J. Dairy Sci. 88:2341-2347
© American Dairy Science Association, 2005.
Use of an 
-Galactosidase Gene as a Food-Grade Selection Marker for Streptococcus thermophilus
S. Labrie*,
C. Bart,
C. Vadeboncoeur and
S. Moineau
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, Quebec City, 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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The 
-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the -galactosidase of L. raffinolactis is thermolabile and inoperative at 42°C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this -galactosidase at 42°C was only 3% of that at 30°C, whereas the enzyme retained 23% of its activity at 37°C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37°C or below. The transformed cells possessed 6-fold more -galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.
Key Words: Lactococcus raffinolactis • melibiose • raffinose • lactic acid bacteria
Abbreviation key: Cm = chloramphenicol, BCP = bromocresol purple, LAB = lactic acid bacteria, OD600nm = optical density at 600 nm.
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INTRODUCTION
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Streptococcus thermophilus is a lactic acid bacterium (LAB) used to manufacture yogurt and a number of cheeses. The extensive commercial use of this bacterium has led to several fundamental and applied studies aimed at increasing our understanding of this LAB, to select better strains or to improve them through genetic modification (Turgeon et al., 2004; Vadeboncoeur and Moineau, 2004). Very few molecular tools are available for the genetic engineering of Strep. thermophilus. Such tools should be food-grade and applicable under laboratory and industrial conditions (Hansen, 2002). Food-grade systems are defined as an association of DNA that originates exclusively from "generally recognized as safe" organisms, including the selection marker (Johansen, 1999; Hansen, 2002; Kondo and Johansen, 2002). The criteria used for the classification as a food-grade marker include the safety of the genetic material transferred in the host, food compatibility, the absence of antibiotic resistance markers, the nonuse of harmful compounds, and the applicability on an industrial scale or in food products (de Vos, 1999; Hansen, 2002). For a recent review on food-grade vectors, readers are referred to Shareck et al. (2004).
Two strategies are generally used to introduce genetic material into relevant LAB: 1) integration into the host genome by homologous recombination (Biswas et al., 1993; Gosalbes et al., 2000; Henrich et al., 2002; Sasaki et al., 2004) or phage-mediated integration (MacCormick et al., 1995; Lillehaug et al., 1997; Martín et al., 2000); or 2) introduction of autonomously replicating plasmids (Kok et al., 1984; Boucher et al., 2002; El Demerdash et al., 2003; Wong et al., 2003). Plasmids are often preferred because many copies of the target gene may be needed to obtain the desired phenotype, most notably for phage-resistance systems (O’Sullivan et al., 1995; Bouchard et al., 2002; Émond et al., 1997, 1998).
Few food-grade markers are available for Strep. thermophilus. Cadmium resistance is a dominant selection marker, encoded by the cadA and cadC genes of L. lactis, and was successfully used in Strep. Thermophilus (Liu et al., 1997; Wong et al., 2003). A ß-galactosidase gene was proposed as a complementation marker for lactose-negative Strep. thermophilus strains (Herman and McKay, 1986). An shsp gene encoding a putative small heat-shock protein conferring heat and acid resistance was also used as a food-grade selection marker in Strep. thermophilus (El Demerdash et al., 2003). Finally, thymidilate synthase genes from Strep. thermophilus or Lactobacillus delbrueckii spp. bulgaricus were shown to be suitable as selection markers in thymidine-requiring mutants of Strep. thermophilus (Sasaki et al., 2004).
The limitation of dominant selection markers is linked to the natural occurrence of the phenotype in Strep. thermophilus, whereas complementation markers may be inconvenient to use as they require prior isolation of appropriate mutants (Boucher et al., 2002). A dominant selection marker that takes advantage of the inability of a number of LAB strains to ferment melibiose was recently developed (Boucher et al., 2002). The dominant marker is based on the aga gene of Lactococcus raffinolactis, which is expressed using its own constitutive promoter (Boucher et al., 2002, 2003). This gene codes for an -galactosidase that catalyzes the hydrolysis of the 1–4 link of melibiose and the release of 2 monosaccharides, glucose and galactose. This gene is able to convert the mesophilic species Lactococcus lactis and Pediococcus acidilactici from melibiose-negative to melibiose-positive phenotypes. This marker was not functional in Strep. thermophilus (Boucher et al., 2002). This result was unexpected because the LacS transporter of Strep. thermophilus, which is partially constitutive (Vaughan et al., 2001), is able to transport melibiose as well as lactose (Poolman et al., 1992; Veenhoff and Poolman, 1999). The presence of a functional -galactosidase in Strep. thermophilus should allow its growth on melibiose by the metabolism of the glucose residue via the glycolytic Embden–Meyerhof–Parnas pathway. In this work, we show that the aga gene of L. raffinolactis can indeed be used as a dominant food-grade selection marker in Strep. thermophilus when the cells are grown at 37°C or below.
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MATERIALS AND METHODS
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Bacterial Strain, Bacteriophage, Plasmids, and Media
The bacterial strain, plasmids, and phage used in this study are listed in Table 1
. Unless mentioned otherwise, Strep. thermophilus was grown on M17 medium (Quélab, Montréal, Québec, Canada) at 42°C. The carbohydrate fermentation assay was carried out in bromocresol purple (BCP) medium (2% tryptone, 0.5% yeast extract, 0.4% NaCl, 0.15% Na-acetate, 40 mg/L of bromocresol purple) (McKay et al., 1972; Boucher et al., 2002). Cultures used for transcriptional analyses were grown in the same medium but without BCP. Sugars were filter-sterilized (0.22 µm) and added at a final concentration of 0.5% in BCP or M17 media. The plasmid pRAF301 used in this study is a 2.5-kb PCR product composed of the aga gene of L. raffinolactis ATCC 43920 (GenBank accession number AY164273) cloned into the shuttle vector pNZ123 (Boucher et al., 2002). Plasmid pNZ123 (de Vos, 1987) carries a chloramphenicol (Cm) acetyl transferase (cat) gene used for clone selection in both Escherichia coli and Strep. thermophilus hosts. When needed, Cm was added to the growth medium at a concentration of 5 µg/mL. The virulent bacteriophage DT1, which is capable of propagating in Strep. thermophilus RD733, was used in a spot assay to confirm the identity of the transformants (Tremblay and Moineau, 1999). Sugar fermentation patterns were determined using API 50 CH strips with API 50 CHL medium (BioMérieux, St-Laurent, Québec, Canada) incubated at either 37 or 42°C.
Plasmid Isolation and Electrotransformation
Streptococcus thermophilus plasmid DNA was isolated using a silica-based method as described previously (Émond et al., 2001), with the following modifications: the lysozyme concentration was increased to 60 mg/mL and the corresponding incubation was extended to 30 min at 37°C. The protocol used for the electrotransformation of Strep. thermophilus was modified from the glycine-shock protocol reported elsewhere (Buckley et al., 1999). Streptococcus thermophilus cells were first grown at 42°C in 100 mL of M17 medium containing 0.5% lactose. When the culture reached an optical density at 600 nm (OD600nm) of 0.5, 100 mL of M17 containing 0.8 M sorbitol, 20% glycine, and 0.5% lactose was added to the medium. The culture was incubated at 42°C for an additional 60 min. The cells were collected by centrifugation and washed 3 times in cold electroporation buffer (0.4 M sorbitol, 10% glycerol) and resuspended in a final volume of 1 mL. The cells were either used immediately or quickly frozen in an isopropanol (80%) bath maintained at –80°C. For electroporation, the cells were thawed on ice, and 40 µL of cells were combined with 1 µg of plasmid DNA and transferred to a prechilled 0.2-cm electroporation cuvette (BioRad, Mississauga, Ontario, Canada). The electroporation was performed using a BioRad Gene Pulser apparatus with a BioRad pulse controller set at 2.5 kV, 200 ohms, and 25 µF. The cells were then immediately recovered in 1 mL of cold recuperation medium (M17 with 0.4 M sorbitol, 20 mM MgCl2, 2 mM CaCl2, and 0.5% lactose). After resting on ice for 5 min, the cells were incubated at42°C for 2 h. Aliquots were then plated on appropriate selective media.
-Galactosidase Activity
The -galactosidase activity was measured as previously described (Boucher et al., 2002, 2003) with the following modifications: strains were grown in medium with 0.5% of the appropriate sugar but without BCP until the OD600nm reached 0.3. Pellets from 10-mL cultures were washed twice with 1 mL of sodium phosphate buffer (50 mM, pH 7.0) and lysed with glass beads. The cell lysates (0.5 mL) were cleared by centrifugation and dialyzed (6000- to 8000-kDa pores) against 250 mL of sodium phosphate buffer for 15 min as described previously (Boucher et al., 2002). The protein concentrations of the cell extracts were determined using the BioRad DC protein assay. The -galactosidase activity was assayed at 30, 37, and 42°C at pH 7.0 using p-nitrophenyl--D-galactopyranoside (Sigma-Aldrich, Oakville, Ontario, Canada) as the substrate.
Transcriptional Analysis
Total RNA was isolated from Strep. thermophilus as reported elsewhere (Boucher et al., 2002), with the following modifications: 1 mL of a cell culture (OD600nm of 0.3) was mixed with 100 µL of rifampicin (2.5 mg/mL in methanol) and pelleted. The cells were gently resuspended in 100 µL of a lysozyme solution (60 mg/mL) diluted in 20% sucrose and incubated at 37°C for 15 min. Total RNA was extracted using the RNeasy kit (Qiagen, Chatsworth, CA). Following the RNAse-free DNAse treatment, a slot-blot analysis was performed on a positively charged nylon membrane (Roche Diagnostic, Laval, Québec, Canada) using a BioDot SF apparatus (BioRad). One microgram of total RNA from each sample was denatured and applied to the slot-blot apparatus (Sambrook and Russell, 2001). After fixing by UV exposure, the RNA was detected by a probe generated using a PCR digoxigenin labeling kit and corresponding to an 882-bp internal fragment of the L. raffinolactis ATCC 43920 aga gene (Primers: Raf66 5'-GCC CGC ATT TGC GCT GTA AT-3'and Raf69 5'-GGG ATG GCA CCA GTT GTC AT-3').
Growth Properties and Plasmid Stability
The growth of Strep. thermophilus strains in M17 medium supplemented with 0.2% lactose and Cm (5 µg/mL), when applicable, was monitored by following the OD660nm at 42°C. Generation times were calculated for cultures in exponential growth phase by plotting the logarithm of the OD660nm against time. The values represent the means of 3 separate experiments. For growth in milk, Strep. thermophilus strains were inoculated at 5% (vol/vol) from an overnight culture (LM17) in 10 mL of pasteurized milk (Natrel) and an aliquot was immediately taken for pH reading at time zero. The tubes were then incubated at 42°C for 7 h. A second pH reading was taken and the 
pH was obtained by subtracting both values. The milk acidification assay was also performed in triplicate. To demonstrate the stability of the plasmid carrying the aga gene from L. raffinolactis, Strep. thermophilus RD733 carrying pNZ123 and Strep. thermophilus RD733 carrying pRAF301 were repeatedly cultivated in LM17 at 42°C for 7 d without Cm. One hundred colonies of each strain were picked at random and tested for their resistance to Cm and, when applicable, growth on melibiose (at 37°C).
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RESULTS AND DISCUSSION
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The aga Gene of L. raffinolactis ATCC 43920 can confer a Melibiose Fermentation Phenotype on Strep. thermophilus
We previously reported that Strep. thermophilus transformants containing pRAF301, a plasmid carrying the L. raffinolactis aga gene and its endogenous promoter are unable to grow on melibiose (Boucher et al., 2002). However, the ability of these transformants to grow on melibiose was only tested at 42°C, the optimal growth temperature for Strep. thermophilus (but a non-permissive growth temperature for L. raffinolactis). When the incubation temperature for Strep. thermophilus was decreased to 37°C, the industrial Strep. thermophilus RD733 strain carrying pRAF301 was able to grow on BCP medium containing melibiose. To confirm that melibiose fermentation by the Strep. thermophilus transformant was temperature-dependent, API strips were inoculated either with Strep. thermophilus RD733 containing the shuttle-vector pNZ123 or with Strep. thermophilus RD733 containing pRAF301 and incubated at 37 or 42°C. Unlike the Strep. thermophilus strain carrying pNZ123, which was unable to ferment melibiose at either temperature, the strain carrying pRAF301 was able to ferment melibiose and raffinose at 37°C but not at 42°C. These results suggest that the -galactosidase of L. raffinolactis is thermolabile or that the aga gene is not transcribed at 42°C, or both.
Temperature Sensitivity of the L. raffinolactis -Galactosidase
Streptococcus thermophilus RD733 carrying pRAF301 was grown at 30°C in a melibiose-containing medium, and a cell extract was obtained as described in Materials and Methods. The -galactosidase activity of the cell extract was determined at 30°C, the optimal growth temperature of L. raffinolactis, as well as at 37 and 42°C (Table 2). When the temperature of the enzymatic assay was set at 42°C, only 3% of the -galactosidase activity found at 30°C was detected in the cell extract. Likewise, only 24% of the -galactosidase activity was retained at 37°C.
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Table 2. Effect of temperature on -galactosidase activity in cell extracts of Strep. thermophilus RD733 containing pRAF301, and grown on melibiose or lactose.
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Effect of Lactose and Temperature on aga Expression
To determine the effect of lactose, the principal sugar found in milk, on the expression of -galactosidase, Strep. thermophilus RD733 cells carrying pRAF301 were grown on lactose at 30°C and -galactosidase activity was measured in cell extracts at 37°C. The -galactosidase activity was 6-fold lower in lactose- than in melibiose-grown cells (Table 2
). To determine whether the lactose repression occurred at the transcriptional level, total RNA was isolated during the exponential growth of Strep. thermophilus RD733 containing pRAF301 or pNZ123. An internal portion of the aga gene was used as a probe to detect specific mRNA (Figure 1). The amount of aga mRNA in Strep. thermophilus carrying pRAF301 was much lower in lactose-than in melibiose-grown cells, which is consistent with the levels of -galactosidase activities. As expected, no aga mRNA was detected in Strep. thermophilus RD733 carrying the cloning vector pNZ123. The decrease in aga mRNA levels and -galactosidase activity caused by growth on lactose suggested that expression of aga is subject to repression by lactose. In Strep. thermophilus, lactose caused partial repression of the lactose operon by a mechanism involving the transcriptional regulator CcpA (van den Bogaard et al., 2000). This regulatory protein recognizes a specific DNA sequence called cre (catabolite-responsive element) in the promoter region of target operons (Hueck et al., 1994; Miwa et al., 2000). A cre sequence encompasses the –35 region of the aga promoter (Boucher et al., 2002), suggesting a possible regulatory role for CcpA during growth on lactose.
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Figure 1. Slot-blot hybridization using an 882-bp PCR-digoxigenin-labeled internal section of the Lactococcus raffinolactis aga gene. Total RNA was isolated from Strep. thermophilus RD733 containing pRAF301 grown in the presence of melibiose at 37°C (lane 1), lactose at 42°C (lane 2), and lactose at 37°C (lane 5). Total RNA was isolated from Strep. thermophilus RD733 carrying pNZ123 grown in the presence of lactose at 37°C (lane 3) and lactose at 42°C (lane 4) and used as negative controls. Streptococcus thermophilus containing pRAF301 does not grow at 42°C in the presence of melibiose.
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To determine whether the growth temperature had an effect on the transcription of aga, we compared the amounts of aga mRNA in cells grown on lactose at 37°C and 42°C. The transcription of aga was similar at both temperatures (Figure 1, lanes 2 and 5), indicating that the inability of the transformants to grow on melibiose at 42°C most likely resulted from the heat sensitivity of the L. raffinolactis -galactosidase rather than inefficient transcription of aga.
The aga Gene as a Dominant Selection Marker on BCP Medium
The efficacy of the aga gene as a dominant selection marker was determined by electroporating pRAF301 into the industrial Strep. thermophilus strain RD733. Bromocresol purple medium was used to select melibiose-positive transformants on solid media because, unlike M17, it does not support residual growth without an external carbon source (data not shown). The ability to recover transformants on melibiose-containing medium using aga as a selection marker was also compared with the Cm resistance marker (Cmr), which is also present on pRAF301. Following electroporation, Strep. thermophilus RD733 cells were plated on BCP-Mel, BCP-Mel-Cm, and BCP-Lac-Cm (Table 3). Similar electroporation efficiencies were obtained with each medium, showing that selection using melibiose is equivalent to selection using Cm. Fifty colonies were randomly picked from lactose plates containing Cm and streaked on melibiose medium. Forty-nine clones were able to readily grow on melibiose, confirming the presence and stability of the aga gene.
Plasmid Stability and Growth Properties
Plasmid stability assays showed that pNZ123 and pRAF301 were highly stable in Strep. thermophilus RD733. Indeed, 98 to 99% of the colonies tested retained their ability to grow on Cm and melibiose (for pRAF301) even after repeated culturing without selective pressure (Table 4). The generation times (at 42°C in LM17 medium) of the wildtype strain Strep. thermophilus RD733 as well as Strep. thermophilus RD733 carrying pNZ123 were identical at 29 min (Table 4). Under the same conditions, Strep. thermophilus carrying pRAF301 had a slightly longer generation time (35 min) in LM17 medium. The 3 strains were also grown in pasteurized milk, and milk acidification was monitored by measuring the difference in pH following a 7-h incubation at 42°C. The milk assay showed that all 3 strains decreased the pH at the same level (Table 4). These data suggest that the presence of pRAF301 has no effect on metabolic activities associated with milk acidification.
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CONCLUSIONS
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The aga gene was isolated from a "generally recognized as safe" organism, and a plasmid containing aga was readily introduced into one industrial Strep. thermophilus strain. The -galactosidase activity is inefficient at 42°C, the optimum growth temperature of Strep. thermophilus, and its expression is down-regulated when the cells are cultivated on lactose, the main sugar in milk. This marker can thus be used in the laboratory to select recombinant Strep. thermophilus strains at 37°C on a melibiose-containing medium. The marker did not interfere with the acidification of milk during fermentation. We are currently investigating the replication machinery of several natural Strep. thermophilus plasmids (Turgeon and Moineau, 2001; Turgeon et al., 2004) to identify several replicons that could be used in combination with the aga gene of L. raffinolactis to construct novel food-grade vectors.
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ACKNOWLEDGEMENTS
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We would like to thank M. Couture, D. Tremblay, and K. Vaillancourt for helpful discussions, and G. Bourgeau for editorial assistance. We are grateful to Danisco for providing Strep. thermophilus RD733. This work was supported by the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT), Novalait Inc., and the Natural Sciences and Engineering Research Council of Canada (NSERC).
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FOOTNOTES
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* Present address: Nutraceuticals and Functional Foods Institute (INAF), Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec City, Québec, Canada, G1K 7P4. 
Received for publication September 22, 2004.
Accepted for publication March 23, 2005.
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ABSTRACT
INTRODUCTION
