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PubMed2001

Different physiological roles of ATP- and PP(i)-dependent phosphofructokinase isoenzymes in the methylotrophic actinomycete Amycolatopsis methanolica.

Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827-6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK. The two A. methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. The A. methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, the A. methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest that A. methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.

Actinomycetales↗
PubMed2005

Characterization of a truncated lipoarabinomannan from the Actinomycete Turicella otitidis.

Lipoarabinomannan (LAM) lipoglycans have been characterized from a range of mycolic acid-containing actinomycetes and from the amycolate actinomycete Amycolatopsis sulphurea. To further understand the structural diversity of this family, we have characterized the lipoglycan of the otic commensal Turicella otitidis. T. otitidis LAM (TotLAM) has been determined to consist of a mannosyl phosphatidylinositol anchor unit carrying an (alpha 1-->6)-linked mannan core and substituted with terminal-arabinosyl branches. Thus, TotLAM has a novel truncated LAM structure. Using the human monocytic THP-1 cell line, it was found that TotLAM exhibited only minimal ability to induce tumor necrosis factor alpha. These findings contribute further to our understanding of actinomycete LAM diversity and allow further speculation as to the correlation between LAM structure and the immunomodulatory activities of these lipoglycans.

Actinobacteria↗
PubMed1967

Flagellated actinomycetes.

Shadowed motile elements from actinomycetes were observed with an electron microscope. Included were three strains of Actinoplanes, two of Ampullariella, two of Dermatophilus, two of Spirillospora, and four of "Nocardia" turbata. In addition, three types of previously undescribed actionmycetes were represented: (i) the C(4) group (four strains) forming substrate mycelium breaking into motile rods; (ii) strain 9-41, forming Microellobosporia-like sporangia with motile spores; and (iii) strain P(2), forming aerial hyphae releasing motile cocci when put in water. All the known chemical cell wall types of actinomycetes except the Nocardia asteroides type and the Actinomyces israeli type were represented in this array of motile actinomycetes. Motile elements were, depending on the genus, cocci, rods (often curved), or pyriform. Flagella were always in tufts (or single), never peritrichous. A relationship seems to exist between the location of the tuft and the cell wall composition. The spores of one strain of Actinoplanes were herniated, thus resembling plasmoptysis forms of bacteria.

Actinomycetales↗
PubMed1997

Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

Actinomyces↗
PubMed2004

Sequence-based identification of aerobic actinomycetes.

We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the genera Streptomyces, Gordonia, and Tsukamurella and 10 taxa of Nocardia were studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65 hsp gene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing of Nocardia species revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences of Nocardia transvalensis, N. otitidiscaviarum, and N. nova isolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within the Nocardia spp. are unnamed at present. A major unresolved issue is the type strain of N. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.

Actinobacteria↗
PubMed1994

The molecular phylogeny and systematics of the actinomycetes.

Sequences of 16S ribosomal RNA have provided actinomycetologists with a phylogenetic tree that allows the investigation of the evolution of actinomycetes and also provides a basis for classification. The origin of actinomycetes and, except for bifidobacteria, the order by which the main sublines evolved, cannot yet be determined with certainty. However, calibration of rRNA sequence divergence with palaeochemical data, and previously published substitution rates of endosymbiotic bacteria, suggest that the main radiation occurred less than 1 billion years ago. Within this radiation, several phylogenetically homogeneous, but sometimes phenotypically heterogeneous, clades appear to have diverged over a short evolutionary period. The resolution of the 16S rRNA molecule appears to be insufficient to clearly determine the branching patterns between clades in this area of the phylogenetic tree. The distribution of some morphological and chemotaxonomic traits such as types of peptidoglycan, menaquinone, phospholipids, cell wall sugars, and fatty acids facilitate the phenotypic delineation of genera within each clade. At higher taxonomic levels, e.g. at the family level, phenotypic similarities are unpredictable and tend to be less conserved. With the exception of mycolic acids, most traits are polyphyletic--hence they are unreliable indicators per se of phylogenetic relationships. Nevertheless, combinations of phenotypic properties are invaluable for predicting whether a new organism is likely to be a member of an established or a novel taxon. Current knowledge about the phylogenetic structure of the actinomycetes provides not only a sound basis for future taxonomic work but also a framework for the rational exploration of their ecology and biotechnological potential.

Actinomycetales↗
PubMed2005

Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes: isolation, characterization and generation of novel glycosylated derivatives.

Many bioactive natural products synthesized by actinomycetes are glycosylated compounds in which the appended sugars contribute to specific interactions with their biological target. Most of these sugars are 6-deoxyhexoses, of which more than 70 different forms have been identified, and an increasing number of gene clusters involved in 6-deoxyhexoses biosynthesis are being characterized from antibiotic-producing actinomycetes. Novel glycosylated compounds have been generated by modifying natural deoxysugar biosynthesis pathways in the producer organisms, and/or the simultaneous expression in these strains of selected deoxysugar biosynthesis genes from other strains. Non-producing strains endowed with the capacity to synthesize novel deoxysugars through the expression of engineered deoxysugar biosynthesis clusters can also be used as alternative hosts. Transfer of these deoxysugars to a multiplicity of aglycones relies upon the existence of glycosyltransferases with an inherent degree of 'relaxed substrate specificity'. In this review, we analyze how the knowledge coming out from isolation and characterization of deoxysugar biosynthesis pathways from actinomycetes is being used to produce novel glycosylated derivatives of natural products.

Actinobacteria↗
PubMed1976

The frequency of precipitins to trichloroacetic acid-extractable antigens from thermophilic actinomycetes in farmer's lung patients and asymptomatic farmers.

The hypothesis that the trichloroacetic acid (TCA)-extractable antigen fractions prepared from Micropolyspora faeni cells and other thermophilic actinomycetes are better diagnostic immunodiffusion reagents for the detection of farmer's lung disease than the crude extracellular antigen concentrates has been tested. The frequency of serum precipitins to the TCA-extractable antigens has been compared in symptomatic farmer's lung patients and in healthy farmers having positive precipitin tests to the crude antigen concentrates of the analogous thermophilic actinomycetes. Sixty-eight per cent of the symptomatic group were found to have precipitins to the M. faeni TCA-extractable antigens compared with only 22 per cent of the asymptomatic group. With regard to the other thermophilic actinomycetes tested, no significant differences in frequencies of precipitins to TCA-extractable antigens between the symptomatic and asymptomatic groups were found. However, the total number of patients available for testing with each of these antigens was small. Sufficient chemical characterization of the TCA-extractable antigens was carried out to show that they were chemically similar to the TCA-extractable antigens prepared by other workers.

Antigens, Bacterial↗
PubMed2002

Isolation and characterization of dibenzofuran-degrading actinomycetes: analysis of multiple extradiol dioxygenase genes in dibenzofuran-degrading Rhodococcus species.

Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edil to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.

Actinobacteria↗
PubMedDate not supplied

Farmer's lung: thermophilic actinomycetes as a source of "farmer's lung hay" antigen. 1963.

Mouldy hay was produced in the laboratory by sterilising good hay, inoculating with aqueous suspensions of microorganisms, and incubating at 40 degrees or 60 degrees C. Extracts were tested for presence of farmer's lung hay (F.L.H.) antigen by agar-gel double-diffusion and immunoelectrophoresis tests against sixteen to twenty sera from patients with farmer's lung. F.L.H. antigen developed in hay after: (1) inoculating with mixed microbial suspensions from antigenically active hay; (2) inoculation with mixed suspensions of pure cultures of thermophilic actinomycetes, after raising the pH of the hay to 70 either by prior inoculation with fungi or by infiltration with ammonia vapour; and (3) inoculation at pH 70 with pure cultures of Thermopolyspora polyspora or with Micromonospora vulgaris. F.L.H. antigen did not develop in hay after inoculation with fungi only, or with six other actinomycetes tested, or after prior heating (though some sera reacted to fungal antigens in all these extracts). T. polyspora is the richest source yet found of F.L.H. antigen, and inhalation of an extract by affected subjects produces some of the features of farmer's lung. Pure cultures can produce F.L.H. antigen on artificial media without hay. Spores and mycelium are rich in F.L.H. antigen, and inhalation of the spores may play a part in farmer's lung disease. Other antigens relevant to farmer's lung may be found in other actinomycetes, not yet cultured.

Air Pollutants, Occupational↗
PubMed1983

Multiple resistance to aminoglycoside antibiotics in actinomycetes.

Actinomycetes were characterized in terms of resistance to 11 different aminoglycoside antibiotics (AGs). Strains freshly isolated in AG containing media showed wide varieties of multiple AG resistance, while the majority of ISP (International Streptomyces Project) cultures and the actinomycete strains isolated in an AG free medium were susceptible to all or most of the AGs tested. Marked characteristics were noted in multiple AG resistance of gray and yellow colored actinomycetes and AG-producing strains. In gray colored isolates, multiple resistance to kanamycin A, dibekacin, ribostamycin, butirosin A, istamycin A and neamine was often observed. Yellow colored isolates having multiple AG resistance were mostly resistant to neamine, ribostamycin and streptomycin and, to a lesser extent, istamycin A, dibekacin and butirosin A. Most of the AG producers tested showed unique multiple AG resistance patterns.

Actinomycetales↗
PubMedDate not supplied

[Population dynamics of oligosporous actinomycetes in Chernozem soil].

Investigation of the dynamics of an oligosporous actinomycete population in chernozem soil in the course of succession induced by soil wetting allowed us to reveal the time intervals and conditions optimal for the isolation of particular oligosporous actinomycetes. Saccharopolysporas and microbisporas proved to be best isolated in the early and late stages of succession, whereas actinomycetes of the subgroup Actinomadura and saccharomonosporas could be best isolated in the early and intermediate stages of succession.

Actinomycetales↗
PubMedDate not supplied

[Lipoamino acids in the composition of actinomycete polar lipids].

The capacity for substitution of phospholipids, in particular phosphatidyl ethanolamine, by a phosphorus-lacking lipoamino acid was studied in different groups of actinomycetes. In the conditions of phosphorus deficiency, most cultures were found to be capable of synthesizing a phosphorus-lacking nitrogen-containing lipoamino acid. Its characteristics (Rf) in TLC are similar to those of ornithinolipid. This fact confirms, for the first time and for different groups of actinomycetes, the hypothesis concerning interchangeability of membranous lipids. In the case of Actinomyces olivaceus, phosphatidyl ethanolamine and ornithine containing a phosphorus-lacking lipid can be regarded, this being corroborated by comparative data about their quantitative content. The detection of the phosphorus-lacking nitrogen-containing lipid in the composition of the polar fraction in different actinomycetes in the conditions of phosphorus deficiency in the medium demonstrates the possibility of adaptive changes in the composition of membranous lipids in microorganisms depending on their habitat.

Chromatography, Thin Layer↗
PubMed2002

Increasing the efficiency of heterologous promoters in actinomycetes.

An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.

Actinobacteria↗
PubMed1975

[Hybridization in Actinomycetes].

Ways of transferring genetic information in Actinomycetes and their use for the transmission of genetic material in intervarietal crosses are discussed. The producing of hybrids is shown to be an efficient method to obtain Actinomycetes strains with new properties. Properties of recombinants obtained in intervarietal crosses Streptomyces coelicolor A3(2)XS. lividans 66 are shown to possess new antibiotic properties differing from those of parental strains: they suppress a number of Actinomycetes and bacterial strains which are not affected by parental strains. It is found that at least two groups of genes located on chromosomes of S. coelicolor A3(2) and S. lividans 66 participate in the synthesis of the antibiotic. The obtaining of recombinants between S. coelicolor A3(2) and S. griseus 15 makes possible to select variants which are capable to produce the antibiotic grisine and are resistant to actinophages, specifically attacking S. griseus. Properties of recombinants between S. coelicolor A3(2) and S. griseus 15 make possible to localize on parental chromosomes regions containing genes which control the synthesis of the antibiotic, the formation of a receptor for the adsorption of actinophages, and genes controlling the restriction and t he modification of actinophage development in S. coelicolor and S. griseus. A sex plasmid (SGP1) is found in S. griseus 15.

Actinomyces↗
PubMedDate not supplied

[Variety-specific actinomycete complexes associated with barley roots in soddy podzolic soil].

The root-colonizing actinomycete complexes of genotypically different barley plants grown in soddy podzolic soil were found to contain streptomycetes of the sections Cinereus (series Chromogenes, Achromogenes, and Aureus) and Roseus (series Fradiae), dominant being streptomycetes of the section Cinereus ser. Chromogenes. The abundance and diversity of soil streptomycetes in the barley rhizoplane increased in the order: var. 999-93 < var. Kumir < var. Novichok < var. 889-93. Experiments revealed functional specificity in the root-associated actinomycete complexes of different barley varieties. The actinomycete complex colonizing the barley var. 999-93 roots was distinguished by a wide range of utilizable root exudate metabolites and a low occurrence rate of antagonistic species.

Actinobacteria↗
PubMedDate not supplied

Bioactive actinomycetes from Krishna River sediments of Andhra Pradesh.

Sediment samples from Krishna River at Nagayalanka of Andhra Pradesh, India were investigated as a source of actinomycetes to screen for the production of novel bioactive compounds. During our investigation on fresh water actinomycetes from 5 different river sediment samples, a total of 80 actinomycetes were isolated. Out of these 80 isolates, 30 isolates which showed distinct macromorphological characteristics were selected. The antimicrobial and enzymatic activities were studied for all the 30 isolates. The preliminary study for antimicrobial activity by cross streak method indicated that 16 isolates (53.3%) have excellent antagonistic properties. All these 16 isolates were subjected to detailed submerged fermentation studies. It was observed that 12 isolates (40.0%) exhibited antibacterial activity, 9 isolates (30.0%) showed antifungal activity while 5 isolates (16.6%) showed both antibacterial and antifungal activities. All the 30 isolates were also subjected for the determination of enzymatic activities 25 isolates (83.3%) exhibited amylolytic activity while 27 isolates (90.0%) showed proteolytic activity. Among these isolates, six promising isolates were selected for detailed morphological, cultural, physiological and biochemical studies. It was established that these isolates belong to the Streptomyces genus by virtue of their cell wall composition pattern and were identified as strains of different Streptomyces species like S. rochei, S. alanosinicus, S. erumpens, S. griseoplanus, S. gancidicus and S. nigrogriseolus.

Actinobacteria↗
PubMed2005

Molecular detection of alpha-glucosidase inhibitor-producing actinomycetes.

In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of C7N aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of C7 aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 C7 aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of C7 aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.

Actinobacteria↗