**5. Conclusions**

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

lactose by the IL1403 *ccpA* mutant.

strain to grow on lactose.

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

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

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

mechanisms involved in assimilation of *β*–glycoside sugars was the lack of complex data specifying the sequences of genes potentially involved in the metabolism of these sugars and its regulation. Indeed, recent access to the genomic sequences of some these bacteria greatly advanced the research on the metabolism of various *β*–glycosides. As expected, the results of sequencing of lactococcal genomes and genes annotations confirmed that there are numerous genes encoding potential *β*-glucosidesspecific transport systems and *β*-glucosidases, sometimes with dual activities. And, to complicate the matter even further, the analysis of the list of genes annotated in *L. lactis* leads to over a hundred transcriptional regulators. A relatively large number of them may be related to carbon metabolism control. These regulators, together with signals modulating their activity, and the controlled genes form a regulatory network that is necessary for sensing the environmental conditions and adjusting the catabolic capacities of the cell.

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Detailed knowledge of sugar metabolism and the regulators controlling gene expression in *Lactococcus lactis* may contribute to the improvement of mechanisms controlling significant cellular processes in these bacteria. In the case of industrial microorganisms, acting on the defined regulatory network may drastically affect the properties of the bacteria and have an impact on bioprocesses.

Lastly, is shown as an example that by the use of a simple microbiological screen, it is possible and worthwhile to modify the metabolic potential of lactococcal strains initially unable to assimilate lactose. By inactivation of the *ccpA* gene or induction of particular genes by supplementation of the medium with cellobiose and thus activation of YebF, it is possible to turn on an alternative lactose assimilation pathway in *L. lactis* IL1403. In contrast to plasmid-located *lac*-operons, the *cel-lac* system is within the chromosome, resulting in a stable, highly adapted strain, potentially valuable for the dairy industry.
