Lactococcal plasmid replicon ; vector construction and genetic organization : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North New Zealand

The 5.5 kb high-copy number cryptic plasmid pDI25 from Lactococcus lactis subsp. lactis 5136 was isolated and used as the basis to construct a series of vectors. The vector pFX1 (5.5 kb) was first made by ligating the 4.5 kb HpaII-MboI fragment of the lactococcal plasmid to the 1 kb chloramphenicol transacetylase gene from the staphylococcal plasmid pC194. Plasmid pFX1 was further modified by deleting a non-essential 1.9 kb ClaI region to construct pFX2 (3.6 kb). Deletion analysis showed an essential region for plasmid replication was located within a 1.2 kb CfoI-ThaI-CfoI fragment. The vector pFX3 was constructed by incorporating the α fragment of the Escherichia coli lacZ structural gene, a multiple cloning region and the T7 and T3 promoters from pUBS into pFX2. Recombinant plasmids constructed in E. coli using X-gal selection could be subsequently electroporated into lactococci. pFX3 could also be used directly for transcription studies or DNA sequencing of cloned inserts. A set of lactococcal translational gene-fusion vectors was constructed by incorporating the E. coli lacZ gene fusion system (pNM480,481,482) into pFX2. These constructions, pFX4, pFX5 and pFX6, permit the fusion, of cloned genes to lacZ in all three reading frames. Gene expression can be readily and quantitatively monitored by measuring β-galactosidase activity. All the pFX vectors were efficiently transformed into lactococci and E. coli by electroporation (104-106 cfu/μg DNA in each host) and maintained stably in both organisms (> 95% cells carrying the Cm marker after 100 generations growth without drug selection). A cell-wall bound proteinase from Lactococcus lactis subsp. cremoris H2 was isolated and characterized as a PI type proteinase since it preferentially degraded β-casein. A 6.5 kb HindIII fragment of plasmid pDI21 (63 kb) was initially cloned and expressed this enzymatic activity in E. coli using vector λNM1149. The restriction map of this pDI21 prt gene fragment had minor differences from those of other published lactococcal prt fragments. Using pFX1, the pDI21 prt gene fragment was recloned and directly electroporated into lactococci where it was efficiently expressed. The effectiveness of pFX3 was demonstrated by initially cloning a pDI1 4.4 kb EcoRI tagatose 1,6-bisphosphate aldolase gene fragment into E. coli from where it was electroporated into lactococci. Using the translational fusion vectors pFX4, pFX5 and pFX6, the 6.5 kb HindIII prt gene fragment of pDI21 was identified as having two promoters with opposite orientations. The pDI21 2.0 kb EcoRI galactose-6-phosphate isomerase gene fragment was shown to carry a promoter and the direction of gene transcription was determined. The complete DNA sequence of the lactococcal portion of pFX2 (2508 bp) was determined and the genetic organization analyzed. A lactococcal plasmid plus origin and two replication protein coding regions (repA and repB) were located. RepA had an αhelix-turn-αhelix motif, a geometry typical of DNA-binding proteins. RepB showed high homology to the plasmid replication initiation proteins from other Gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Upstream of the plus origin site, and in a region nonessential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with the pE194 group of plasmids