Transduktion in Streptococcus thermophilus
Because of commercial losses due to phage infection, development of phage resistant starter strains is important for dairy industry. For this purpose directed genetic exchange is one option. Transduction, a phage mediated gene transfer, is an alternative to standard methods as transformation and conjugation, which are not very efficient in Streptococcus thermophilus. This work characterizes transduction in S. thermophilus and its use as a gene transfer system avoiding genetic engineering. Several derivatives of native S. thermophilus plasmids were successfully transduced with S. thermophilus cos-type phages P1109, P53, a10/J9 and PST. Plasmid DNA was packaged as concatemers into the phage head. Amount of plasmid containing phage particles was up to 50% in comparison to infective particles. All transducable plasmids and several native plasmids of S. thermophilus share homologies to cos-regions and ori-regions of S. thermophilus phages. On the basis of DNA alignments with cos-regions from S. thermophilus phages and plasmids, highly conserved sequences were characterized, containing direct and indirect repeats typical for cos-regions. Conserved sequences showed homologies to the cos-regions of the Lactococcus lactis phage BK5-T and the Lactobacillus gasseri phage adh. Cloning of the cos-region of P53 and transduction experiments with deletion derivatives of pAGJ34E and pAG106AE showed that in contrast to ori-regions homologies to cos-regions were responsible and sufficient for high transduction frequencies. Derivatives of S. thermophilus St11 harbouring plasmids pAG106AE, pSt04 or pAGS4E2 were less sensitive against infection by phage P1109. This was due to a reduced burst of infective phage particles. For strain a10, reduction of phage sensitivity was only observed for plasmid pAG106AE. Transduction were successfully done with several host and non-host strains of S. thermophilus. Several host and non-host strains failed as recipients even in spite of high adsorption. Restriction-/modification systems were detected in strains 55n and DC5. The occurrence of prophage TP-J34 reduces the transduction frequency 50fold compared to a prophage-cured derivative of S. thermophilus J34. L. lactis was also suitable as an recipient in transduction with S. thermophilus phages, showing the impact of transduction on horizontal gene transfer. For plasmids pAGJ34E and pAG106AE, an optimised transduction protocol was applied to detect transductants of S. thermophilus St11 and a10 without use of selective marker. Important parameters were the use of high phage concentrations, adapted adsorption times, use of logarithmic pre-cultures, low cell concentrations and a calcium clorid concentration of 100 mM.