Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes.

Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.     

Positive selection of antibiotic-producing soil isolates.

Stepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing a collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produce antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0.1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7.7-8.0. Growth on proline (1%) and humic acid (0.1%) as sole carbon/nitrogen sources, growth on asparagine as a nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic-producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.     

Gene transfer between streptomycetes in soil.

The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.     

Biodegradation of alachlor by soil streptomycetes

Streptomycetes resistant to the herbicide alachlor [2-chloro-2,6-diethyl-N-(methoxymethyl) acetanilide] were used in degradation assays to characterize the products of alachlor biodegradation. Of six strains tested, Streptomyces sp. LS166, LS177, and LS182 were able to grow at an alachlor concentration of 144 mg l^–1 and degraded approximately 60–75% of the alachlor in 14 days, as evaluated by high performance liquid chromatography. The alachlor biodegradation products were identified by gas chromatography-mass spectrometry based on mass spectral data and fragmentation patterns. All compounds detected in these assays were similar for all streptomycetes strains tested, and involved dechlorination with subsequent N-dealkylation and cyclization of the remaining N-substituent with one of the ethyl groups to produce indole and quinoline derivatives. The enzymatic pathway used by Streptomyces sp. LS182 did not generate DEA (2,6-diethylaniline), a carcinogenic derivative of alachlor reported in other studies. Given the high degradation rates observed here, the Streptomyces strains tested may be useful in the degradation/detoxification processes of alachlor.     

Method for isolating cyanobacteria-lysing streptomycetes from soil

A new technique is described for the isolation and enumeration of cyanobacteria-lysing Streptomyces spp from soil or water. Two cyanobacteria, Anabaena cylindrica (ACTT 27899) and Tolypothrix tenuis (ATCC 27914) were used as the test organisms. Ten-day-old cyanobacterial cultures were vacuum-filtered to form a lawn on a 7 cm Whatman No. 1 filter paper which was then supported on Allen's agar. When the lawn was inoculated with dilutions of a heavy clay prairie soil and incubated at 27 PT 1dEC under constant illumination, white or grey colonies of streptomycetes grew. These colonies were surrounded by zones where a yellowing and lysis of the algal cells occurred. Streptomyces achromogenes was identified as a major lytic species within a population of 5 times 10₃ plaque-producing streptomycetes/g (dry weight) soil.     

Spontaneous sector formation in soil streptomycetes colonies

A model of the formation of sectors during the growth and zone formation of a colony of the soil radiant fungus streptomycetes was developed. It was shown that the basic parameters determining the shape of the sector border are the correlations between the growth rates of the sector of mycelium hyphae and the remainder of the colony and between the frequency and the numbers of branching order of these hyphae. Based on the long-standing observations, a polyseasonal dependence of spontaneous sector formation in streptomycetes zone-forming colonies was demonstrated.     

Biosystematic studies on novel streptomycetes from soil

Members of three putatively novel Streptomyces species, designated Streptomyces groups A, B and C, were repeatedly isolated from environmental samples taken from four hay meadow plots at Cockle Park Experimental Farm, Northumberland (UK). Representative isolates were found to have properties consistent with their classification in the genus Streptomyces and were recovered in three taxa using different phenotypic criteria, namely morphological and pigmentation properties, rapid enzyme tests, and whole-organism fatty acid, protein electrophoretic and pyrolysis mass-spectrometric data. The isolates were rapidly characterised as three taxonomic groups using pyrolysis mass spectrometry. The three taxa were also distinguished from one another and from validly described species of Streptomyces using rapid enzyme tests based on the fluorophores 7-amino-methylcoumarin and 4-methylumbelliferone, and computer-assisted identification procedures. The results indicate that selective isolation and rapid characterisation of streptomycetes using pyrolysis mass spectrometry provide a practical way of determining the phenotypic species diversity of streptomycetes in natural habitats. The experimental data also indicate that representative sampling of cultivable streptomycetes from soil can best be achieved using a multi-step extraction procedure coupled with the use of selective isolation procedures.     

Genetic determinants of bone mass.

A genetic contribution to bone mass determination was first described in the early 70s. Elucidation of gene contribution to this has since been attempted through studies analyzing associations between bone mass acquisition and/or maintenance and polymorphic variations of several genes. The first to be described was the vitamin D receptor gene (VDR), initially claimed to contribute to almost 75% of the genetic variation in bone mineral density (BMD) in twin and general population studies. Not all of the studies published to date conclude that a clear relationship exists between polymorphic VDR alleles and BMD, and the molecular basis for the VDR gene polymorphisms influence on bone mineralization has not yet been clarified. Since then, other genes with a significant role in bone metabolism such as estradiol receptor, collagen type 1alpha1, TGF-beta1, interleukin-6, calcitonin receptor, alpha2-HS-glycoprotein, osteocalcin, calcium-sensing receptor, interleukin-1 receptor antagonist, beta3-adrenergic receptor, apolipoprotein E, PTH, IGF-I and glucocorticoid receptor have been analyzed. Some polymorphic variations in these genes have been associated in some works with significant differences in BMD, with even more significant contributions when associations of different gene polymorphisms were analyzed. Again, the molecular basis for the contribution of these alleles to bone mass determination has not yet been described. A different approach has been attempted by linkage analysis of loci involved in bone density in pedigrees with low BMD using BMD as a quantitative trait. Recent results do not confirm, in these families, any association with any of the previously reported genes, but rather with other as yet unidentified genes. The genetic contribution to mild variations in the general population, as a result of environmental and endogenous individual influences, probably differs completely from that providing a pathologic BMD.     

Genetic factors that influence moenomycin production in streptomycetes

Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.     

Use of polyvalent phage for reduction of streptomycetes on soil dilution plates.

An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.     

Determination of metabolic activity of streptomycetes in soil microcosms

Two Streptomyces griseus strains were isolated from different soil types. S. griseus CAG17 strain was isolated from an agricultural area with low organic matter but rich in phosphorus content and S. griseus 26K strain was isolated from a forest area rich in organic matter with a low phosphorus content. The survival and metabolic activity of these isolates were studied in dynamic sterile soil microcosm systems. The fitness of each isolate was studied by re-inoculation in a soil type different from its origin. Maximum percentage of germination and respiration rates occurred within the first 48 h after each soil turnover (removal and addition of certain soil volumes). Data suggested that S. griseus CAG17 survived better independently of the soil type in comparison with S. griseus 26K which sporulated within the first 12 h after inoculation. Incubation temperatures did affect the lifecycles in relation to soil type. For example, the lowest temperature tested, 22 degrees C, was more favourable for extended germination and adaptation in general but revealed lesser spore numbers in the 'foreign' soil environment. Monitoring metabolic activity by estimation of urease, phosphatases and dehydrogenase-specific activities, between 18 and 35 degrees C incubation temperatures, was a reliable method for studying the survival and growth of streptomycete populations in soil. Results also confirmed that respiration rate and enzyme-specific activity corresponded with spore counts in long-term experiments which were designed for the investigation of survival and growth of S. griseus CAG17. Under selective pressure by heavy metals, in soil microcosm systems, metabolic activity proved a useful tool for the investigation of streptomycete activity. These methods could also be applied in agricultural field studies for monitoring microbial populations under conditions where various 'pollutants' are present in soil samples.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.     

Production of SacI and SacII isoschizomers by soil streptomycetes

Five fresh soil Streptomyces spp. strains were isolated, phylogenetically characterized on the basis of 16S rDNA sequences and analyzed for the presence of restriction modification systems. Three type II site-specific endonucleases were detected and partially purified. Two isolated enzymes were isoschizomers of SacI restriction endonuclease recognizing 5′-GAGCTC-3′ sequence; the third one recognised 5′-CCGCGG-3′ sequence and it was an isoschizomer of SacII. SacII like modification was observed in other two isolates having no detectable restriction activity. The lack of correlation between restriction and modification phenotypes and phylogenetic classification of the isolates indicates efficient gene transfer mechanism in the Streptomyces genus.     

Biodegradation of the herbicide diuron by streptomycetes isolated from soil

The diuron degrading activity of 17 streptomycete strains, obtained from agricultural and non-agricultural soils, was determined in the laboratory. All strains were identified as Streptomyces sp. by phenotypic characteristics and PCR-based assays. The strains were cultivated in liquid medium with diuron (4 mg L⁻¹) at 25 °C for 15 days. Biodegradation activity was determined by high-performance liquid chromatography. The results indicated that all strains were able to degrade diuron, but to different amounts. Twelve strains degraded the herbicide by up to 50% and four of them by up to 70%. Strain A7-9, belonging to S. albidoflavus cluster, was the most efficient organism in the degradation of diuron, achieving 95% degradation after five days of incubation and no herbicide remained after 10 days. Overall, the strains isolated from agricultural soils exhibited higher degradation percentages and rates than those isolated from non-agricultural soils. Given the high degradation activity observed here, the streptomycete strains show a good potential for bioremediation of soils contaminated with diuron.     

Biodegradation of the herbicide diuron by streptomycetes isolated from soil

The diuron degrading activity of 17 streptomycete strains, obtained from agricultural and non-agricultural soils, was determined in the laboratory. All strains were identified as Streptomyces sp. by phenotypic characteristics and PCR-based assays. The strains were cultivated in liquid medium with diuron (4 mg L⁻¹) at 25 °C for 15 days. Biodegradation activity was determined by high-performance liquid chromatography. The results indicated that all strains were able to degrade diuron, but to different amounts. Twelve strains degraded the herbicide by up to 50% and four of them by up to 70%. Strain A7-9, belonging to S. albidoflavus cluster, was the most efficient organism in the degradation of diuron, achieving 95% degradation after five days of incubation and no herbicide remained after 10 days. Overall, the strains isolated from agricultural soils exhibited higher degradation percentages and rates than those isolated from non-agricultural soils. Given the high degradation activity observed here, the streptomycete strains show a good potential for bioremediation of soils contaminated with diuron.     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.