Molecular analysis of the lactose metabolising genes from Lactococcus lactis : a thesis presented in partial fulfilment of the requirement for the degree of PhD in Biotechnology at Massey University, New Zealand
Two lactococcal strains possessing different lactose metabolism systems were chosen for the molecular analysis of lactose metabolising genes from Lactococcus lactis. These were Lactococcus lactis ssp. cremoris strain H2 and Lactococcus lactis ssp. lactis strain ATCC 7962. An attempt was made to sequence a previously cloned 4.4 kb EcoRI fragment of pDI21 reported to encode D-tagatose 1,6-bisphosphate aldolase. A comparison of sequence data generated from this fragment with DNA sequences in the GenEMBL data base revealed that the clones provided for this study were not lactococcal DNA but were chromosomal DNA of E. coli which encoded genes involved in purine biosynthesis. This part of the research programme was therefore abandoned. The aim of the second part of this study programme was to clone and characterize the β-galactosidase gene from plasmid pDI3, an uncharacterized plasmid of Lactococcus lactis ssp. lactis strain ATCC 7962. This was of interest as most lactococcal bacterial strains metabolize lactose by way of the Lac-PEP:PTS system and possess high phospho-β-galactosidase activity, whereas strain 7962 metabolizes lactose by way of the lactose permease system and possesses high β-galactosidase activity and low phospho-β-galactosidase activity. Previous plasmid curing experiments indicated that the β-galactosidase activity of strain 7962 is associated with pDI3. DNA hybridization work between pDI3 and a previously cloned DNA fragment containing the β-galactosidase gene of another Gram-positive genus, Clostridium acetobutylicum (cbgA) showed that pDI3 contains DNA sequence that is to some extent homologous to β-galactosidase sequence. Initial experiments were carried out to confirm the involvement of pDI3 on strain 7962's β-galactosidase activity. Strain ATCC 7962 wildtype containing four plasmids and the strain cured of three other plasmids (i.e. derivative strain of 7962 containing only pDI3) exhibited a Lac+ phenotype, while the strain cured of all four plasmids exhibited a Lac- phenotype. As part of the mapping strategy and the attempt to clone the β-galactosidase gene from strain 7962, various fragments of pDI3 were cloned. Hybridization experiments using the cloned pDI3 fragments as DNA probes were also carried out to confirm the arrangement of pDI3 fragments. A physical map of pDI3 was constructed using the restriction enzymes BamHI, PstI, and SalI. The size of pDI3 was confirmed to be 70 kb and it may contain some small repeat sequences. Some EcoRI restriction sites contained in pDI3 were also determined. Several approaches were used to localize and identify the β-galactosidase gene on pDI3. Southern hybridizations were first carried out using the cbgA gene. The cbgA gene showed weak homology to a 4.3 kb EcoRI doublet from pDI3. Two redundant oligonucleotide probes were designed from the highly conserved domain of deduced amino acid sequences of the available β-galactosidase sequences from other closely related Gram-positive bacteria as well as some deduced amino acid sequences derived from β-galactosidase sequences of Gram-negative bacteria. The 4.3 kb EcoRI doublet and a 4.3 kb HindIII doublet exhibited weak homology to these probes. On the basis of these results one of the 4.3 kb EcoRI fragments was subsequently cloned and transformed into a Lac- E. coli strain, MC1022. The cloned 4.3 kb EcoRI fragment (the 4.3a fragment) was shown to cross hybridize to one of the oligonucleotide probes, but did not show β-galactosidase activity. The 4.3a kb EcoRI fragment was also cloned into pBR322 and transformed into another Lac- E. coli host (JM109) and no β-galactosidase activity was detected. Based on the pDI3 physical map constructed, the 4.3a kb EcoRI fragment was shown to overlap a 13.6 kb SalI fragment. This fragment was cloned into pBR322 and the plasmid designated pSY303, and this was transformed into E. coli JM109. Introduction of pSY303 into E. coli JM109 gave a Lac+ phenotype. Lac+ phenotypes were also found for other Lac- E. coli host strains including E. coli PB2959. It was found that pSY303 expressed β-galactosidase constitutively, however the addition of 0.1% (w/v) lactose into the medium gave a higher level of expression. An inducer for β-galactosidase, IPTG, was not required for expression. Glucose had no repression effect on β-galactosidase. Some instability of the Lac+ phenotype was observed. Transformants that were initially Lac+ manifested phenotypic segregation into Lac+ and Lac- colonies. Both were found to retain the intact pSY303 plasmid and there was no difference in pSY303 DNA isolated from representative Lac+ colonies and from representative Lac- colonies. Some stable Lac+ colonies were observed that became dark blue. Analysis of the plasmid from these colonies showed that pSY303 had undergone a deletion. The generation of these deletion derivatives may be a consequence of small repeat sequences. In conclusion, a physical map of pDI3 has been constructed and the β-galactosidase gene from pDI3 of Lactococcus lactis ssp. lactis strain ATCC 7962 was cloned and was found to express constitutively in E. coli, and at a higher level in the presence of lactose.