Isolation And Characterisation Of Par141, A Cryptic Lactococcus Lactis Plasmid, And Its Development Into An Expression Vector

Lactococcus lactis is one of the best characterised lactic acid bacteria (LAB). It is widely used in traditional biotechnology as dairy starter culture for the production of cheese, butter and buttermilk. Its applications have been further expanded in modern biotechnology. L. lactis has been used as an alternative to Escherichia coli as a cell factory for the production of chemicals, pharmaceuticals and neutraceuticals. It has also been engineered to be a live oral vaccine. To engineer the cells, plasmids serve as vectors for the introduction of foreign DNA into the hosts, and hence giving the hosts unique features for special purposes. The basic knowledge and understanding of the plasmid is significant for the development of a prominent cloning and expression system. However, there are limited information and no commercially available vectors for L. lactis in the market currently. In this study, the Gram-positive cocci isolated from cow’s milk were identified by comparing the partial 16S ribosomal RNA (rRNA) gene sequences to Internet databases. Among these cocci eight strains were identified as L. lactis subspecies lactis and were further characterised by plasmid profiling, antibiotic resistance pattern and antimicrobial activity. Two of the strains, L. lactis M12 and M14, were found to carry multiple plasmids of various sizes, ranging from 1.6 kilo base pair (kb) to approximately 46 kb. However, they exhibited the same antibiotic resistance pattern as the plasmidless strain L. lactis MG1363. These two strains were able to inhibit the growth of other lactococcal strains isolated from the same source. The smallest plasmid from L. lactis M14, designated as pAR141, was chosen for further analysis. The restriction enzyme-digested fragments of the plasmid were cloned and sequenced. The sequence analysis of pAR141 indicated that it replicated via rolling circle (RC) mechanism. This 1,594-base pair (bp) cryptic plasmid carried essential genes required for its own replication and control, which included the transcriptional repressor repA and replication initiator repB genes, in a single operon. Other elements such as the putative coding region of a small countertranscribed RNA (ctRNA), the double strand origin (dso) and single strand origin (sso) of replication, were also identified. A constitutive expression vector, pAR1411, was constructed by cloning the erythromycin resistance marker (ery), P32 promoter and a multiple cloning site (MCS) into pAR141. The functionality of the new vector was verified by using the chloramphenicol acetyltransferase (cat) gene as the reporter gene. The cat gene was successfully cloned into pAR1411 and expressed in L. lactis MG1363. In conclusion, the small lactococcal cryptic RC replicating plasmid, pAR141, was isolated and characterised. The newly developed pAR1411 could be used as an expression vector for L. lactis.

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