Die molekulargenetische Charakterisierung von Milchsäurebakterien und Phagen in instabilen Quarkfermentationen

Abstract

Single cell fluorescence-in situ-hybridisation (FISH) with rRNA targeted oligonucleotide probes was used to exclude that contaminants are the reason for the acidification failure in milk fermentations. Population analysis showed, that at the breakdown of acidification the amount of Lactococcus lactis ssp. cremoris decreases to 1% of the total population, compared to 98% at the beginning of the fermentation. To characterise the genetic potential of organisms involved in milk fermentations the appearance of 34 phage resistance genes was investigated within seven starter cultures, 282 isolates of L. lactis ssp. cremoris and four samples of fresh cheese (quark). Lysogenic phage particles might be the source for phages of the species P335, whereas phages of the species 936 are introduced to fermentations by the milk or polluted fermentation devices. Therefore P482, a newly isolated member of this species, was completely sequenced. The genome consists of 30945 bp and the 55 ORFs are organised in an early, a middle and a late transcription unit. Genes for a terminase subunit, several structural proteins, holin and lysin, the subunits for the DNA-polymerase, HNH-endonucleases and an enzyme with methyltransferase activity was identified. The variety of Lactococcus phages is rather an effect of point mutations, but arises from the exchange of genes or gene segments as described in the modular theory. For the simultaneous analysis of these parameters DNA-Chip hybridisations offer an interesting future perspective. Experiments with enzymatic labelling of the extracted DNA from milk fermentations showed an average labelling rate of one fluorophore per 30-40 bases. Since enzymatic labelling seems to be too expensive for a routine application of microarray technologies, techniques using chemical labelling should be the methods of choice in the future.