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PubMed1991

Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and Streptomyces badius 252.

Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, lignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column.

Blotting, Western↗
PubMed2003

Gamma-butyrolactone autoregulators and receptor proteins in non- Streptomyces actinomycetes producing commercially important secondary metabolites.

The presence of gamma-butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non- Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non- Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.

4-Butyrolactone↗
PubMed2005

Identification of a lipoarabinomannan-like lipoglycan in the actinomycete Gordonia bronchialis.

The cell envelopes of actinomycetes contain lipidated macroamphiphiles, of which the most extensively characterised are the lipoarabinomannans of mycobacteria and related bacteria. We have investigated the mycolic acid-containing actinomycete Gordonia bronchialis and identified the presence of a lipoarabinomannan-like lipoglycan. The extraction and purification procedures recovered a second amphiphilic fraction with properties suggesting a phosphatidylinositol mannoside, consistent with studies of other Gordonia species.

Fatty Acids↗
PubMed2001

Motility Responses and Desiccation Survival of Zoospores from the Actinomycete Kineosporia sp. Strain SR11.

During investigations into the effects of water availability on the ecology of leaf litter actinomycetes, a motile-spored strain (SR11) was isolated and identified as a Kineosporia sp. Observations of spore motility revealed they could achieve speeds up to 160 mm s?1. Chemotaxis was not observed toward several organic compounds, although K, Mg, and Ca salts of phosphate, sulfate, and halides elicited a positive response. The inability of all the corresponding Na salts, and KNO3, to act as attractants suggested that chemoattraction of SR11 spores toward inorganic compounds may be dependent on specific cations and anions. An unusual motility response to CO2 was also observed and a laminar-flow microchamber was designed and constructed to investigate this phenomenon. Increasing the CO2 concentration by as little as 0.3% (v/v) caused a rapid, synchronous arrest of spore motility. A 1% (v/v) decrease in CO2 concentration caused a similar response, although the duration of the pause was much longer. The survival responses of strain SR11 spores were observed following desiccation at various water potentials. At the highest water potential tested (-9 MPa), spore viability declined minimally during the first 24 hr but continued to decrease over 10 days. In contrast, desiccation at -87 MPa and below resulted in a 50% loss in viability within 2 hr and subsequent survival at this level of viability. Continued desiccation at -302 MPa indicated that spores were capable of long-term survival at low water potentials. Many of these responses are likely to be significant in determining the ecology of these motile-spored actinomycetes in leaf litter.

Journal Article↗
PubMed2004

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila.

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg(-1). The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4-9) and temperature range (40-80 degrees C) and has a half-life of 1.2 h at 70 degrees C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K(m) values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the D,L-tryptophanamide and D,L-methioninamide were converted to the corresponding D,L-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.

Actinobacteria↗
PubMed2001

Purification and properties of a beta-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.

Amino Acid Sequence↗
PubMed2000

Multiple peptide synthetase gene clusters in Actinomycetes.

Two oligonucleotide probes derived from conserved motifs in peptide synthetases were hybridized with a cosmid library of Planobispora rosea genomic DNA. Detailed characterization of the physical organization of the positive cosmids indicated the existence of at least eight unlinked contigs containing multiple fragments that hybridized to both probes. Partial sequences of PCR products from the positive cosmids confirmed the existence of peptide synthetase genes. The combined results of hybridizations and physical mapping indicate that, in all likelihood, the isolated P. rosea contigs encode over 40 putative peptide synthetase modules. Similar results were obtained on screening a cosmid library of Actinoplanes teichomyceticus DNA. Furthermore, Southern hybridizations with several actinomycete strains, belonging to different genera, indicate that most strains contain multiple hybridizing bands well in excess of the number expected from the structure of the oligopeptides produced by these strains. Even strains not reported to produce oligopeptides gave clear positive signals when examined with the probes. These results strongly suggest that actinomycetes devote a notable fraction of their genomes to the non-ribosomal synthesis of peptides, and that most strains have the genetic potential to produce more oligopeptides than are currently described.

Actinomycetales↗
PubMed2006

Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench.

Thirty-eight actinomycetes were isolated from sediment collected from the Mariana Trench (10,898 m) using marine agar and media selective for actinomycetes, notably raffinose-histidine agar. The isolates were assigned to the class Actinobacteria using primers specific for members of this taxon. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Dermacoccus, Kocuria, Micromonospora, Streptomyces, Tsukamurella and Williamsia. All of the isolates were screened for genes encoding nonribosomal peptide and polyketide synthetases. Nonribosomal peptide synthetase sequences were detected in more than half of the isolates and polyketide synthases type I (PKS-I) were identified in five out of 38 strains. The Streptomyces isolates produced several unusual secondary metabolites, including a PKS-I associated product. In initial testing for piezotolerance, the Dermacoccus strain MT1.1 grew at elevated hydrostatic pressures.

Actinobacteria↗
PubMed2006

Antibiotic production by actinomycetes: the Janus faces of regulation.

This manuscript reviews some of the common regulatory mechanisms that control antibiotic production in actinomycetes. These ubiquitous bacteria, collectively responsible for the earthy smell of soil, are prolific producers of antibiotics and other secondary metabolites. The content of this review is biased towards the author's current research interests, concerning the action of regulatory gene products that control transcription of antibiotic-biosynthetic genes and the associated involvement of low molecular weight signalling molecules of the gamma-butyrolactone family. As a result, much fertile ground remains unturned particularly in the area of environmental monitoring and responses of actinomycetes to stimuli so perceived. Reviews casting a broader net are cited in the text.

Actinobacteria↗
PubMed2005

Isolation and characterization of alachlor-degrading actinomycetes from soil.

Alachlor (2-cloro-N-(methoxymethyl)-N-(2,6-diethylphenyl)-acetamide) is an extremely toxic and highly mobile herbicide that is widely used for pre-emergence control of grasses and weeds in many commercial crops in Brazil. In order to select soil actinomycetes able to degrade this herbicide, fifty-three actinomycete strains were isolated from soil treated with alachlor using selective conditions and subjected to in vitro degradation assays. Sixteen isolates were shown to be tolerant to high concentrations of the herbicide (up to 720 mg L(-1)), and six of these were able to grow and degrade >/= 50 alachlor (72 mg L(-1)) in mineral salts medium. Morphological and phylogenetic analysis enabled the assignment of the alachlor-degrading strains to the genus Streptomyces. Strain LS151 was related to the type strains of Streptomyces capoamus/Streptomyces galbus, whereas strains LS143 and LS153 were related to Streptomyces bikiniensis. The remaining strains, LS166, LS177 and LS182, were similar in morphological features and recovered in a single cluster based on 16S rDNA sequence analysis, but shown to be distinct on the basis of genomic fingerprint data (rep-PCR). Though a definitive taxonomic assignment of alachlor-degrading strains was not possible, these data indicate that ability to degrade this pesticide was detected in different Streptomyces taxa.

Acetamides↗
PubMed2005

Marine actinomycete diversity and natural product discovery.

Microbial natural products remain an important resource for drug discovery yet the microorganisms inhabiting the world's oceans have largely been overlooked in this regard. The recent discovery of novel secondary metabolites from taxonomically unique populations of marine actinomycetes suggests that these bacteria add an important new dimension to microbial natural product research. Continued efforts to characterize marine actinomycete diversity and how adaptations to the marine environment affect secondary metabolite production will create a better understanding of the potential utility of these bacteria as a source of useful products for biotechnology.

Actinobacteria↗
PubMed2005

Exploiting marine actinomycete biosynthetic pathways for drug discovery.

Drug discovery relies on the generation of large numbers of structurally diverse compounds from which a potential candidate can be identified. To this end, actinomycetes have often been exploited because of their ability to biosynthesize an impressive array of novel metabolites particularly polyketides. The genetic organization of polyketide synthases (PKSs) makes them readily amenable to manipulation, and thus re-engineering artificial or hybrid PKSs to produce unnatural natural products is a reality. This review highlights two approaches we have used to generate novel polyketides by manipulating genes responsible for starter unit biosynthesis in the 'Streptomyces maritimus' enterocin type II PKS. Our preliminary investigation into the biosynthesis of neomarinone, a rare marine actinomycete-derived meroterpenoid, is also presented.

Actinobacteria↗
PubMed1976

Home humidifier thermophilic actinomycete isolates.

Twenty-seven thermophilic actinomycete isolates obtained from humidification systems or living-bedroom areas of individuals with suspected but unproved home environment-related respiratory disease were characterized morphologically and biochemically. All isolates were demonstrated to be members of the Thermoactinomyces genus. Two previously misidentified isolates were reclassified as a Thermoacetinomyces sp. Thus, all of our thermophilic actinomycete humidifier isolates studied to date have been identified as either Thermoactinomyces vulgaris or Thermoactinomyces sp. Large numbers of unidentified thermotolerant bacteria have also been isolated from virtually all residual humidifier water samples and their possible role in production of "humidifier-associated" respiratory disease is unknown.

Actinomycetales↗
PubMed1996

Molecular genetic methods for improving secondary-metabolite production in actinomycetes.

The practical applications of genetic engineering to improve secondary-metabolite production in actinomycetes are potentially numerous, but have been limited by: (1) restriction barriers, which can hinder the introduction of DNA into many actinomycetes; (2) self-replicating plasmid-cloning vectors, which generally inhibit secondary-metabolite production; and (3) recombinants containing heterologous DNA, which may be subject to additional regulatory hurdles. Recently, intergeneric conjugation has been used to circumvent host restriction, and integration of cloned DNA into neutral genomic sites prevents product inhibition by self-replicating plasmids, and has enabled construction of recombinant strains lacking heterologous DNA sequences. The rpsL system permits direct selection for gene replacements and gene insertions that can facilitate this process.

Actinomycetales↗
PubMed1992

A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes.

A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes. This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101. The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker. To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts. The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes. Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts. An M13 ori allows the production of single-stranded DNA.

Actinomycetales↗
PubMed1990

Physiology of antibiotic production in actinomycetes and some underlying control mechanisms.

Some of the accumulated information on the physiology and nutritional control of antibiotic production in actinomycetes can now be related to recent discoveries in the field of actinomycete molecular biology. This review focuses on aspects of genetic and metabolic control of antibiotic biosynthesis. It surveys some well established principles in the relationship between primary and secondary metabolism, and summarizes briefly the areas where progress is being made in elucidating the molecular organization of regulatory systems underlying this relationship.

Journal Article↗
PubMed2005

Production of alkaline protease from an alkaliphilic actinomycete.

The repression of alkaline protease synthesis from alkaliphilic actinomycete was studied by using glucose, peptone, yeast extract, KH2PO4 and amino acids; tyrosine, tryptophan, lysine, and arginine. There was a critical limit of stimulation of enzyme production by these components. Crude components such as molasses, wheat flour, and wheat bran were found to be effective for growth and enzyme production. The high level of enzyme production using agro-industrial by-products is commercially significant due to cheap nature of these sources. The findings are quite attractive, as only few actinomycetes, particularly alkaliphilic ones, have so far been explored for their enzymatic potential and regulation of enzyme synthesis.

Actinobacteria↗
PubMed2005

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.

Actinobacteria↗