Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures

Abstract

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while highfrequency transfer (10 ؊3 per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.

The changing face of dairy starter culture research: From genomics to economics

“…Interestingly, the producing strain was isolated from an Irish buttermilk plant used domestically for bread making (Figure 3). To date this plasmid has been transferred to over 30 different lactococcal hosts, many of which are derivatives of commercial starter strains (Ryan et al 1996;Coakley et al 1997). Lacticin 3147 has been shown to be effective in many food systems for the control of food spoilage or pathogenic bacteria Ross et al 1999;Morgan et al 2001;O'Sullivan et al 2006).…”

Plasmids of lactococci – genetic accessories or genetic necessities?

“…The maximal size of the transferred region depends on the process and is the highest for conjugation, which can mediate transfer of genome-size segments (Bolotin et al, 2004). Conjugal plasmids are commonly found in starter lactococci and can be transferred to recipient strains at frequencies as high as 10 À2 (Coakley et al, 1997). It is well known that many conjugative plasmids have been associated with traits that are readily exploitable in the dairy industry, such as bacteriocin production (McKay et al, 1980;Coakley et al, 1997;Mills et al, 2002), phage resistance (Coakley et al, 1997;Mills et al, 2002), proteinase production (Kok, 1990), polysaccharide production (van Kranenburg et al, 2000) and lactose catabolism (Snook & McKay, 1981;Gasson, 1990).…”

“…However, nck202.50 continued to be sensitive to restriction by other lactococcal R/M systems of different specificity from LlaI (Alatossava & Klaenahmmer, 1991). Since then, the approach has been adopted worldwide with considerable success (Jarvis, 1988(Jarvis, , 1989Kelly et al, 1990;Powell et al, 1990;Harrington & Hill, 1991;Gireesh et al, 1992;Ward et al, 1992;Coakley et al, 1997;Pillidge et al, 2000;Trotter et al, 2001;Mills et al, 2002). Conjugal strategies have also been used to combine more than one mechanism in a single strain (Klaenhammer, 1987;Coffey et al, 1989;Klaenahmmer, 1991;Sing & Klaenhammer, 1993;O'Sullivan et al, 1998).…”

“…In many cases, transfer of the selectable marker has added beneficial traits to the starter, as in the case of bacteriocin production and immunity. Coakley et al (1997) successfully transferred the 60.2-kb phage resistance, conjugative plasmid, pMRCO1, to a variety of starters by using the genetic determinants for lacticin 3147 immunity encoded on the plasmid. This resulted in the generation of a number of lactococcal starter cultures, which were phage resistant and bacteriocin-producing.…”

Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese

“…Bacterial strains and culture conditions L. lactis DPC4275, a food-grade lacticin 3147-producing transconjugant strain (Coakley et al 1997) derived from L. lactis DPC4268, a bacteriocin-negative strain, were used in this study to demonstrate live-culture bacteriocinmediated anti-listerial activity. These strains, along with L. lactis HP, an indicator strain used for quantification of bacteriocin activity, were routinely propagated in glucose-M17 medium (G-M17) and lactose-M17 medium (L-M17) (Terzaghi and Sandine 1975), respectively at 30°C.…”