Characterisation of a high copy number mutant pAL5000 origin of replication

[摘要] ENGLISH ABSTRACT:The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacteriumfortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g.M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Mostmycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. Theminimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin ofreplication.Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 originof replication (pORI) and then subjected it to semi-random mutagenesis. A high copynumber mutant was identified (pHIGH) and the causative mutation was tentativelyidentified as a 3bp deletion situated just upstream of repB. This work describes thefurther characterisation of the mutant plasmid.Firstly, it was shown by retransforming M. smegmatis with both the original andmutant plasmids (pORI and pHIGH), that the mutation causing the increased copynumber was plasmid-encoded and not on the chromosome. Following this, it wasdemonstrated by simple subcloning of the region that carries the 3 bp deletion, thatother pAL5000-based vectors could be converted to high copy number. In addition tothis, the subcloned region was sequenced and the nature of the mutations wasconfirmed. The subcloning experiment confirmed that the 3bp deletion caused thehigh copy number phenotype.Following this, the exact copy number of pHIGH and the relative increase in copynumber was determined. From this, the copy number of pORI could also bedetermined. The plasmid pHIGH has a copy number of approximately 54, comparedto the 8 of pORI (a relative increase by a factor of 7).Because it is important for researchers to know the characteristics of the vectors thatthey use, especially the influence it will have on its host, stability tests and growthcurves were also performed. It was seen that the higher copy number did notmarkedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves,the increased copy number has little effect on the growth of the host M. smegmatis.Possible mechanisms for the increased copy number were then investigated. By usinga promoter probe vector, the possible existence of a promoter situated between thetwo open reading frames of pAL5000 (repA and repB) was investigated. It wasthought that the mutation might have created, or changed an existing promoter,situated between repA and repB. The results showed, however, that in both pORI andpHIGH there might be a very weak promoter upstream of repB, but the mutation didnot cause any change that was measurable by the method that was used.A further possibility was that the mutation caused a change in the RNA secondarystructure, which might then have an effect on the translational efficiency of RepB. Itwas found that the 3bp deletion in pHIGH causes a change in the local RNAsecondary structure around the ribosomal binding site and the start codon, whencompared to pORI (wild type). This change may cause the translation initiation rate ofRepB to be different between pHIGH and pORI. Ultimately it would lead to adifferent ratio of RepA and RepB in the cell.

[发布机构] Stellenbosch University