Pollutants That Replicate: Xenogenetic DNAs.

Pollution is the dissemination of material that has harmful effects. Mobile DNA elements and antibiotic-resistance genes are being disseminated into the environment via human activity, and are increasingly being viewed as serious pollutants. These pollutants differ from conventional contaminants in important ways: they can replicate, and they can evolve.

Chemical, biological, and DNA markers for tracing slaughterhouse effluent.

Agricultural practices, if not managed correctly, can have a negative impact on receiving environments via waste disposal and discharge. In this study, a chicken slaughter facility on the rural outskirts of Sydney, Australia, has been identified as a possible source of persistent effluent discharge into a peri-urban catchment. Questions surrounding the facility's environmental management practices go back more than four decades. Despite there having never been a definitive determination of the facility's impact on local stream water quality, the New South Wales Environment Protection Authority (NSW EPA) has implemented numerous pollution reduction requirements to manage noise and water pollution at the slaughter facility. However, assessment of compliance remains complicated by potential additional sources of pollution in the catchment. To unravel this long-standing conundrum related to water pollution we apply a forensic, multiple lines of evidence approach to delineate the origin of the likely pollution source(s). Water samples collected between 2014 and 2016 from irrigation pipes and a watercourse exiting the slaughter facility had elevated concentrations of ammonia (max: 63,000µg/L), nitrogen (max: 67,000µg/L) and phosphorus (max: 39,000µg/L), which were significantly higher than samples from adjacent streams that did not receive direct runoff from the facility. Arsenic, sometimes utilised in growth promoting compounds, was detected in water discharging from the facility up to ~4 times (max 3.84µg/L) local background values (<0.5µg/L), with inorganic As(∑V+III) being the dominant species. The spatial association of elevated water pollution to the facility could not unequivocally distinguish a source and consequently DNA analysis of a suspected pollution discharge event was undertaken. Analysis of catchment runoff from several local streams showed that only water sampled at the downstream boundary of the facility tested positive for chicken DNA, with traces of duck DNA being absent, which was a potential confounder given that wild ducks are present in the area. Further, PCR analysis showed that only the discharge water emanating from the slaughter facility tested positive for a generalized marker of anthropogenic pollution, the clinical class 1 integron-integrase gene. The environmental data collected over a three-year period demonstrates that the slaughter facility is indisputably the primary source of water-borne pollution in the catchment. Moreover, application of DNA and PCR for confirming pollution sources demonstrates its potential for application by regulators in fingerprinting pollution sources.

Cultivation of fastidious bacteria by viability staining and micromanipulation in a soil substrate membrane system.

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.

Eimeria trichosuri: phylogenetic position of a marsupial coccidium, based on 18S rDNA sequences.

Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E.trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line.

Terminal restriction fragment length polymorphism for identification of Cryptosporidium species in human feces.

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

Use of chromosomal integron arrays as a phylogenetic typing system for Vibrio cholerae pandemic strains.

Approximately 200 serogroups of Vibrio cholerae exist, with only two, O1 and O139, responsible for epidemic and pandemic cholera. Strains from these serogroups have evolved from a common progenitor, with lateral gene transfer largely driving their emergence. These strains are so closely related that separation using single- or multi-locus phylogeny has proven difficult. V. cholerae strains contain a genetic system called the integron that is located in the chromosome and that can integrate and excise DNA elements called mobile gene cassettes (MGCs) by site-specific recombination. Large arrays of MGCs are found in V. cholerae strains. For instance, the O1 El Tor strain N16961 contains 179 MGCs. Since integron arrays are dynamic through recombination and excision of MGCs, it was hypothesized that the MGC composition in a given V. cholerae pandemic strain would be useful as a phylogenetic typing system. To address this, a PCR-based method was used to rapidly characterize the MGC composition of V. cholerae arrays. The results showed that the MGC composition of pandemic V. cholerae cassette arrays is relatively conserved, providing further evidence that these strains have evolved from a common progenitor. Comparison of MGC composition between the V. cholerae pandemic strains was also able to resolve the evolution of O139 from a subgroup of O1 El Tor. This level of differentiation of closely related V. cholerae isolates was more sensitive than conventional single-gene phylogeny or multi-locus sequence analysis. Using this method, novel MGCs from an O1 classical strain and an Argentinian O139 isolate were also identified, and a major deletion in the MGC array in all pandemic O139 strains and a subset of O1 El Tor strains was identified. Analysis of sequenced V. cholerae integron arrays showed that their evolution can proceed by rearrangements and deletions/insertions of large portions of MGCs in addition to the insertion or excision of single MGCs.

Survival of a lacZY-marked strain of Pseudomonas corrugata following a field release.

Abstract Pseudomonas corrugata, strain 2140, a biological control agent of take-all disease of wheat, was originally isolated from an acidic red-brown earth soil in New South Wales, Australia. A spontaneous rifampicin-resistant mutant of this bacterium was marked with the disarmed transposon, Tn7::lacZY. This marked strain (2140RlacZY) was introduced into a calcareous sandy loam soil (pH 8) in South Australia. Up to 4 years after its release, P. corrugata 2140RlacZY cells were re-isolated, single colony purified and stored at -80 degrees C. Re-isolated bacteria, including re-isolates obtained 3 (22 re-isolates) and 4 (3 re-isolates) years after release, were examined for stability of the lacZY insert site and for gross chromosomal changes. Hybridization of a cloned lacZY fragment to DNA extracted from the soil re-isolates did not reveal any major changes to the lacZY insert site. Gross chromosomal changes were further examined by restriction endonuclease fingerprinting and PCR based on repetitive sequences (repetitive extragenic palindromic-, enterobacterial repetitive intergeneric consensus- and BOX-PCR). MspI digests distinguished the lacZY-marked strain from the parental strain. None of the genetic techniques used revealed any polymorphisms between the original 2140RlacZY-marked strain and the soil re-isolates. The results demonstrated that the chromosomal landscape within and around the insertion site of the lacZY construct had not altered in the re-isolated bacteria during the 4 years the organism had been in the field.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.