesis in Pseudomonas aureofaciens ATCC 15926
More LessAssays of enzyme activities suggest that arogenate, the product of prephenate transamination, is an intermediate in the biosynthesis of both phenylalanine and tyrosine in Pseudomonas aureofaciens ATCC 15926. In addition to prephenate dehydratase and prephenate dehydrogenase, arogenate dehydratase and arogenate dehydrogenase activities were demonstrated. This pattern of aromatic amino acid biosynthesis in pseudomonads had previously been demonstrated only in P. aeruginosa. Arogenate dehydrogenase from P. aureofaciens differs from that in P. aeruginosa in its utilization of either NAD+or NADP+as cofactor and its inhibition by l-tyrosine. During ammonium sulphate fractionation, arogenate dehydratase co-precipitated with prephenate dehydratase I activity and not with prephenate dehydratase II. The pattern of regulation of the arogenate route to tyrosine in P. aureofaciens ATCC 15926 differed from that previously reported for strain ATCC 13986.
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Inhibition of Ornithine Carbamoyltransferase by Arginase among Yeasts: Correlation with Energy Production, Subcellular Localization and Enzyme Synthesis
More LessThirty-two yeast species belonging to twelve genera were examined for the occurrence of inhibition of ornithine carbamoyltransferase by arginase (epiarginasic regulation) and related properties. Obligate aerobes were devoid of this regulation. Among fermenting species, Schizosaccharomyces and budding genera had different properties: all Schizosaccharomyces species were devoid of this regulation whereas all species of budding yeasts showing a weak or absent Pasteur effect had this regulation. Strains showing a strong Pasteur effect and taxonomically related to Saccharomyces (Kluyveromyces had the regulation, whereas species classified in genera that include species which are obligate aerobes did not.
We confirm that the absence of epiarginasic regulation is correlated with a mitochondrial localization of ornithine carbamoyltransferase but not with the function of the mitochondria in Schizosaccharomyces japonicus. In this case, compartmentation could serve to channel anabolic and catabolic functions. Although, in general, the arginase of negative epiarginasic species had no affinity for cytosolic ornithine carbamoyltransferase, one exception was found (Hansenula anomala).
Regulation by repression of ornithine carbamoyltransferase and induction of arginase by arginine was the general rule. These types of regulation of enzyme synthesis were only absent in yeasts in which compartmentation is present and could serve as the basis for anabolismcatabolism exclusion.
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- Short Communication
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Polysaccharase Activity in Streptococcus agalactiae (Group B Streptococci)
More LessOf 300 recently isolated strains of Streptococcus agalactiae from human sources, 97% degraded starch. Representative strains also degraded glycogen, pullulan, amylopectin and amylose. The polysaccharase activity is easily detected by clearing around growth on Columbia agar base medium. The activity is weaker than that of some S. pyogenes strains, and it does not appear to produce fermentable products but is inhibited by the presence of easily used sugars.
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- Taxonomy
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Numerical Taxonomy of Psychrotrophic Pseudomonads
More LessThe taxonomy of 218 psychrotrophic pseudomonad strains (200 field strains from meat and 18 type and reference strains) was numerically studied by 174 biochemical and physiological tests. All strains were Gram-negative rods, oxidase positive and motile by means of one or more polar flagella. The strains clustered into 15 groups, of which 9 were regarded as major clusters. The major clusters were designated as Pseudomonas fragi (112 strains), P. fluorescens biotype III (7 strains), P. fluorescens biotype I (16 strains), P. aureofaciens/chlororaphis (3 strains), P. fluorescens biotype II (3 strains), P. putida biotype I (4 strains), Alteromonas putrefaciens (10 strains) and Aeromonas hydrophila biotype I (5 strains). One major cluster, containing 21 strains (cluster 2), was left unassigned. The phenotypic data indicate that this cluster might represent a new species. The P. fluorescens/P. putida complex matched closely the descriptions of Stanier et al. (1966) , but the two largest clusters (1 and 2) were not in agreement with any species described in the eighth edition of Bergey’s Manual of Determinative Bacteriology. Cluster 1 included the type strain (ATCC 4973) of the hitherto incompletely described P. fragi. A simplified scheme for the separation between P. fragi, P. fluorescens, P. putida and cluster 2 is presented.
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The Construction by Computer of a Diagnostic Key to the Genera of Yeasts and Other Such Groups of Taxa
More LessGroups of taxa such as genera, or groups derived from some forms of cluster analysis, may have insufficient test results that are constant within the groups to allow diagnostic keys and tables to be constructed in the usual way. This paper describes how the usual methods can be adapted to allow construction based on information about the individual group members, instead of on the overall group information. A new key to the genera of yeasts is constructed by these modified methods.
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Reclassification of ‘Corynebacterium haemolyticum’ (MacLean, Liebow & Rosenberg) in the Genus Arcanobacterium gen.nov. as Arcanobacterium haemolyticum nom.rev., comb.nov
More Less‘Corynebacterium haemolyticum’ (MacLean, Liebow & Rosenberg) differs to such an extent from the type species of Corynebacterium, C. diphtheriae (Lehmann & Neumann), that it should be removed from this genus. Chemical and numerical phenetic data indicate that C. haemolyticum is a distinct taxon worthy of generic status. A new genus, Arcanobacterium, is described for the species A. haemolyticum (MacLean, Liebow & Rosenberg) nov.rev., comb.nov. The genus is tentatively placed within the coryneform group of bacteria. The type species of the genus is Arcanobacterium haemolyticum and the type strain is ATCC 9345.
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Numerical and Chemical Classification of Nocardia amarae
More LessTwenty-one strains of Nocardia amarae and marker cultures of Mycobacterium, Nocardia, Rhodococcus and the ‘aurantiaca’ taxon were subjected to numerical phenetic analyses using 92 unit characters. The data were examined using the simple matching (S SM), Jaccard (S J) and pattern (D p) coefficients and clustering was achieved using the unweighted average linkage algorithm. Neither cluster nor aggregate cluster composition was markedly affected by the coefficient used or by test error, estimated at 1·5%. The N. amarae strains formed a distinct and homogeneous cluster which showed its highest similarity to phena equated with Nocardia asteroides, Nocardia brasiliensis and Nocardia otitidis-caviarum. The non-hydroxylated fatty acid composition and overall size of the mycolic acids was similar to that found to be characteristic of Nocardia sensu stricto, though the long-chain in the 2-position of the mycolic acids was relatively much richer in monounsaturated components. Nocardia amarae, in containing dihydrogenated menaquinones with nine isoprene units, is clearly distinguished from established representatives of Nocardia.
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Classification of Mycobacterium farcinogenes and Mycobacterium senegalense by Immunodiffusion and Thin-layer Chromatography of Long-chain Components
More LessComparative immunodiffusion studies and thin-layer chromatographic analyses of whole-organism acid methanolysates were performed on 37 strains of Mycobacterium farcinogenes, Mycobacterium senegalense and Nocardia farcinica. The latter were clearly distinguished from the mycobacteria in containing a single mycolic acid methyl ester and showing more precipitinogens with nocardial than with mycobacterial and rhodococcal reference systems. The distribution of precipitinogens showed that M. farcinogenes and M. senegalense were very closely related and that both showed a greater affinity to Mycobacterium fortuitum than to any of the other established species of Mycobacterium tested. The complex pattern of a-mycolates and characteristic polar mycolates found in both M. farcinogenes and M. senegalense has only previously been found in M. fortuitum and Mycobacterium smegmatis.
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