The role of transcription in lactococcal phage replication : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
The involvement of transcription in DNA replication has been demonstrated in mitochondria, bacteria, plasmids and phages. Previous studies indicated that transcription may also be required for the prolate-headed lactococcal phage c2 DNA replication in L. lactis. In this study, the role of transcription in origin of DNA replication function was examined. A model system was used, in which the intergenic region of phage c2, presumably containing the origin of DNA replication, supported the replication of a plasmid in Lactococcus lactis. Within this region there is an active promoter that produces several non-coding transcripts. This allowed the importance of this early promoter 1 (PE1) and the length and sequence (and presumably therefore secondary structure) of the PE1 transcripts in replication to be investigated. It was demonstrated that a functional promoter (but not necessarily wildtype PE1) and a specific length and sequence of the PE1 transcripts are required for c2 origin function. The transcription start site of the PE1 transcripts in the plasmid system was determined by primer extension analysis and was identical to the transcription start site in the phage itself. The PE1 transcripts made in the replicating plasmids were detected and quantified by Northern blots, and processing of the transcripts was shown by RNase protection analysis. However, no transcripts of the expected size were detected in the non-functional origins cloned in a plasmid able to replicate in L. lactis. Possible secondary structures of the wildtype and modified PE1 transcripts were modelled using several different computer programs. Lactococcal proteins were shown to bind to the PE1 transcripts by RNA gel shifts and North-Western blots. Affinity purification and amino-terminal sequencing were used to identify one such protein with similarity to the ribosomal protein SI. Growth curves of L. lactis containing the various replicating plasmids did not show any major differences in the ability of the L. lactis cells to grow. To characterize the plasmids further, the relative amount of plasmid DNA per lactococcal cell was determined. It was also demonstrated, by using a high copy number plasmid that harboured the c2 origin, that the c2 origin does not confer a Per phenotype.