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Fig. 4.9 A typical RFLP DNA fingerprint (kindly provided by Philip Meaden. ICBD. Heriot-Watt University).

of pathogenic bacteria. Similarly in brewing microbiology, PCR is finding favour in the identification of spoilage yeasts and bacteria (for a review see Russell & Dow-hanick, 1996).

In essence, PCR results in the exponential amplification (up to a million-fold) over a few hours of a piece of double-stranded DNA through the repeated copying of each strand. The process is described in Fig. 4.10. The DNA to be amplified is specifically targeted by the use of specific primer sequences of 10-25 nucleotides long that flank the desired sequence. This requires that target DNA sequence is known - as many are now through the yeast genome project - so that the primer DNA sequence can be synthesised. Essentially, PCR is a repeated thermal cycling process where the DNA is initially denatured (94°C), the primer sequence then anneals (55°C) and is then extended by the thermostable DNA polymerase (72°C). Having amplified the DNA, it can be characterised by electrophoresis and visualised using ethidium bromide or UV light. Further discrimination can be sought by cutting the amplified DNA with restriction enzymes and probing the RFLPs (Masneuf et al., 1996).

Target DNA

DNA is denatured (single stranded)

hybridisation of primer sequence

target DNA replicated

Fig. 4.10 The principle behind PCR.

PCR lends itself to the differentiation at the genus or species level. Not surprisingly, its use in the differentiation of strains is limited, as this would depend on changes within the target DNA sequence. However, some success has been reported with brewing yeasts using a defined set of probes (Walmsley, 1994) or a multi-locus primer sequence (8 sequence, Donnelly & Hurley, 1996). Wine yeasts have also been broadly differentiated using primers directed at the highly variable intron splice sites (de Barros Lopes et al., 1996). Subsequent work with this approach (de Barros Lopes et al., 1998) has shown the differentiation of the closely related cluster of species, Sac-charomyces sensu stricto (see Section 4.2.1). However, in another study the use of hypervariable repetitive sequences was unable to successfully differentiate wine, brewing or baking strains of S. cerevisiae (Lieckfeldt et al., 1993). Similarly, mixed success has been reported (Yamagishi et al., 1999) for differentiation of brewing and non-brewing yeasts using PCR targeted to a fragment of the putative flocculation gene, FLOl. Much improved discrimination was reported by digestion of the PCR products with specific restriction enzymes and RFLP analysis (see Section 4.2.6.1). This hybrid approach of PCR and RFLP enabled the differentiation of brewing strains from wild Saccharomyces and non-Saccharomyces yeasts.

An alternative to the targeted probes is the use of randomly designed non-specific DNA probes ('random amplified polymorphic DNA' or RAPD PCR). This approach has the benefit of not requiring any prior knowledge of DNA sequence and can - if the primer sequence is 'right' - result in several PCR products as the random primer bind at several sites. These products are separated and visualised to give a fingerprint. This approach has been successful in the differentiation/identification of brewery bacteria (Tompkins et al., 1996) or ale from lager yeasts (Laidlaw et al., 1996).

Compared to the complexities and hardware of RFLP DNA fingerprinting, PCR techniques are simple, straightforward and rapid. However PCR has suffered somewhat from a 'bad press' inasmuch that 'contamination' can be an issue as can the presence of interfering/inhibitory beer components such as polyphenols (Dowhanick, 1995). In conclusion, the development of PCR in the brewing industry is presently more likely to be driven by the detection and identification of spoilage organisms rather than the differentiation of brewing yeast strains.

4.2.6.3 Genetic fingerprinting - karyotyping. 'Karyotyping' is the determination of chromosomal size and number (Meaden 1990). This approach has arguably been applied more successfully to the differentiation of brewing yeast strains (Casey et al., 1988, 1990; Casey, 1996; Donhauser, 1997; Oakley-Gutowski et al., 1992; Pedersen, 1994; Sheehan et al., 1991) than any other method.

S. cerevisiae is particularly amenable to karyotyping as it has 16 chromosomes (more than other yeast!) which range dramatically in size from 230 kb (chromosome I) to approximately 1.5 Mb (chromosome IV). The size distribution of the 16 chromosomes is shown in Fig. 4.19 (Goffeau et al., 1996). Such complexity is both a strength (karyotypes are likely to vary widely) and a weakness (electrophoretic separation of both big and small chromosomes at the same time is difficult).

The problem (or 'opportunity') of resolving yeast karyotypes was resolved with the development in 1984 of pulsed field gel electrophoresis (PFGE) (Carle & Olson, 1984; Schwartz & Cantor, 1984). This approach supports the separation of chromosomes ranging from 50 kb to 10 Mb in agarose gels when electric fields are applied in different directions. Different pulse times are used to separate only the small chromosomes (< 500 kb, Chromosomes I, III, VI and IX) or all of the chromosomes (Sato et al., 1994; Casey, 1996). Once separated the chromosomes are stained with ethidium bromide and then visualised under UV light. As with RFLP, karyotyping yields a barcode-like fingerprint (Fig. 4.11) which can be used to differentiate between strains.

Inevitably, the PFGE approach has been developed primarily in the area of elec-

Fig. 4.11 A typical karyotype DNA fingerprint (kindly provided by Miles Schofield, BRi).

trode design and consequent electrical field. Two approaches have found particular favour in the brewery applications of karyotyping - CHEF (contour clamped homogeneous electric field) and TAFE (transverse alternating field electrophoresis) (for details see Russell & Dowhanick, 1996).

Although the original PAGE approach has been used (Donhauser, 1997), the majority of reports on brewing yeast karyotytping have favoured the CHEF (Sheehan et al., 1991; Oakley-Gutowski et al., 1992, Pedersen, 1994; Wright et al., 1994) or TAFE (Casey, 1996; Casey et al., 1988, 1990) approach.

Unlike the RFLP approach, karyotyping offers more than strain fingerprinting and differentiation. In a notable paper, Casey (1996) was able to show that changes in chromosomal size and dosage can be associated with changes in cell colony morphology ('smooth' v. 'rough'), culture purity and long-term culture stability. Karyotyping was also able to track the genetic evolution of American lager strains which were shown to have originated 150 years ago from either the Tuborg or Carlsberg breweries in Denmark.

As is described in Section 4.3.2.5, Casey's (1996) observations fit the growing theme that brewing yeasts can be genetically unstable. Instability can be beneficial or detrimental. These so-called 'chromosome-length polymorphisms' (Zolan, 1995) are well-recognised in fungi and can be explained by movements of 'bits of DNA' either through deletions, insertions or translations within and between chromosomes.

An interesting extension of karyotyping has been the use of CHEF to study the ecology of wild S. cerevisiae flora in wine musts in Spain (Briones et al., 1996). In a survey of 14 vats from three cellars, 392 discrete colony types were recovered. Remarkably, after CHEF analysis, 174 different karyotypes were identified which when subject to cluster analysis reduced to four major groups. Intriguingly, 'the majority of S. cerevisiae strains in cellars A and C possess the karyotype characterizing Groups 1 and 2; while in cellar B the strains correspond to three Groups: 1, 3 and 4' (Briones et al., 1996).

4.2.6.4 Genetic fingerprinting - AFLP. Amplified fragment length polymorphism (AFLP) is described (Savelkoul et al., 1999) as the 'newest and most promising method' for the identification and typing of organisms at the DNA level. First described by Vos et al. (1995), AFLP can simply be viewed as a hybrid method comprising RFLP (see Section 4.2.6.1) and PCR (see Section 4.2.6.2). The extracted DNA is digested with two restriction enzymes, one with average cutting frequency (e.g. £coRI) and one with a higher cutting frequency (e.g. MseI). The fragments are selectively amplified by PCR which, after gel electrophoresis, results in 'highly informative' (Savelkoul et al., 1999) fingerprints with 40-200 bands. As with all molecular fingerprinting approaches, the complex AFLP patterns require measurement and software driven data capture of the digitised images. For an insight into the many commercial packages used in fingerprinting, see Savelkoul et al. (1999).

AFLP has been used with a diversity of yeast species including those of the Sac-charomyces sensu stricto cluster (see Section 4.2.1) (de Barros Lopes et al., 1999). As the yeast strains used in this work mirror those used previously by this group with PCR (de Barros Lopes et al., 1998), it is possible to compare the performance of the two approaches. The previous work (de Barros Lopes et al., 1998), had been unable to differentiate two environmental isolates of S. bayanaus which could be differentiated using AFLP. This and other observations suggest that although more labour intensive, AFLP offers greater sensitivity, is more reproducible and is more 'discriminatory' than PCR.

However, as with many molecular approaches, AFLP has its limitations. Whilst particularly effective in demonstrating genetic relatedness and accordingly differentiation of domesticated industrial strains of S. cerevisiae, AFLP cluster analysis failed to identify the known genomic relationships in the closely related species found in the Saccharomyces sensu stricto group.

4.2.6.5 Pyrolysis mass spectroscopy. The demands of clinical microbiology have spawned a variety of approaches that examine aspects of cell composition. Initially, pyrolysis gas chromatography (PyGC) and, now the more sophisticated, pyrolysis mass spectroscopy (PyMS) are increasingly being used to identify and differentiate micro-organisms to the level of genus, species and subspecies (for reviews see

Goodacre, 1994; Goodacre & Kell, 1996; Gutteridge & Priest, 1996). Perhaps the secret of PyMS's success is its simplicity and speed. Typically, sample time is less than two minutes with preparation limited to the careful transfer of a colony from a plate or drying down a few microlitres of a liquid culture. Indeed, Goodacre (1994) noted that a British daily newspaper had reported that PyMS was 'so simple that a chimpanzee could be trained to do it!'

In essence, pyrolysis involves the thermal degradation in an inert atmosphere of- in this case - microbial biomass. Typically the 'Curie point' pyrolysis temperature is 530°C which has been shown to give an ideal balance between the fragmentation of microbial polysaccharides and proteins (Goodacre, 1994). The ensuing volatile, low-molecular-weight fragments are typically separated using a mass spectrometer (PyMS) (see Fig. 4.12). This powerful approach has superseded separation using a gas chromatograph.

Sample

Volatile organic fragments t --

Singly positively charged fragments t ^-

Ions of specific mass h-

Detector

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