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Biomedical subjects

E M Wellington

Publications and source records attributed to E M Wellington.

At least 19 recordsLinked to original sources
PubMed2001

Phylogeny of Streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters.

The phylogenetic relationships of a collection of streptomycete soil isolates and type strains were resolved by sequence analysis of trpB, a housekeeping gene involved in tryptophan biosynthesis. The analysis confirmed that two isolates were recipients in a gene transfer event, demonstrated by phylogenetic incongruency between trpB and strB1 trees. One strain had acquired the entire streptomycin biosynthetic cluster, whilst the other contained only strRAB1, the resistance gene and two flanking genes from the cluster. Sequence analysis of trpB, as part of a polyphasic approach, was a useful tool in determining intra-generic relationships within the genus Streptomyces.

Bacterial Proteins↗
PubMed2001

Assessment of chitin decomposer diversity within an upland grassland.

The breakdown of chitin within an acidic upland grassland was studied. The aim was to provide a molecular characterisation of microorganisms involved in chitin degradation in the soil using soil microcosms and buried litter bags containing chitin. The investigation involved an examination of the effects of liming on the microbial communities within the soil and their chitinolytic activity. Microcosm experiments were designed to study the influence of lime and chitin enrichment on the grassland soil bacterial community ex situ under controlled environmental conditions. Bacterial and actinomycete counts were determined and total community DNA was extracted from the microcosms and from chitin bags buried at the experimental site. PCR based on specific 16S rRNA target sequences provided products for DGGE analysis to determine the structure of bacterial and actinomycete communities. Chitinase activity was assessed spectrophotometrically using chitin labelled with remazol brilliant violet. Both liming and chitin amendment increased bacterial and actinomycete viable counts and the chitinase activity. DGGE band patterns confirmed changes in bacterial populations under the influence of both treatments. PCR products amplified from DNA isolated from chitin bags were cloned and sequenced. Only a few matched known species but a prominent coloniser of chitin proved to be Stenotrophomonas maltophilia.

Actinomycetales↗
PubMed2001

The taxonomy of Streptomyces and related genera.

The streptomycetes, producers of more than half of the 10,000 documented bioactive compounds, have offered over 50 years of interest to industry and academia. Despite this, their taxonomy remains somewhat confused and the definition of species is unresolved due to the variety of morphological, cultural, physiological and biochemical characteristics that are observed at both the inter- and the intraspecies level. This review addresses the current status of streptomycete taxonomy, highlighting the value of a polyphasic approach that utilizes genotypic and phenotypic traits for the delimitation of species within the genus.

Base Sequence↗
PubMed2001

Biotransformation of selamectin with Streptomyces lydicus SX-1298 using a novel static agar fermentation system with Reemay mesh.

A novel approach to biotransformation is described using a solid medium matrix and Reemay mesh that gives efficient biotransformation of compounds with minimal matrices in the ensuing gum solids. Using this approach with a newly isolated biotransforming organism, Streptomyces lydicus SX1298, a series of hydroxylations and an O-demethylation is described for selamectin the first endectocide for cats and dogs.

Antiparasitic Agents↗
PubMed2000

Kyanomycin, a complex of unusual anthracycline-phospholipid hybrids from Nonomuria species.

A blue secondary metabolite complex, named kyanomycin (2), in addition to epsilon-rhodomycinone (1), was detected in the mycelium extract of Nonomuria sp. NN 22303 by HPLC-diode array and HPLC-electrospray mass spectrometry screening. The chemical structures of the novel compounds were determined to be unusual anthracycline-phosphatidylethanolamine hybrids by spectroscopic and chemical degradation experiments.

Actinomycetales↗
PubMed2000

Molecular detection of bacterial and streptomycete chitinases in the environment.

Sets of PCR primers were designed to amplify bacterial chitinases at different levels of specificity. The bacterial chitinase group primers were successful in targeting enzymes classified within the group A glycosyl hydrolases of family 18. The widespread occurrence of group A bacterial chitinases in actinomycetes was demonstrated. Streptomycete chitinase specific primers were designed and a collection of type strains of species changed in the genes Streptomyces were screened and shown to have at least one and usually multiple chitinase genes. The presence of the gene for the chitin binding protein was also demonstrated within the streptomycete type strains. These data indicate that streptomycetes are well equipped to degrade chitin. The detection of group A chitinases in total community DNA is described and a sandy soil shown to contain more than 10 different genes using DGGE to indicate genetic diversity.

Actinomycetales↗
PubMed2000

Interspecific transfer of Streptomyces giant linear plasmids in sterile amended soil microcosms.

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.

Blotting, Southern↗
PubMed2000

Mathematical analysis of growth and interaction dynamics of streptomycetes and a bacteriophage in soil.

We observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its host Streptomyces lividans TK24 in sterile soil microcosms. Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts. The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens. The interaction of S. lividans and KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments. This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics. Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates. There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (>150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean [SEM], 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage. We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil.

Bacteriophages↗
PubMed2000

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt.

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.

Antibiosis↗
PubMed2000

Novel streptopyrroles from Streptomyces rimosus with bacterial protein histidine kinase inhibitory and antimicrobial activities.

A series of halogenated pyrrolo [2,1-b] [1,3] benzoxazines (1 approximately 9) was isolated from fermentations of an actinomycete strain X10/78/978 (NCIMB40808), identified as Streptomyces rimosus, during a microbial extract screening programme to identify inhibitors of bacterial histidine kinase. The structures of these compounds were elucidated by spectroscopic methods including the HMQC, HMBC and INADEQUATE NMR experiments. The structure of 1 was confirmed by X-ray crystallographic studies. Compounds 5 and 6 were produced in fermentations in the presence of NaBr and NaI respectively. The most abundant member of the series, streptopyrrole, 1, inhibited the nitrogen regulator II (NRII) histidine kinase from Escherichia coli with an IC50 of 20 microM and exhibited antimicrobial activity against a range of bacteria and fungi.

Anti-Bacterial Agents↗
PubMed1999

Comparison of different methods for the isolation and purification of total community DNA from soil.

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.

DNA↗
PubMed1998

Evidence for transfer of antibiotic-resistance genes in soil populations of streptomycetes.

Phylogenetic analysis was used to evaluate the hypothesis of gene transfer in streptomycetes, many of which are antibiotic producers. The diversity and possible origins of streptomycin-resistance genes was investigated for a population of Streptomyces strains isolated from a site in Brazil where antibiotic production had previously been implicated The analysis provides compelling evidence for the transfer of these genes. Examination of other Streptomyces-type strains also reveals a scattered distribution of streptomycin producers with respect to the overall phylogeny. These results suggest that horizontal gene transfer may be an important factor in the evolution of antibiotic genes in streptomycetes.

Anti-Bacterial Agents↗
PubMed1998

Phenotypic, genotypic and pathogenic variation among streptomycetes implicated in common scab disease.

This study examines the diversity of streptomycetes causing potato scab using phenotypic identification, 16S rRNA sequence, and pathogenicity measurements. Pathogenicity did not correlate well with taxonomic identification, nor with phylogenetic position. This result suggests that neither phenotypic nor 16S characteristics are diagnostic for scab-causing strains. Additionally, phenotypic characterizations were not fully consistent with phylogenetic relationships. This incongruency highlights the difficulties of taxonomy within the streptomycetes and affirms the need for better integration of genetic and phenotypic characters in the systematics of this group.

DNA, Bacterial↗
PubMed1998

Transfer of streptomycin biosynthesis gene clusters within streptomycetes isolated from soil.

Streptomyces strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.

Chromosome Mapping↗
PubMed1997

Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.

Amino Acid Sequence↗
PubMed1997

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.

Actinomycetales↗
PubMed1996

Physical map of the genome of Planctomyces limnophilus, a representative of the phylogenetically distinct planctomycete lineage.

A physical map of the chromosome of Planctomyces limnophilus DSM 3776T was constructed by pulsed-field gel electrophoresis techniques. A total of 32 cleavage sites for the rare-cutting restriction endonucleases PacI, PmeI, and SwaI were located on the chromosome, which was shown to be circular and approximately 5.2 Mbp in size. An extrachromosomal element was detected but was found not to be cleaved by any of the enzymes used in the analysis of the chromosome. The order of the fragments on the chromosome was determined by hybridization of excised, labelled restriction fragments to Southern blots of pulsed-field gel electrophoresis-separated restriction digests. Seven genetic markers, rrs, rrl, atpD, tuf, gyrB, rpoD, and dnaK, on the chromosome were located by hybridization. Probes for all genetic markers were obtained by PCR. For five of these markers, probes were constructed by PCR with degenerate primers targeting conserved sequences. The arrangement of the genetic markers was compared with that found in other bacteria.

Base Sequence↗