**4. Alternative lactose utilization system and its interconnection with cellobiose assimilation**

The existence in several lactococcal strains devoid of *lac*-plasmids of cryptic lactose transport and catabolism systems has already been suggested in earlier studies (Anderson & McKay, 1977; Cords & McKay, 1974; de Vos & Simons, 1988; Simons et al*.*, 1993). The presence in *L. lactis* of chromosomally-encoded lactose permease has been proposed since introduction of the *E. coli lacZ* gene into a lactose-deficient *L. lactis* strain restored its ability to utilize lactose (de Vos & Simons, 1988). Moreover, P-*β*-galactosidase activities have also been detected in strains cured of their lactose plasmids, suggesting the presence of chromosomally-encoded cryptic *lac*-PTS(s) (Anderson & McKay, 1977; Cords & McKay, 1974). However, it was suggested that these PTSs are not specific for lactose, but rather for the translocation of other sugars (e.g., *β*-glucosides), and lactose could be transported alternatively. This hypothesis was supported by observations suggesting that a putative P-*β*-glucosidase, involved in cellobiose hydrolysis, is probably also involved in lactose-6-P cleavage in *L. lactis* strain ATCC7962 (Simons et al*.*, 1993). This seems reasonable, as according to http://www.tcdb.org/, PTS lactose transporters belong to the Lac family (TC No. 4.A.3) and porters of this family have broad substrate specificity. Besides lactose, they can also transport aromatic *β*-glucosides and cellobiose.

Until recently (Aleksandrzak et al*.*, 2000; Aleksandrzak-Piekarczyk et al., 2005, 2011; Kowalczyk et al., 2008), little information on the organization in *L. lactis* strains of chromosomal alternative lactose utilization genes has been available. It was shown that in *lac*-plasmid-free, and thus lactose-negative *L. lactis* IL1403, the ability to assimilate lactose can be induced in two ways: (i) by the presence of cellobiose or (ii) by inactivation of CcpA (Aleksandrzak et al*.*, 2000; Aleksandrzak-Piekarczyk et al., 2005). The CcpA protein is a member of the LacI-GalR family of bacterial repressors and exists only in Gram-positive bacteria (Weickert & Adhya, 1992). It exerts its regulatory role in carbon catabolite repression (CCR) by binding to DNA sites called *cre*s, which occur in the vicinity of CcpAregulated genes (Weickert & Chambliss, 1990). In *L. lactis* the known targets of CcpA are the *gal* operon for galactose utilization (Luesink et al., 1998), the *fru* operon for fructose utilization (Barrière et al., 2005), the *ptcABC* operon for cellobiose utilization (Zomer et al., 2007), and *cel-la*c genes for cellobiose and lactose utilization (Aleksandrzak-Piekarczyk et al., 2011). Thus, one could speculate that in *L. lactis* IL1403 cellobiose-inducible chromosomal

alternative lactose utilization genes are under the negative control of CcpA, and, therefore, inactivation of the *ccpA* gene could result in their derepression and ability to assimilate lactose by the IL1403 *ccpA* mutant.

Lactose and *β*-Glucosides Metabolism and Its Regulation in *Lactococcus lactis*: A Review 477

**Figure 1.** Schematic representation of the proposed mechanism of chromosomally-encoded lactose, cellobiose-inducible lactose and *β*-glucosides metabolism and of its regulation in *L. lactis* IL1403. In this model the key elements are the CelB, PtcB, PtcA, BglS and PtbA proteins. In the presence of glucose, IL1403 is unable to assimilate either lactose or *β*-glucosides. Under these conditions, these catabolic systems are either repressed by the CcpA protein and/or are not induced by the BglR activator.

Besides cellobiose, other *β*-glucosides like arbutin, esculin and salicin are transported by the PtbA-mediated PTS system. In the absence of any of these three sugars, *ptbA* expression is not induced by the inactive the phosphorylated BglR antiterminator protein. Once a *β*glucoside is available, BglR becomes dephosphorylated and active, inducing the expression of the *ptbA* gene. The PtbA protein transports, with concomitant phosphorylation, arbutin, esculin and salicin, which are then probably hydrolyzed by BglH, a P-*β*-glucosidase,

It is also proposed in this model that LacS is not engaged in lactose internalization and its

Despite the fact that the metabolism of lactose and *β*-glucosides is very important for the biotechnological processes catalysed by *L. lactis*, thorough studies of the chromosomally encoded features enabling use of these carbon sources were so far rather scarce. The reason for this could be the fact that *L. lactis* demonstrates a very large and complex metabolic capability towards carbohydrates used as carbon and energy sources, and, moreover, that this genetic potential is tightly regulated by various environmental and intracellular factors. It seems that the main obstacle in studies on the complicated

encoded by a gene located downstream of and in the same operon as the *ptbA* gene.

function is limited to galactose transport.

**5. Conclusions** 

Further studies of Aleksandrzak-Piekarczyk et al. (2005, 2011) and Kowalczyk et al*.* (2008) provided details on interconnected metabolism of *β*-glucosides (cellobiose) and *β*galactosides (lactose) and its variable regulation in *L. lactis* IL1403. Several genes have been implicated in coupled cellobiose and lactose assimilation in *L. lactis* IL1403, such as *bglS* and *celB, ptcA* and *ptcB,* encoding proteins homologous to P-*β*-glucosidase and EII components of cellobiose-specific PTS, respectively (Fig. 1). It has been shown that in *L. lactis* IL1403 the cellobiose-specific PTS system, comprising of *celB, ptcB* and *ptcA*, is also able to transport lactose because cellobiose-specific permease CelB has also an affinity for lactose, and, moreover, is the only permease involved in lactose uptake (Aleksandrzak-Piekarczyk et al., 2011). Furthermore, internalized lactose-P is hydrolyzed exclusively by BglS – an enzyme with dual P-*β*-glucosidase and P-*β*-galactosidase activity, and high affinity for cellobiose (Aleksandrzak-Piekarczyk et al., 2005) (Fig. 1). Thus, BglS activity generates glucose and galactose-P molecules. Glucose enters the Embden-Meyerhof-Parnas glycolytic pathway through phosphorylation by glucokinase, whereas galactose-P requires dephosphorylation performed by an unidentified phosphatase or phosphohexomutase, before entering the Leloir pathway (Neves et al., 2010) (Fig. 1). Moreover, this alternative lactose utilization system has been shown to be tightly controlled by CcpA-directed negative regulation (Fig. 1), since inactivation of the *ccpA* gene led to derepression of *bglS*, *celB, ptcA* and *ptcB* and *L. lactis* IL1403 *ccpA* mutant ability to assimilate lactose (Aleksandrzak-Piekarczyk et al., 2011). In addition to CcpA-mediated repression, the *celB* and *bglS* genes are specifically activated by cellobiose, as its presence leads to an increase in their transcription. This phenomenon has not been observed when other sugars, such as glucose, galactose or salicin, were used as carbon sources (Aleksandrzak-Piekarczyk et al., 2011). Preliminary results suggest that a hypothetical transcriptional regulator, namely YebF, could be engaged in this cellobiosedependent activation of *celB* and *bglS* (Aleksandrzak-Piekarczyk et al., 2011; unpublished personal analysis) (Fig. 1). The YebF protein belongs to the RpiR family of phosphosugar binding proteins (Sorensen & Hove-Jensen, 1996), and, in addition to its sugar binding domain (SIS), it has a putative helix-turn-helix (HTH) DNA-binding domain. In addition to *yebF* mutant ferment lactose inability (Aleksandrzak-Piekarczyk et al., 2005), inactivation of the *yebF* gene in IL1403 resulted in inability to grow on cellobiose (unpublished personal analysis), suggesting the gene's requirement in both cellobiose and lactose assimilation. Further studies on this phenomenon in *L. lactis* are needed to address it in greater detail.

When cellobiose is available, it activates the cellobiose-specific PTS transport system, comprising CelB, PtcB and PtcA proteins, and *L. lactis* IL1403 is able to grow on cellobiose and lactose. This growth is supported by the activity of cellobiose-inducible BglS protein, which splits lactose-P into galactose-P and glucose. Then, after the dephosphorylation step, galactose is further metabolized through the Leloir pathway, while glucose enters glycolysis. Therefore, inactivation of the *ccpA* gene results in derepression of the cellobiosespecific PTS transport system and also of the *bglS* gene, which in turn enable the IL1403 strain to grow on lactose.

**Figure 1.** Schematic representation of the proposed mechanism of chromosomally-encoded lactose, cellobiose-inducible lactose and *β*-glucosides metabolism and of its regulation in *L. lactis* IL1403. In this model the key elements are the CelB, PtcB, PtcA, BglS and PtbA proteins. In the presence of glucose, IL1403 is unable to assimilate either lactose or *β*-glucosides. Under these conditions, these catabolic systems are either repressed by the CcpA protein and/or are not induced by the BglR activator.

Besides cellobiose, other *β*-glucosides like arbutin, esculin and salicin are transported by the PtbA-mediated PTS system. In the absence of any of these three sugars, *ptbA* expression is not induced by the inactive the phosphorylated BglR antiterminator protein. Once a *β*glucoside is available, BglR becomes dephosphorylated and active, inducing the expression of the *ptbA* gene. The PtbA protein transports, with concomitant phosphorylation, arbutin, esculin and salicin, which are then probably hydrolyzed by BglH, a P-*β*-glucosidase, encoded by a gene located downstream of and in the same operon as the *ptbA* gene.

It is also proposed in this model that LacS is not engaged in lactose internalization and its function is limited to galactose transport.
