Biomedical subjects

M R Gillings

Publications and source records attributed to M R Gillings.

12 recordsLinked to original sources

PubMed2001

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Amino Acid Sequence↗

PubMed2001

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.

Anti-Infective Agents↗

PubMed2001

Phylogenetic structure of unusual aquatic microbial formations in Nullarbor caves, Australia.

The nature of unusual aquatic microbial formations in flooded passages of cave systems in the Nullarbor region of Australia was investigated using electron microscopy and DNA analysis. The caves are located in a semiarid region but intersect the watertable at depths of approximately 100 m below the surface. Throughout submerged portions of the caves divers have noted the presence of unusual microbial formations. These 'microbial mantles' comprise sheets or tongues of mucoid material in which small crystals are embedded. Examination of the biomass revealed it to be primarily composed of densely packed, unbranched filaments, together with spherical-, rod- and spiral-shaped cells, and microcrystals of calcite in a mucoid matrix. Molecular phylogenetic analysis of the community structure revealed approximately 12% of clones showed high similarity to autotrophic nitrite-oxidizing bacteria (Nitrospira moscoviensis). The remainder of the clones exhibited a high proportion of phylogenetically novel sequence types. Chemical analysis of water samples revealed high levels of sulphate and nitrate together with significant nitrite. The community structure, the presence of nitrite in the water, and the apparent absence of aquatic macrofauna, suggest these microbial structures may represent biochemically novel, chemoautotrophic communities dependent on nitrite oxidation.

Australia↗

PubMed2001

Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

Bacteria↗

PubMed2000

Heteroduplex mobility assay as a tool for predicting phylogenetic affiliation of environmental ribosomal RNA clones.

Heteroduplex mobility assay (HMA) of partial 16S rRNA gene fragments was tested as a tool for predicting bacterial phylogenetic relationships. Approximately 400-bp fragments were amplified from a selection of cloned environmental DNAs representing a range of sequence identities and phylogenetic relationships. Heteroduplexes between pairs of sequences were formed by mixing equal amounts of PCR products, denaturing and annealing. Annealed mixes were separated on 8% polyacrylamide gels and silver stained. Heteroduplexes were readily distinguished from reannealed homoduplex and unannealed fragments in all sequences where percentage identity was less than 95%. The heteroduplexes showed retarded electrophoretic migration with respect to homoduplexes. The relative retardation was strongly correlated to the percentage sequence identity between the two strands. The HMA is a useful tool for screening environmental clone libraries to systematically select clones representative of the phylogenetic diversity within the sample, or to selectively retrieve members of a particular phylogenetic group for more detailed study.

Bacteria↗

PubMed2000

Novel forms of ring-hydroxylating dioxygenases are widespread in pristine and contaminated soils.

Ring-hydroxylating dioxygenases (RHDs) are of central importance to bacterial recycling of aromatic hydrocarbons, including anthropogenic pollutants. The database of presently characterized RHDs is biased towards those from organisms readily isolated on anthropogenic substrates. To investigate the extent to which RHDs from extant organisms reflect the natural diversity of these enzymes, we developed a polymerase chain reaction (PCR) method for retrieval of RHD gene fragments from environmental samples. Gene libraries from two contaminated and two pristine soil samples were constructed. None of the inferred peptides from clones examined were identical to previously described RHDs; however, all showed significant sequence homology and contained key catalytic residues. On the basis of sequence identity, the environmental clones clustered into six distinct groups, only one of which included known RHDs. One of the new sequence groupings was particularly widespread, being recovered from all soil samples tested. Comparison of inferred peptide sequences of the environmental clones and known RHDs showed the former to have greater sequence variation at sites thought to influence accessibility of the active site than that seen between currently known RHDs. We conclude that presently characterized RHDs do not adequately represent the diversity of function found in in situ forms.

Amino Acid Sequence↗

PubMed2000

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes.

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.

Animals↗

PubMed1999

Sphingomonas paucimobilis BPSI-3 mutant AN2 produces a red catabolite during biphenyl degradation.

The biphenyl degradation pathway of Sphingomonas paucimobilis BPSI-3 was investigated using a degradation-deficient mutant generated by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) mutagenesis. The mutant, designated AN2, was confirmed as originating from BPSI-3 through the use of ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR and by detection of the diagnostic pigment, nostoxanthin, in cellular methanol extracts. Mutant AN2 produced a yellow followed by red extracellular substance when grown in the presence of biphenyl. In the presence of 2,3-dihydroxybiphenyl, yellow followed by red then yellow compounds were formed over time. This colour change was consistent with the characteristics of a quinone, 1-phenyl-2,3-benzoquinone, which could arise from the oxidation of 2,3-dihydroxybiphenyl. A quinone was synthesised from 2,3-dihydroxybiphenyl and compared to the red compound produced by mutant AN2. Gas chromatography-mass spectrophotometry (GC-MS) confirmed that a similar quinone (4,5-dimethoxy-3-phenyl-1,2-benzoquinone) compared to the structure of the proposed biogenic compound, had been formed. This compound was also found after GC-MS analysis of mutant AN2 culture extracts. Spectrophotometric analysis of the quinone synthesised and the red product produced revealed almost identical spectral profiles. A likely inference from this evidence is that the mutant AN2 is blocked, or its activity altered, in the first gene cluster, bphA to C, of the biphenyl degradation pathway.

Journal Article↗

PubMed1998

Methods for microbial DNA extraction from soil for PCR amplification.

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

Journal Article↗

PubMed1997

PCR amplification of crude microbial DNA extracted from soil.

A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes.

Base Sequence↗

PubMed1992

Differentiation of cucumber mosaic virus isolates using the polymerase chain reaction.

A procedure based on the polymerase chain reaction (PCR) has been developed to classify cucumber mosaic cucumovirus (CMV) isolates accurately into two subgroups. Two CMV-specific primers that flank the CMV capsid protein gene were used to amplify a DNA fragment of approximately 870 bp. Restriction enzyme analysis of this fragment produces distinct restriction patterns that assign the CMV isolate into one of two subgroups. These two restriction groups correlate with the previously established CMV subgroupings; this PCR-based method may provide a simple alternative to the serological assays used for typing CMV isolates.

Base Sequence↗

PubMed1992

Differentiation of biologically distinct cucumber mosaic virus isolates by PAGE of double-stranded RNA.

Electrophoresis on 5% polyacrylamide was used to analyze dsRNAs of 26 cucumber mosaic virus isolates propagated in Nicotiana tabacum. There was variation between isolates in the migration of each of the dsRNAs 1, 2, and 3. Comparison of the dsRNA profiles enabled each isolate to be allocated to 1 of 7 distinct dsRNA profile types. Two distinct and readily distinguishable isolates were mixed in planta and dsRNA from these infected plants compared with in vitro mixtures of them. All bands from both types were present, indicating that the differences were real and reproducible. This method is of value as a means of classifying cucumber mosaic virus isolates, as it more closely reflects a range of biological characteristics than do other methods currently used.

Electrophoresis, Polyacrylamide Gel↗