**1.2. Genus** *Lactococcus*

Lactococci are homofermentative, mesophilic LAB that basically inhabit two natural environments, milk and plants, of which plants seem to constitute the primary niche. Occasionally, there have been reports that *L. lactis* was also isolated from soil, efuent water, the skin of cattle (Klijn et al., 1995), insects (leafhoppers, termites) (Bauer et al., 2000;

© 2013 Aleksandrzak-Piekarczyk, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Latorre-Guzman et al., 1977; Schultz & Breznak, 1978) and fish (Itoi et al., 2008, 2009; Pérez et al., 2011). Adaptation of lactococcal strains from plants to the dairy environment has caused the loss of some functions, resulting in smaller chromosomes and acquisition of genes (often plasmidic) important for growth in milk (Kelly et al., 2010).

Lactose and *β*-Glucosides Metabolism and Its Regulation in *Lactococcus lactis*: A Review 469

pathogenic and spoilage bacteria, it supports curd formation, and creates optimal conditions for ripening. Further, due to their proteolytic activity and amino acid conversion, lactococci contribute to the final texture (moisture, softness) and flavour of dairy products (Smit et al., 2005). Many of lactococcal functions vital for successful fermentations are borne on plasmids, which are a common feature in lactococci, even in strains isolated from non-dairy sources (Davidson et al., 1996). For example, specific plasmid-borne genes encode proteins involved in lactose transport and metabolism and in hydrolysis and utilization of casein (Davidson, et al., 1996; McKay, 1983). Hence, there is considerable selective pressure on dairy strains to retain these plasmids, since plasmid-cured derivatives grow poorly in milk. Since plasmids are mobile elements, they can be readily exchanged among different strains

Due to its industrial importance *L. lactis* has become the best studied LAB, and although most studies have been performed on a small number of laboratory strains of dairy origin, it is regarded as a model organism for this bacterial group. A number of genome sequences of *L. lactis* strains are available, including strains from *L. lactis* subsp. *lactis*, such as IL1403, KF147 and CV56, as well as strains from *L. lactis* subsp. *cremoris*, such as MG1363, A76, NZ9000 and SK11 (according to http://www.ncbi.nlm.nih.gov/genome/). Among them, *L. lactis* subsp. *lactis* IL1403 (Chopin et al*.*, 1984) and *L. lactis* subsp. *cremoris* MG1363 (Gasson, 1983) are the most important laboratory strains, and they can be distinguished by differences in specific DNA sequences, including those encoding 16S rRNA (Godon et al*.*, 1992), and by their genome organization (Le Bourgeois et al*.*, 1995). These two strains are plasmid-cured derivatives of the dairy starter strains IL594 (IL1403) and NCDO 712 (MG1363) respectively, and due to their industrial importance, their metabolism, physiology and genetics have been extensively studied over the past years. Both belong to *L. lactis* subsp. *lactis* phenotypically, but the parent strain of IL1403 has a citrate permease plasmid (Górecki et al., 2011) and is able to metabolize citrate, placing it with *L. lactis* subsp. *lactis* biovar diacetylactis, whereas MG1363 has a *lactis* phenotype and a *cremoris* genotype (Kelly et al., 2010). Despite their physiological and 16S rRNA gene sequence similarities, they share only about 85% chromosomal sequence identity, which is comparable to the genetic distance between *Escherichia coli* and *Salmonella typhimurium* (McClelland et al., 2001; Salama et al., 1991; Wegmann et al., 2007). A derivative of MG1363 was created by the integration of the *nisRK* genes (involving the "NICE" system for nisin-controlled protein overexpression) into

Most microorganisms have adapted to growth in milk habitat due to acquisition of the ability to the use its most abundant sugar, lactose, as a carbon source. This disaccharide consists of a galactose moiety linked at its C1 via a *β*-galactosidic bond to the C4 of glucose. Because of the efficiency and economic importance of its fermentation, a large number of

Uptake of lactose into a bacterial cell can be mediated by several pathways, such as the lactose-specific phosphotransferase system (*lac*-PTS), ABC protein-dependent systems and

(via conjugal transfer) (Gasson, 1990).

the *pepN* gene, yielding *L. lactis* NZ9000 (Kuipers et al., 1998).

studies have focused on the utilization of lactose by LAB.

**2. Lactose metabolism** 

Since *Lactococcus lactis* was first described in 1919 (Orla-Jensen, 1919), its taxonomy has changed repeatedly and still is confusing in some aspects. This group of bacteria, previously designated lactic streptococci, was placed in the new *Lactococcus* taxon in 1985 (Schleifer *et al.,* 1985). The current taxonomy of *L. lactis* is based on phenotype and includes four subspecies (*lactis,* c*remoris, hordniae,* and the newly identied subsp. *tructae*) and one biovar (subsp. *lactis* biovar diacetylactis) (Schleifer et al., 1985; van Hylckama Vlieg et al., 2006; Pérez et al., 2011; Rademaker et al., 2007). Among them, only *L. lactis* subsp. *hordniae* and subsp. *tructae* have never been isolated from dairy products. The *lactis* and *cremoris* phenotypes are distinguished on the basis of several basic criteria, such as: arginine and maltose utilization, decarboxylation of glutamate to *γ*-aminobutyric acid (GABA), and 40°C, 4% NaCl and pH 9.2 tolerance. *L. lactis* subsp. *cremoris* strains are reported to be negative for all of these features (Nomura et al., 1999; Schleifer et al*.,* 1985). Moreover, the biovar diacetylactis strains are able to metabolize citrate, which is converted to diacetyl, an important aroma compound. Additionally, numerous genetic studies (DNA–DNA hybridization, 16S rRNA and gene sequence analysis) of *L. lactis* isolates of dairy and plant origin have revealed the existence among them of two main genotypes that have also been called *L. lactis* subsp. *lactis* (*lactis* genotype) and *L. lactis* subsp. c*remoris* (*cremoris* genotype)*.*  Furthermore, it has been demonstrated that the genotype and phenotype do not always correspond within one isolate, thus introducing a degree of disorder into the taxonomy of this species (Tailliez et al., 1998). It has been observed that within the group of *cremoris* genotype, strains with both *lactis* (MG1363) and *cremoris* (SK11) phenotypes may occur, and, likewise, within the group of *lactis* genotype there are ones with *lactis* (KF147) as well as biovar diacetylactis (IL594) phenotypes (Bayjanov et al., 2009; Kelly et al., 2010; Nomura et al., 2002; Rademaker et al., 2007; Tanigawa et al., 2010). Hence, the *L. lactis* has an atypical taxonomic structure with two phenotypically distinct groups, such as *L. lactis* subsp. *lactis* and *L. lactis* subsp. *cremoris*, which may belong to two distinct genotype groups. As a result, in order to sufficiently describe the individual strains, it is necessary to specify both the genotype (*cremoris* or *lactis*) and the phenotype (*cremoris, diacetylactis*, or *lactis*).

Strains belonging to *L. lactis* subsp. *lactis* and *L. lactis* subsp. *cremoris* together with a diverse assortment of other LAB are widely used as dairy starters for the production of a vast range of fermented dairy products, including various types of cheeses, sour cream, buttermilk and butter (Daly, 1983; Davidson et al., 1996). In the dairy industry, the *lactis* subspecies are better for making soft cheeses and the *cremoris* subspecies for the hard ones. Overall, it is generally accepted that the *L. lactis* subsp. *cremoris* strains make better quality products than *L. lactis* subsp. *lactis* because of their important contribution to flavour development via their unique metabolic mechanisms (Salama et al*.*, 1991; Sandine, 1988).

During growth in milk, the primary function of *L. lactis* is rapid conversion of lactose to lactic acid, which provides preservation of the fermented product by preventing growth of pathogenic and spoilage bacteria, it supports curd formation, and creates optimal conditions for ripening. Further, due to their proteolytic activity and amino acid conversion, lactococci contribute to the final texture (moisture, softness) and flavour of dairy products (Smit et al., 2005). Many of lactococcal functions vital for successful fermentations are borne on plasmids, which are a common feature in lactococci, even in strains isolated from non-dairy sources (Davidson et al., 1996). For example, specific plasmid-borne genes encode proteins involved in lactose transport and metabolism and in hydrolysis and utilization of casein (Davidson, et al., 1996; McKay, 1983). Hence, there is considerable selective pressure on dairy strains to retain these plasmids, since plasmid-cured derivatives grow poorly in milk. Since plasmids are mobile elements, they can be readily exchanged among different strains (via conjugal transfer) (Gasson, 1990).

Due to its industrial importance *L. lactis* has become the best studied LAB, and although most studies have been performed on a small number of laboratory strains of dairy origin, it is regarded as a model organism for this bacterial group. A number of genome sequences of *L. lactis* strains are available, including strains from *L. lactis* subsp. *lactis*, such as IL1403, KF147 and CV56, as well as strains from *L. lactis* subsp. *cremoris*, such as MG1363, A76, NZ9000 and SK11 (according to http://www.ncbi.nlm.nih.gov/genome/). Among them, *L. lactis* subsp. *lactis* IL1403 (Chopin et al*.*, 1984) and *L. lactis* subsp. *cremoris* MG1363 (Gasson, 1983) are the most important laboratory strains, and they can be distinguished by differences in specific DNA sequences, including those encoding 16S rRNA (Godon et al*.*, 1992), and by their genome organization (Le Bourgeois et al*.*, 1995). These two strains are plasmid-cured derivatives of the dairy starter strains IL594 (IL1403) and NCDO 712 (MG1363) respectively, and due to their industrial importance, their metabolism, physiology and genetics have been extensively studied over the past years. Both belong to *L. lactis* subsp. *lactis* phenotypically, but the parent strain of IL1403 has a citrate permease plasmid (Górecki et al., 2011) and is able to metabolize citrate, placing it with *L. lactis* subsp. *lactis* biovar diacetylactis, whereas MG1363 has a *lactis* phenotype and a *cremoris* genotype (Kelly et al., 2010). Despite their physiological and 16S rRNA gene sequence similarities, they share only about 85% chromosomal sequence identity, which is comparable to the genetic distance between *Escherichia coli* and *Salmonella typhimurium* (McClelland et al., 2001; Salama et al., 1991; Wegmann et al., 2007). A derivative of MG1363 was created by the integration of the *nisRK* genes (involving the "NICE" system for nisin-controlled protein overexpression) into the *pepN* gene, yielding *L. lactis* NZ9000 (Kuipers et al., 1998).

### **2. Lactose metabolism**

468 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

Latorre-Guzman et al., 1977; Schultz & Breznak, 1978) and fish (Itoi et al., 2008, 2009; Pérez et al., 2011). Adaptation of lactococcal strains from plants to the dairy environment has caused the loss of some functions, resulting in smaller chromosomes and acquisition of

Since *Lactococcus lactis* was first described in 1919 (Orla-Jensen, 1919), its taxonomy has changed repeatedly and still is confusing in some aspects. This group of bacteria, previously designated lactic streptococci, was placed in the new *Lactococcus* taxon in 1985 (Schleifer *et al.,* 1985). The current taxonomy of *L. lactis* is based on phenotype and includes four subspecies (*lactis,* c*remoris, hordniae,* and the newly identied subsp. *tructae*) and one biovar (subsp. *lactis* biovar diacetylactis) (Schleifer et al., 1985; van Hylckama Vlieg et al., 2006; Pérez et al., 2011; Rademaker et al., 2007). Among them, only *L. lactis* subsp. *hordniae* and subsp. *tructae* have never been isolated from dairy products. The *lactis* and *cremoris* phenotypes are distinguished on the basis of several basic criteria, such as: arginine and maltose utilization, decarboxylation of glutamate to *γ*-aminobutyric acid (GABA), and 40°C, 4% NaCl and pH 9.2 tolerance. *L. lactis* subsp. *cremoris* strains are reported to be negative for all of these features (Nomura et al., 1999; Schleifer et al*.,* 1985). Moreover, the biovar diacetylactis strains are able to metabolize citrate, which is converted to diacetyl, an important aroma compound. Additionally, numerous genetic studies (DNA–DNA hybridization, 16S rRNA and gene sequence analysis) of *L. lactis* isolates of dairy and plant origin have revealed the existence among them of two main genotypes that have also been called *L. lactis* subsp. *lactis* (*lactis* genotype) and *L. lactis* subsp. c*remoris* (*cremoris* genotype)*.*  Furthermore, it has been demonstrated that the genotype and phenotype do not always correspond within one isolate, thus introducing a degree of disorder into the taxonomy of this species (Tailliez et al., 1998). It has been observed that within the group of *cremoris* genotype, strains with both *lactis* (MG1363) and *cremoris* (SK11) phenotypes may occur, and, likewise, within the group of *lactis* genotype there are ones with *lactis* (KF147) as well as biovar diacetylactis (IL594) phenotypes (Bayjanov et al., 2009; Kelly et al., 2010; Nomura et al., 2002; Rademaker et al., 2007; Tanigawa et al., 2010). Hence, the *L. lactis* has an atypical taxonomic structure with two phenotypically distinct groups, such as *L. lactis* subsp. *lactis* and *L. lactis* subsp. *cremoris*, which may belong to two distinct genotype groups. As a result, in order to sufficiently describe the individual strains, it is necessary to specify both the

genes (often plasmidic) important for growth in milk (Kelly et al., 2010).

genotype (*cremoris* or *lactis*) and the phenotype (*cremoris, diacetylactis*, or *lactis*).

unique metabolic mechanisms (Salama et al*.*, 1991; Sandine, 1988).

Strains belonging to *L. lactis* subsp. *lactis* and *L. lactis* subsp. *cremoris* together with a diverse assortment of other LAB are widely used as dairy starters for the production of a vast range of fermented dairy products, including various types of cheeses, sour cream, buttermilk and butter (Daly, 1983; Davidson et al., 1996). In the dairy industry, the *lactis* subspecies are better for making soft cheeses and the *cremoris* subspecies for the hard ones. Overall, it is generally accepted that the *L. lactis* subsp. *cremoris* strains make better quality products than *L. lactis* subsp. *lactis* because of their important contribution to flavour development via their

During growth in milk, the primary function of *L. lactis* is rapid conversion of lactose to lactic acid, which provides preservation of the fermented product by preventing growth of Most microorganisms have adapted to growth in milk habitat due to acquisition of the ability to the use its most abundant sugar, lactose, as a carbon source. This disaccharide consists of a galactose moiety linked at its C1 via a *β*-galactosidic bond to the C4 of glucose. Because of the efficiency and economic importance of its fermentation, a large number of studies have focused on the utilization of lactose by LAB.

Uptake of lactose into a bacterial cell can be mediated by several pathways, such as the lactose-specific phosphotransferase system (*lac*-PTS), ABC protein-dependent systems and secondary system transporters like lactose-galactose antiporters and lactose-H+ symport systems (de Vos & Vaughan, 1994). While ABC protein-dependent lactose transport has been demonstrated only in non-LAB, Gram-negative *Agrobacterium radiobacter* (Williams et al*.*, 1992), the *lac*-PTS as well as secondary lactose transport systems have been described for many LAB species.

Lactose and *β*-Glucosides Metabolism and Its Regulation in *Lactococcus lactis*: A Review 471

are chimeric in nature: at their carboxy terminal end they contain an approximately 160 amino acid hydrophilic extension homologous to the EIIA domains of PTS. Thus, lactose transport is controlled by HPr-dependent phosphorylation (Gunnewijk et al*.*, 1999; Gunnewijk & Poolman, 2000a; Gunnewijk & Poolman, 2000b). Due to this additional domain these lactose permeases are larger than the other carriers from the GPH family, which are generally about 500 amino acids in length. Depending on the organism, LacS can mediate lactose transport coupled to proton symport or by antiport with galactose. Following its import, lactose is hydrolyzed by *β*-galactosidase (David et al*.*, 1992; Vaughan et al*.*, 1996) yielding glucose and galactose. The glucose moiety is further metabolized via glycolysis, whereas the galactose moiety follows different pathways depending on the particular LAB. While some thermophilic strains of LAB (*e.g., Lactobacillus bulgaricus* and *Streptococcus thermophilus*) are known to release the galactose moiety of lactose into the medium, other LAB (*e.g., Lactobacillus helveticus, Leuconostoc lactis* and *Streptococcus salivarius*) metabolize this saccharide via the Leloir pathway (de Vos, 1996; Poolman, 1993; Vaughan et al*.*, 2001). This pathway was one of the first central metabolic pathways to be discovered, by L. F. Leloir and coworkers in the early 1950s. It includes the key enzyme galactokinase (GalK), and hexose-1-P uridylyltransferase (GalT) plus UDP-glucose 4-epimerase (GalE), all of which are involved in the conversion of galactose to glucose-1P. The generated glucose-1P, after conversion to glucose-6P by phosphoglucomutase, enters the glycolytic pathway. Aldose-1-epimerase, a mutarotase (GalM), is an additional, more recently characterized enzyme required for rapid galactose metabolism (Bouffard et al*.*, 1994; Mollet & Pilloud, 1991; Poolman et al*.*, 1990). GalM catalyses the interconversion of the *α*- and *β*-anomers of galactose. This enzyme was found to be essential for efficient lactose utilization in *E. coli* since cleavage of this *β*-galactoside by *β*-galactosidase yields glucose and *β*-D-galactose, the

The existence of genes encoding components of the lactose permease-*β*-galactosidase system seems to be limited among the *L. lactis* strains as they have been identified only in the genomes of the dairy-derived strain IL1403 (Bolotin et al., 2001), non-dairy NCDO2054 (Vaughan et al., 1998) and KF147 isolated from mung bean sprouts (Siezen et al., 2010). Remarkably, in addition to galactose genes of the Leloir pathway cluster, these strains contain genes needed for lactose assimilation, such as *lacZ* (*β*-galactosidase) and *lacA* (thiogalactoside acetyltransferase), arranged in an identical layout. Directly upstream of the aforementioned genes required for lactose hydrolysis and subsequent galactose conversion,

Some details concerning the role of the lactose permease-*β*-galactosidase system in lactose utilization have been reported for the slow lactose fermenter - *L. lactis* NCDO2054 (Vaughan et al., 1998), and for the devoid of the *lac*-plasmid, essentially lactose-negative *L. lactis* IL1403 strain (starts to utilize lactose slowly after approximately 40 h of incubation) (Aleksandrzak-Piekarczyk et al., 2005). Since these strains possess the complete lactose permease-*β*galactosidase system and an active Leloir pathway, it seems odd that they are barely capable of lactose metabolism. In the case of *L. lactis* NCDO2054, which can accumulate a high intracellular concentration of lactose-6-phosphate by using an efcient *lac*-PTS and

latter being the sole substrate for GalK (Bouffard et al*.*, 1994).

there is the gene encoding the LacS permease for sugar uptake.
