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Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23.

The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, alpha and beta, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit alpha-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit beta. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits alpha and beta, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-beta-D-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits alpha and beta of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.

Amidohydrolases↗

Heterologous expression of biphenyl dioxygenase-encoding genes from a gram-positive broad-spectrum polychlorinated biphenyl degrader and characterization of chlorobiphenyl oxidation by the gene products.

The bphA1A2A3A4 gene cluster, encoding a biphenyl dioxygenase from Rhodococcus globerulus P6, a gram-positive microorganism able to degrade a wide spectrum of polychlorobiphenyls (PCBs), was expressed in Pseudomonas putida, thereby allowing characterization of chlorobiphenyl oxidation by this enzyme. While P6 biphenyl dioxygenase activity was observed in P. putida containing bphA1A2A3A4, no activity was detected in Escherichia coli cells containing the same gene cluster. In E. coli, transcription of genes bphB and bphCl, located downstream of bphA1A2A3A4, was shown to be driven solely by a vector promoter, which indicated that the lack of biphenyl dioxygenase activity was not due to a lack of mRNA synthesis. Radioactive labelling of bph gene products in E. coli implied inefficient translation of the bphA gene cluster or rapid degradation of the gene products. The biosynthesis of functional P6 biphenyl dioxygenase in P. putida cells containing the same plasmid construct that yielded no activity in E. coli emphasizes the importance of the host strain for heterologous expression and shows that synthesis, correct folding, and assembly of a Rhodococcus biphenyl dioxygenase can be achieved in a gram-negative organism. Dioxygenation of six mono- and dichlorinated PCB congeners by P. putida containing the P6 bphA gene cluster indicates the following ring substitution preference for this reaction (from most to least preferred): un-, meta-, para-, and ortho-substitution. No indications were found for dioxygenation of meta/para carbon pairs, or for hydroxylation of chlorinated carbons at any position of a monochlorinated ring, suggesting a strict specificity of this biphenyl dioxygenase for attack at nonhalogenated ortho/meta vicinal carbons. This contrasts the properties of an analogous enzyme from Pseudomonas sp. strain LB400, which can both dioxygenate at meta and para positions and dehalogenate substituted ortho carbons during ortho and meta dioxygenation.

Biodegradation, Environmental↗

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil.

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.

Arctic Regions↗

Bacterial composition of the biofilm on the surface of course sediment of the Danube: with special reference to the clinically important bacteria.

On monthly intervals and over a period of 14 months (November, 1993-December, 1994) biofilm samples from sediments taken at the Szentendre Island on the Danube were culturel and the isolated organisms were examined macromorphologically and micromorphogically and tested for oxidase and catalase production and their ability to oxidise and ferment glucose. The majority (85%) of the strains isolated were catalase positive. 43% were oxidase positive, 38% were glucose oxidisers and only 19% fermented glucose. In the sediment upmost biofilm layers Gram-negatives dominate (10 samples out of 14). A representative set strains has been subjected to complete identification using the Biolog automated identification system. The coryneform bacteria (e.g. Arthrobacter spp.) and members of the genus Rhodococcus dominated the Gram-positive bacteria while the dominant Gram-negative bacteria were facultative H2 autotrophs, methylotrophs (e.g. Xanthobacter flavus, Hydrogenophaga spp.) and different Aeromonas, Enterobacter and Pseudomonas species. Because the water supply of Budapest (the capital of Hungary) comes from bankwall filtred Danube water resources, the presence of clinically important microorganisms (i.e. Aeromonas, Enterobacter, Pseudomonas and Rhodococcus spp) in the Danube-sediments may pose a public health problem to the users (in particular the immunocompromised) of such water supplies.

Aeromonas↗

[Low-temperature microbial degradation of crude oil products differing in the extent of condensation].

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.

Benzene↗

[Comparison of the desulfurization activity among several bacteria and analysis of the conservation of their desulfurization genes].

Several bacteria, Bacillus brevis R-6, Pseudomonas delafleldii R-8, Nocardia globerula R-9, Bacillus sphaericus R-16, Rhodococcus erythropolis LSSE8-1 and Gordonia nitida LSSEJ-1, which can convert dibenzothiophene into 2-hydroxybiphenyl and sulfate, were investigated. Desulfurization products were quantitively determined by HPLC. Result revealed that each of these bacteria desulfurize DBT at a different rate. In order to obtain more information, the fragments encoding desulfurizing enzymes were studied. Desulfurization genes of R-6 and R-8 were separately amplified via PCR with specific primers based on the related sequences of Rhodococcus sp. IGTS8. Both sequences areminimally 99% related to IGTS8 sequence. Afterwards, dsz operon of LSSEJ-1 and R-9 were amplified and cloned. Sequences are also highly conservative. Data shows that identity of dszA between R-9 and IGTS8 is 99.6%, and identity of dszA between LSSEJ-1 and IGTS8 is 99.9%; dszB sequence of R-9 and LSSEJ-1 is 99.6% similarity to their counterpart sequence from IGTS8;Identity of dszC between R-9 and IGTS8 is 99.9%, and identity of dszC between LSSEJ-1 and IGTS8 is 99.1% . It may be deduced that the origins of desulfurization genes from mesophilic bacteria are the same.

Bacillus↗

Acquired immunodeficiency in a seven-year-old horse.

A 7-year-old horse with no previous history of illness was determined to have a systemic infection of Rhodococcus equi. Rhodococcus equi was isolated from blood, tracheal fluid, and feces. Lymphopenia, failure to respond to concanavalin A and phytohemagglutinin lymphocyte stimulation testing, decreased concentrations of immunoglobulin (Ig)M, IgA, and IgG, low R equi antibody titer, histologic depletion of lymphoid tissue, and a failure to respond to antigenic stimulation led to the conclusion that both humoral and cell-mediated immunity were compromised. No cause for the acquired immunodeficiency could be determined.

Actinomycetales Infections↗

[Susceptibility of bacterial isolates from the equine respiratory tract to trimethoprim, sulfadoxine, sulfadimethoxine and combinations of these compounds].

Using a broth microdilution technique, the in vitro susceptibility of bacterial isolates from the equine respiratory tract to trimethoprim, sulfadoxine, sulfadimethoxine, and combinations of these compounds was determined. The bacterial strains (n = 88) isolated recently from horses with respiratory symptoms belonged to the following species: Streptococcus equi subsp. zooepidemicus (n = 34), Streptococcus equi subsp. equi (n = 22), Staphylococcus aureus (n = 9), Klebsiella pneumoniae (n = 7), Rhodococcus equi (n = 4), Pseudomonas spp. (n = 3) and Escherichia coli (n = 3). In addition, two isolates of Enterobacter spp. and one isolate of Streptococcus equisimilis, Staphylococcus intermedius, Proteus mirabilis and Serratia marcescens were examined. For determination of susceptibility of an organism the following minimal inhibitory concentrations (MIC) were fixed as limiting values: Trimethoprim < or = 0.5 microgram/ml, sulfadoxine < or = 32 micrograms/ml, sulfadimethoxine < or = 32 micrograms/ml, trimethoprim/sulfadoxine < or = 0.5/32 micrograms/ml, trimethoprim/sulfadimethoxine < or = 0.5/32 micrograms/ml. As expected, Rhodococcus-equi-isolates were resistant to the antimicrobials tested. However, most of the clinically more common isolates showed a high degree of susceptibility to the combinations. The fractional inhibitory concentration (FIC) indices indicated synergism of the combination-partners in a wide range. According to these in vitro results, application of trimethoprim/sulfonamide combinations for the initial therapy of equine respiratory tract infections can be recommended.

Animals↗

Uncommon manifestations of opportunistic infections in an HIV infected patient.

A case of an HIV infected patient complicated with Penicillium marneffei and Rhodococcus equi infection is reported. He presented with chronic ulcer as pyoderma gangrenosum-like on his right calf and submandibular lymphadenitis as scrofuloderma-like. Penicillium marneffei and Rhodococcus equi were isolated from the ulcer and lymph node respectively.

AIDS-Related Opportunistic Infections↗

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN.

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.

Drug Design↗

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa.

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 microgram/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C44 or C46 ) was inhibited more significantly than shorter homologues such as C38 or C40 . In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 micrograms/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of alpha-alkyl chain, but to the inhibition of chain elongation of C28 to C32 straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di- mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 micrograms/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C44 or C46 specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C28 or C30.

Actinomycetales↗

Parallel antitumor, granuloma-forming and tumor-necrosis-factor-priming activities of mycoloyl glycolipids from Nocardia rubra that differ in carbohydrate moiety: structure-activity relationships.

Multiple intravenous injections (30 micrograms, ten times) in ICR mice of trehalose dimycolate and glucose monomycolate from Nocardia rubra, containing C36-48 mycolic acids, showed a prominent antitumor effect on a subcutaneously implanted sarcoma-180, an allogeneic sarcoma of mice with a significant granuloma formation in lungs, spleen and liver. On the other hand, mycoloyl glycolipids other than glucose monomycolate and trehalose dimycolate, such as mannose or fructose mycolate, showed no significant activity for tumor regression or granuloma formation in mice. Trehalose dimycolate and glucose monomycolate from N. rubra, and glucose monomycolate with C56-60 mycolic acids from Rhodococcus terrae also showed a distinctive priming activity for tumor necrosis factor (TNF), when lipopolysaccharide from Escherichia coli was administered as an eliciting agent. The TNF activity in the sera of mice was abrogated almost completely by anti-(murine TNF alpha) antibody with protein-A-agarose. Again in contrast, mannose and fructose mycolate from N. rubra and glucose monomycolate with C30-34 mycolic acids from Rhodococcus equi did not show such activities in mice. Meth-A, a syngeneic fibrosarcoma of BALB/c mice, was less sensitive to administration of glycolipids than sarcoma-180. These results indicated that the existence of a glucose or trehalose molecule was necessary for the expression of immunomodifying activities among various mycoloyl glycolipids differing in carbohydrate structure. However, since the administration of lipopolysaccharide was essentially required as an eliciting agent for the induction of TNF, while no eliciting agent was required for the antitumor activities, TNF does not seem to contribute directly to the antitumor activities of mycoloyl glycolipids in our systems. There was, however, a parallel structure-activity relationship among granuloma-forming, antitumor and TNF-priming activities, indicating that the structures of both the carbohydrate moiety and the mycoloyl residues influenced an initial step, such as macrophage activation, commonly and profoundly.

Animals↗

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds.

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 microgram/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Agar↗

Triacylglycerols in prokaryotic microorganisms.

Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.

Bacteria↗

Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria.

The enzymatic hydrolysis of the nitrile group of different 2-acetoxynitriles was investigated in order to obtain catalysts that chemoselectively hydrolyse nitriles in the presence of ester groups. The biotransformation of four 2-acetoxynitriles [2-acetoxybutenenitrile (ABN), 2-acetoxyheptanenitrile (AHN), 2-acetoxy-2-(2-furyl)acetonitrile (AFN), and 2-acetoxy-2,3,3-trimethylbutanenitrile (ATMB)] by different bacterial strains that synthesise nitrilases or nitrile hydratases was studied. ABN, AHN and AFN were converted by various microorganisms belonging to different bacterial genera (e.g. Pseudomonas or Rhodococcus) expressing either nitrilase or nitrile hydratase activities. In contrast, no metabolism of the sterically hindered substrate ATMB was observed. All wild-type strains investigated formed considerable amounts of cyanide and aldehydes from the 2-acetoxynitriles. This indicated the presence of esterases converting the 2-acetoxynitriles to 2-hydroxynitriles, which then spontaneously decomposed to the corresponding aldehydes and cyanide. In order to suppress unwanted side-reactions, biotransformations were performed with recombinant Escherichia coli strains that heterologously expressed nitrilase activities originating from Pseudomonas, Rhodococcus, or Synechocystis strains. The attempted conversion of the 2-acetoxynitriles to almost stoichiometric amounts of the corresponding 2-acetoxycarboxylic acids was finally achieved by using either a recombinant E. coli strain that highly overexpressed the nitrilase gene from the pseudomonad or the purified enzyme derived from this strain.

Acetonitriles↗

Analysis of the relative abundance of different types of bacteria capable of toluene degradation in a compost biofilter.

A microbial community of a compost biofilter treating toluene vapors was investigated using serum-bottle assays and mineral-agar plates. Toluene was not consumed in the absence of oxygen. However, filter-bed extracts exposed to toluene vapor as the only carbon source produced distinct colony types (phenotypic groups) that were counted separately. Strains from each group were isolated and checked for toluene-degradation activity in serum bottles. Only 15% of colonies were true toluene degraders. This population was divided into 11 genotypic groups based on DNA fingerprints. Identification of a member of each group using 16S rRNA gene-sequence comparison showed that they belonged to seven genera: Acinetobacter, Azoarcus, Mycobacterium, Nevskia, Pseudomonas, Pseudonocardia and Rhodococcus. Together, members of the genera Pseudonocardia and Rhodococcus were 34 times more numerous than all the others. We hypothesized that these two organisms are K-strategists (adapted to a resource-restricted and crowded environment) and that the compost biofilter is a K-environment. This would explain why they are not outnumbered by faster growers like Pseudomonas or Acinetobacter species, which would be r-strategists (adapted to a resource-abundant and uncrowded environment).

Bacteria↗

Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function.

Epoxide hydrolases are vital to many organisms by virtue of their roles in detoxification, metabolism and processing of signaling molecules. The Mycobacterium tuberculosis genome encodes an unusually large number of epoxide hydrolases, suggesting that they might be of particular importance to these bacteria. We report here the first structure of an epoxide hydrolase from M.tuberculosis, solved to a resolution of 2.5 A using single-wavelength anomalous dispersion (SAD) from a selenomethionine-substituted protein. The enzyme features a deep active-site pocket created by the packing of three helices onto a curved six-stranded beta-sheet. This structure is similar to a previously described limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis and unlike the alpha/beta-hydrolase fold typical of mammalian epoxide hydrolases (EH). A number of changes in the mycobacterial enzyme create a wider and deeper substrate-binding pocket than is found in its Rhodococcus homologue. Interestingly, each structure contains a different type of endogenous ligand of unknown origin bound in its active site. As a consequence of its wider substrate-binding pocket, the mycobacterial EH is capable of hydrolyzing long or bulky lipophilic epoxides such as 10,11-epoxystearic acid and cholesterol 5,6-oxide at appreciable rates, suggesting that similar compound(s) will serve as its physiological substrate(s).

Amino Acid Sequence↗

Immunodiffusion and lipid analyses in the classification of "Mycobacterium album" and the "aurantiaca" taxon.

Comparative immunodiffusion studies were performed on representative strains of "Mycobacterium album", the "aurantiaca" taxon and an "aurantiaca"-like group of actinomycetes, using reference systems representing 25 taxa assigned to the genera Arthrobacter, Corynebacterium, Kurthia, Mycobacterium, Nocardia, Rhodococcus, Streptomyces and the "aurantiaca" taxon. Thin-layer chromatographic analyses of whole-organism methanolysates were carried out on single strains of "Mycobacterium album" and the "aurantiaca"-like taxon. Both the distribution of precipitinogens and the type of mycolic acids determined showed that the "Mycobacterium album" and "aurantiaca" strains were closely related to one another, but not to the "aurantiaca"-like strains. The aggregate "aurantiaca" taxon, containing "Mycobacterium album" and the "aurantiaca" taxon, can be distinguished from the established mycolic-acid containing taxa, Corynebacterium, Mycobacterium, Nocardia and Rhodococcus, using both serological and lipid data. Further studies on additional strains are needed to determine whether the aggregate "aurantiaca" taxon merits generic status.

Actinomycetales↗