A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.Brettanomyces is a well-recognized wine spoilage yeast that causes an undesirable flavor. The sensory character of this "Bretty" flavor is often described as mousiness, barnyard, horse sweat, or Band-Aid (5, 9). Current methods for identification and enumeration of Brettanomyces contamination take 1 to 2 weeks and rely on growth on a semiselective culture medium, followed by final identification by biochemical and physiological analysis and morphology as determined by microscopic examination (3). Morphological characterization of Brettanomyces is somewhat subjective, and there have been various morphological descriptions, such as bud scars, bullet shape, and Mickey Mouse-like. Newer techniques for rapid detection and identification of Brettanomyces, such as an enzyme-linked immunosorbent assay (7) and, more recently, PCR (6), have also been described.The nomenclature of Brettanomyces used in the wine industry differs from that of the recently revised taxonomy of yeasts (11,12). Enologists refer to the spoilage organism as Brettanomyces or "Brett" or, in some publications, by the species names Dekkera intermedia and Brettanomyces intermedius (3), Brettanomyces lambicus (3), Brettanomyces custersii, or Dekkera bruxellensis (6). Today, only D. bruxellensis is an accepted species name, and the other names are considered synonyms.Peptide nucleic acid (PNA) molecules are pseudopeptides which are able to hybridize to complementary nucleic acid targets (RNA and DNA) obeying Watson-Crick base pairing rules (2, 10). Due to their uncharged, neutral backbone, PNA probes exhibit favorable hybridization characteristics, such as high specificity, strong affinity, and rapid kinetics resulting in improved hybridization to highly structured targets, such as rRNA (13). In addition, the relatively hydrophobic character of PNAs compared to DNA oligonucleotides makes PNA probes capable of penetrating the hydrophobic cell wall following mild fixation conditions that do not lead to disruption of cell morphology (14). These unique characteristics of PNA have opened new possibilities for molecular diagnostic assays.The D1-D2 region of 26S ribosomal DNA (rDNA) of eucaryotic organisms shows a high degree of species variation and has been used for identification and taxonomy of yeast species (1,8). In this study, 26S rDNA sequence information was used to design speci...

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SummaryProduction of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coli, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and ␤-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectantinduced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three-to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.

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