Abstract
BackgroundMany plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.ResultsSeveral bacterial strains were isolated from cow's milk and eight of those were identified as Lactococcus lactis by 16S rRNA sequence analysis. Antibiotic susceptibility tests that were carried out showed that 50% of the isolates had almost identical antibiotic resistance patterns compared to the control strains MG1363 and ATCC 11454. Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4. Competent L. lactis M4 and MG1363 were prepared and electrotransformed with several lactococcal plasmids such as pMG36e, pAR1411, pAJ01 and pMG36e-GFP. Plasmid isolation and RE analyses showed the presence of these plasmids in both M4 and the control strain after several generations, indicating the ability of M4 to maintain heterologous plasmids. SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.ConclusionsBased on the 16S rRNA gene molecular analysis, eight Gram-positive cocci milk isolates were identified as L. lactis subsp. lactis. One of the strains, L. lactis M4 was able to maintain transformed low molecular weight plasmid vectors and expressed the GFP gene. This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.
Highlights
Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources
E. coli recombinants were screened by the addition of 0.004% (w/v) of 5-bromo-4-chloro-3-indolylb-D-galactopyranoside (X-gal) while the L. lactis transformants were screened based on the antibiotic resistance selection marker
The results of this study showed that eight of the 14 milk strains randomly selected from the laboratory collection were identified as L. lactis subsp. lactis, while the other six strains were found with high sequence similarity with Enterococcus species
Summary
Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Lactococcus lactis is a lactic acid bacterium possessing the status of generally regarded as safe (GRAS). It is usually used in the dairy and fermented food industry since many decades. Kleerebezem et al [5] highlighted several examples of metabolic engineering of L. lactis for the production of compounds such as diacetyl, alanine and exopolysaccharides, which are important for food industry. These authors reviewed a number of elegant genetic tools used to reroute carbon metabolism in L. lactis. This bacterium is proved to be an efficient cell factory for the production of food ingredient [6], nutraceuticals [7], heterologous proteins [8,9] and vaccine delivery [10,11]
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