Peter D. Turner

The nitrilase family of CN hydrolysing enzymes - a comparative study.

The enzymes nitrilase, cyanide dihydratase and cyanide hydratase are a group of closely related proteins. The proteins show significant similarities at the amino acid and protein structure level but the enzymes show many differences in catalytic capability. Nitrilases, while catalysing the hydration of nitrile to the corresponding acid, vary widely in substrate specificity. Cyanide dihydratase and cyanide hydratase use HCN as the only efficient substrate but produce acid and amide products, respectively. The similarities of all these enzymes at the amino acid level but the functional differences between them provide a rich source of material for the study of structure/function relationships in this biotechnologically important group of enzymes. This review provides an overview of current understanding of the genetics and biochemistry of this interesting group of enzymes.

Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris

SummaryXanthomonas campestris pv. campestris was tested for its ability to maintain various plasmids after they had been transferred by conjugation from Escherichia coli donors. Broad host-range plasmids belonging to incompatibility groups P and Q could be maintained but X. campestris was unable to support replication of narrow host-range ColE1, pACYC184 and pBR325 replicons. Delivery systems based on E. coli donors of suicide plasmids and on X. campestris Hfrs were used to introduce Tn7 and Tn5 into X. campestris. Tn7 insertions were recovered at high frequency while Tn5 transposed at low frequency. Three auxotrophic Tn5 insertions were isolated but transposition of Tn7 into the X. campestris genome did not generate any auxotrophs. DNA hybridization analysis showed that Tn7 had inserted into the same “hot spot(s)” in all cases tested.

Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene

The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66). This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide. The enzyme was purified from F. lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE). mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated. This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible. Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone. A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E. coli using the expression vector pGEX-2T. Features of F. lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented.

Efficient species identification of pine marten (Martes martes) and red fox (Vulpes vulpes) scats using a 5′ nuclease real-time PCR assay

Monitoring wildlife species by DNA identification of samples collected non-invasively is an important tool in conservation management. DNA identification of species from faecal (scat) samples is problematic due to the small quantities and poor quality of the DNA isolated from such samples. This study demonstrates the use of real-time PCR technology in the identification of red fox (Vulpes vulpes) and pine marten (Martes martes). It is shown that real-time PCR can be used to identify fox and pine marten by either melting curve analysis (Tm determination) with SYBR Green 1 detection or by the use of species specific fluorogenic probes. The technique is shown to work efficiently with scat DNA.

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes, L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

Non‐invasive genetic identification of small mammal species using real‐time polymerase chain reaction

DNA identification of non‐invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species‐specific real‐time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.

Non-invasive multi-species monitoring: real-time PCR detection of small mammal and squirrel prey DNA in pine marten (Martes martes) scats

DNA identification of mammal species occurring in the diet of a predator is potentially a useful approach to remotely monitor the distribution of multiple species. This is important in Ireland, where it has been shown that the combined presence of the introduced bank vole and greater white-toothed shrew impact the distribution of the indigenous small mammals, the wood mouse and pygmy shrew. Direct monitoring of these species and their interactions requires trapping, a labour-intensive and costly approach. In this study, we applied an indirect method by genetically testing the presence of small mammals in pine marten scats collected during the National Pine Marten Survey (2005–2007) to map their distribution. We also included additional scats to investigate if less common prey items, the red squirrel and grey squirrel, could also be detected. This study demonstrates that all target species were genetically detected from pine marten scats. This strategy could be implemented as a monitoring programme for indigenous and introduced mammal species.

Molecular comparison of historical and contemporary pine marten (Martes martes) populations in the British Isles: evidence of differing origins and fates, and implications for conservation management

We investigated the origins and persistence of European pine marten (Martes martes) populations across the British Isles by identifying mitochondrial DNA (mtDNA) sequences from contemporary populations (sampled since 1981) and comparing these with those of older ‘historical’ museum specimens (pre-1981) originally collected from the same geographic areas. Excluding Scotland, where the haplotype composition of populations appears to be unchanged, haplotypes found in contemporary and historical marten populations elsewhere differed both temporally and geographically. While these data suggest that the contemporary Irish population is descended from a relict population that passed through an early to mid 1900s bottleneck, the historical and contemporary English and Welsh populations differ in their abundance of specific mtDNA control region haplotypes. These data appear to suggest that particular haplotypes may have been lost from England and Wales at some point in the early to mid 1900s, but further nuclear DNA work is required to determine whether this shift has occurred by rapid genetic drift in the mtDNA control region or whether relict populations have been replaced by pine martens from elsewhere. If the reported shifts in mtDNA haplotypes reflect population extirpation events, historical pine marten populations of England and Wales would appear to have become extinct in the twentieth century (in Wales after 1950 and in England after 1924). Additionally, the recent occurrence of haplotypes originating from continental Europe, and of M. americana, suggest that relict populations of England and Wales have been replaced by, or hybridised with, occasional released, escaped and/or translocated animals. The implications of these results for pine marten conservation, and particularly reintroduction, are discussed.

Development of novel real-time TaqMan(®) PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring.

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.