Quantitative PCR reveals the frequency and distribution of 3 indigenous yeast species across a range of specialty cheeses.

Indigenous microorganisms are important components of the complex ecosystem of many dairy foods including cheeses, and they are potential contributors to the development of a specific cheese's sensory properties. Among these indigenous microorganisms are the yeasts Cyberlindnera jadinii, Pichia kudriavzevii, and Kazachstania servazzii, which were previously detected using traditional microbiological methods in both raw milk and some artisanal specialty cheeses produced in the province of Québec, Canada. However, their levels across different cheese varieties are unknown. A highly specific and sensitive real-time quantitative PCR assay was developed to quantitate these yeast species in a variety of specialty cheeses (bloomy-rind, washed-rind, and natural-rind cheeses from raw, thermized, and pasteurized milks). The specificity of the quantitative PCR assay was validated, and it showed no cross-amplification with 11 other fungal microorganisms usually found in bloomy-rind and washed-rind cheeses. Cyberlindnera jadinii and P. kudriavzevii were found in the majority of the cheeses analyzed (25 of 29 and 24 of 29 cheeses, respectively) in concentrations up to 104 to 108 gene copies/g in the cheese cores, which are considered oxygen-poor environments, and 101 to 104 gene copies/cm2 in the rind. However, their high abundance was not observed in the same samples. Whereas C. jadinii was present and dominant in all core and rind samples, P. kudriavzevii was mostly present in cheese cores. In contrast, K. servazzii was present in the rinds of only 2 cheeses, in concentrations ranging from 101 to 103 gene copies/cm2, and in 1 cheese core at 105 gene copies/g. Thus, in the ecosystems of specialty cheeses, indigenous yeasts are highly frequent but variable, with certain species selectively present in specific varieties. These results shed light on some indigenous yeasts that establish during the ripening of specialty cheeses.

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.