Replication and stability of the linear plasmid pBSSB2
Plasmid pBSSB1 is a 27 kb linear DNA with proteins attached at the 5’ termini. It encodes the H: z66 flagellar antigen in Salmonella enterica serovar Typhi (S. Typhi) isolated from Indonesia. Together with the H: j or H: d flagellar antigen encoded by the host chromosome, pBSSB1 renders expression of the flagellar antigen biphasic in S. Typhi. Following the discovery of pBSSB1, initial bioinformatic analyses were carried out. However, no genetic analysis of replication and stability functions was conducted. Such studies form the basis of the present work. Plasmid pBSSB2, that contains a kanamycin cassette inserted at position 1295 bp of pBSSB1, was used in the present investigation.
The first objective of the work was to develop a method of purification for the linear plasmid. Conventional plasmid extraction methods which had been used previously were found to produce a very poor yield of plasmid DNA. It was shown in the present study that a proteinase-K treatment was essential for the removal of the linear plasmid terminal proteins to avoid loss of the plasmid in the phenol-chloroform-isoamylalcohol treatment which removes cellular proteins from the plasmid DNA.
The region containing the basic replicon of pBSSB2 was identified by screening for a region that was able to support replication in E. coli of a ColE1-like plasmid in a polA host (in which it would not normally replicate). This identified a 2831 bp fragment encompassing nucleotides 12820 to 15649 of pBSSB2. It was expected that this would encode an initiator of replication such as a Rep protein. However, mutagenesis studies showed that none of the annotated ORFs in this fragment was essential for replication. Candidate ORFs, not identified in the original annotation, have been suggested that remain to be tested as possible candidates for the rep encoding gene. The possibility of an alternative RNA primed initiation of replication has also been hypothesized. An adjacent region was found to exert strong incompatibility against pBSSB2, suggesting that it might encode a repressor of replication. The minimum region conferring incompatibility was 179 bp, encompassing nucleotides 10840 bp to 11018 bp of pBSSB2. A six base pair imperfect repeat, (G/T) (G/A) TGTT was found within this sequence. It is hypothesized that these imperfect repeats may function as iterons that titrate a Rep protein and regulate pBSSB2 replication.
A 1023 bp region (nucleotides 7236 to 8258 of pBSSB2) was found to confer stability in E. coli upon an otherwise unstable circular plasmid. Mutational analysis showed that an annotated ORF within this region (ORF09) was required for plasmid stabilisation. When expressed independently from an expression vector ORF09 killed host cells. It is proposed that the stability function acts as a toxin-antitoxin system, although the antitoxin has not yet been identified. A candidate promoter for a putative countertranscript and two potential ORFs as candidates for encoding the antitoxin have been suggested for future work to identify the antitoxin.
The preliminary functional characterization of pBSSB2 has contributed to our general understanding of the replication and stability functions of linear plasmids. However, further work will be required to achieve a complete understanding of the molecular basis of these functions in pBSSB2.