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Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire Université Laval, Québec, Canada, G1K 7P4
Corresponding author:
S. Moineau; e-mail:
Sylvain.Moineau{at}bcm.ulaval.ca.
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ABSTRACT |
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Key Words: exopolysaccharide • Lactococcus lactis • restriction fragment length polymorphism
Abbreviation key: EPS = exopolysaccharide, EPS+ = ability to produce exopolysaccharides, RFLP = restriction fragment length polymorphism
Lactococcus lactis is a gram-positive lactic acid bacterium used to acidify milk during the manufacture of several fermented milk products. Most Lactococcus strains contain plasmids that encode industrial phenotypes such as lactose utilization, proteinase activity, phage resistance and, in some cases, production of extracellular polysaccharides (EPS). The production of EPS by these cultures increases the viscosity, decreases the syneresis, and improves the texture of dairy products. Because these polysaccharide-producing strains impart desirable properties, they are increasingly used by the food industry (De Vuyst and Degeest, 1999).
Several lactococcal EPS have already been analyzed for their chemical compositions as well as the location, sequence, and organization of the genes involved in their biosynthesis (Nakajima et al., 1990; Gruter et al., 1992; De Vuyst and Degeest, 1999; van Casteren et al., 2000a and 2000b). For various purposes (i.e., novel food applications, enhanced EPS production, need of phage-unrelated EPS+ strains), there is an ongoing search for lactococcal strains producing novel EPS. However, given that the EPS must be extracted, purified, and chemically analyzed, the classical method for characterizing EPS is rather fastidious (Gruter et al., 1992; Cerning, 1995; van Kranenburg et al., 1999). Moreover, Lactococcus strains produce a low quantity of EPS. Thus, the isolation of new wild-type Lactococcus strains producing unique EPS is a daunting task. Restriction fragment length polymorphism (RFLP) can be used to distinguish strains by the analysis of their DNA restriction patterns. Recently, van Kranenburg et al. (1999) proposed to differentiate lactococcal EPS+ strains based on RFLP of the eps operon. The grouping was based on the size of two SstI fragments that hybridized with an epsB (gene involved in chain length determination) and epsD (priming glycosyltransferase gene) probes which correlated with the monosaccharide composition of the repetitive unit of the EPS. These analyses led to the grouping of most of the lactococcal strains into three major clusters (van Kranenburg et al., 1999 and Table 1
). The polymer produced by strains of group I is made of glucose, galactose, and rhamnose. The EPS synthesized by group II strains contain only galactose. Finally, strains of group III make exopolysaccharides composed of glucose and galactose (van Kranenburg et al., 1999).
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Although the three reference strains (MLT3, H414, MLT2) showed the expected hybridization patterns (van Kranenburg et al., 1999), three of the four lactococcal strains from our laboratory could not be classified by RFLP (Figure 1, Table 1
). Two disadvantages were found with the proposed RFLP method. First, the SacI (isoschizomer of SstI) fragments that hybridized with the epsB and epsD probes were often too large (>12 kb) for accurate size estimation in Southern analysis (Figure 1). But most importantly, a critical SacI site is located upstream (outside) the eps operon (the other SacI site is within the operon), indicating that the DNA sequence flanking the operon will dictate the size of the hybridizing fragment rather than the eps operon (Figure 2). Thus, the hybridization signals are reflecting the variability of the nucleotide sequence upstream of the operon rather than the variability of the operon itself. These findings led to the modification of the classification procedure.
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). The restriction fragments were then separated by electrophoresis and transferred onto a membrane. The epsD probe was used for the hybridization because it is the most conserved gene within the eps operon of the three groups. The hybridization results are presented in Figure 2 and summarized in Table 1. The reference strain MLT3 (group I) had a 9.5-kb AcyI fragment as well as a 3.5-kb HindII fragment that hybridized with the epsD probe (Figure 3, panels A and B, lane 8). Identical results were obtained with the strains SMQ-419 and SMQ-575 (Figure 3, panels A and B, lanes 3 and 5, respectively). The strain H414 (group II) had unique hybridization patterns with a 3.0-kb AcyI and a 2.7-kb HindII fragments that hybridized with the epsD probe (Figure 3, panels A and B, lane 4). The reference strain MLT2 (group III) and SMQ-420 had identical hybridization signals with the epsD probe, which included a 7.0-kb AcyI fragment and a 5.0-kb HindII fragment (Figure 3, panels A and B, lanes 2 and 7, respectively). Finally, the strain SMQ-461 also had unique hybridization patterns with a 2.0-kb AcyI and a 4.7-kb HindII fragments that hybridized with the epsD probe (Figure 3, panels A and B, lane 6), confirming that it belongs to a fourth and novel group (Deveau et al., 2002). The classification of the seven EPS+ L. lactis strains by this new RFLP procedure is in agreement with the previous analysis of the monosaccharide composition of the EPS (Deveau et al., 2002). Moreover, the eps operon of SMQ-575 was recently sequenced, and it is 100% identical at the nucleotide level to the eps operon of the strain NIZO B40 (GenBank accession number AF036485), which is also a member of group I, confirming the classification of SMQ-575 into this group (H. Deveau and S. Moineau, unpublished).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
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Received for publication July 26, 2002. Accepted for publication October 15, 2002.
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