To understand how microbes utilize carbohydrates, we must determine the genetic and biochemical bases for sugar transport into the cell, and identify the regulatory networks involved in transcription of genes encoding transporters. Carbohydrate transport and catabolism are well orchestrated in LAB, so as to utilize carbohydrate sources optimally. The regulatory mechanism for global carbohydrate utilization is carbon catabolite repression (CCR).
Carbon catabolite repression (CCR) is a mechanism widely distributed amongst Gram-positive bacteria, usually mediated in cis by catabolite response elements (cre)
(Weickert and Chambliss, 1990; Miwa et al., 2000), and in trans by repressors of the LacI family, responsible for transcriptional repression of genes encoding unnecessary saccharolytic components (Weickert and Chambliss, 1990; Viana et al., 2000; Muscariello et al, 2001; Titgemeyer and Hillen, 2002; Warner and Lolkema, 2003). Cre
sequences (Weickert and Chambliss, 1990) are well conserved amongst Gram-positive bacteria and found in most LAB in the promoter-operator of many genes involved in carbohydrate utilization (Barrangou et al., 2003), including: L. plantarum (Muscariello et al., 2001), L. pentosus (Mahr et al., 2000). CCR controls transcription of proteins involved in transport and catabolism of carbohydrates (Miwa et al., 2000), as to transcribe genes encoding the transport and enzymatic machinery of a particular substrate, exclusively when it is present in the environment. This regulatory system allows cells to coordinate the utilization of diverse carbohydrates, as to focus primarily on preferred energy sources (Poolman, 2002). Understanding carbon catabolite repression is critical to describing how microbes adapt their uptake machinery to changing nutrients in their environment.
CCR is able to control both PTS, ABC and GPH transporters. Specifically, ABC transporters of the MsmEFGK family have been shown to be repressed by glucose in a manner consistent with CCR, in S. pneumoniae (Rosenow et al., 1999; Barrangou et al., 2003). Similarly, genes of the galactose operon seem to be regulated via CCR in S. salivarius (Vaillancourt et al., 2002).
The L. acidophilus genome encodes a large variety of genes related to carbohydrate utilization. In particular, many members of the ABC and PTS families of transporters were found. Additionally, the members of the general carbohydrate
utilization regulatory network were identified, namely HPr (ptsH), E1 (ptsI), CcpA (ccpA) and HPrK/P (ptsK). Similarly, all those genes were identified in S. pneumoniae
(Tettelin et al., 2001). Those genes are involved in an active regulatory network based on sugar availability. The regulatory networks involved in sugar utilization are not well documented in lactobacilli and bifidobacteria, whereas they have been characterized in streptococci (Vadeboncoeur and Pelletier, 1997). Nevertheless, previous work has indicated involvement of CcpA in repression of specific operons in L. casei, and L. plantarum (Viana et al., 2000; Muscariello et al., 2001) and L. pentosus (Mahr et al., 2000). Specifically, the pepQ-ccpA locus has been identified in L. pentosus, L. delbrueckii, L. casei, S. mutans and L. lactis (Mahr et al., 2000), and in most cases, a cre
sequence is found in the promoter-operator region of ccpA. The PTS is characterized by a phosphate transfer cascade involving PEP, EI, HPr, and various EIIABCs, whereby a phosphate is ultimately transferred to the carbohydrate substrate (Saier, 2000; Titgemeyer and Hillen, 2002; Warner and Lolkema, 2003). HPr is a key component of CCR, which is regulated via phosphorylation by enzyme I (EI) and HPr kinase/phosphatase (HPr K/P). While HPr is the primary regulator of CCR, HPr K/P is the sensor enzyme of CCR in Gram positive bacteria (Nessler, et al., 2003). HPrK/P has been found in a variety of LAB, including L. casei, L. brevis, L. delbrueckii, L. gasseri, L. acidophilus, L. lactis, Streptococcus bovis, S. mutans, S. salivarius, S. pneumoniae, S. pyogenes, S. agalactiae
and Leuconostoc mesenteroides (Warner and Lolkema, 2003; Altermann et al., 2004). Similarly, HPr has also been found in a variety of LAB, including L. casei, L. sakei, L. acidophilus, L. gasseri, L. brevis, L. mesenteroides, L. lactis. E. Faecalis, S. mutans, S. salivarius, S. bovis, S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae and
Oenococcus oeni (Warner and Lolkema, 2003; Altermann et al., 2004). The HPr-HPrK/P complex has been characterized structurally (Fieulaine et al., 2002). When HPr is phosphorylated at His15, the PTS is on (Poolman, 2002), and carbohydrates transported via the PTS are phosphorylated via EIIABCs. In contrast, when HPr is phosphorylated at Ser46, the PTS machinery is not functional (Vadeboncoeur and Pelletier, 1997; Mijakovic et al., 2002,; Nessler et al., 2003). HPr-Ser46 acts as a co-repressor by binding to CcpA (Fieulaine et al., 2002; Nessler et al., 2003). Ultimately, CcpA binds to cre
sequences in the promoter-operator region of operons encoding carbohydrate transporters and hydrolases, and prevents their transcription (Hueck and Hillen, 1995; Poolman, 2002).
HPr has been identified in E. faecalis (Vadeboncoeur and Pelletier, 1997), S. pyogenes (Deutscher and Saier, 1983; Vadeboncoeur and Pelletier, 1997), and L. lactis
(Luesink et al., 1999a).
CcpA-dependent repression and activation is well documented in a variety of LAB, including enterococci, lactobacilli, lactococci and streptococci, especially with regard to repression of the genes involved in utilization of galactosides (Titgemeyer and Hillen, 2002).
The interaction between HPr and LacS has been shown in S. salivarius (Lessard et al., 2003). It happens between HPr-His and EIIALacS, although LacS is not a member of the PTS system. Since HPr is the primary regulator of CCR, the interaction between HPr and LacS illustrates the likely regulation of the GPH system by CCR. In S. thermophilus, the control of LacS by CCR has been illustrated, likely via interaction between CcpA and
two cre sequences found in the promoter-operator region of the lacSZ. Operon (van den Bogaard et al., 2000).
Although the phosphorylation cascade suggests regulation at the protein level, studies in LAB report both transcriptional modulation and constitutive expression of
ccpA and ptsHI. Specifically, in S. thermophilus, CcpA production is induced by glucose (can den Bogaard, 2000). Similarly, in other bacteria, the carbohydrate source modulates
ptsHI transcriptional levels (Luesink et al., 1999a). In contrast, expression levels of ccpA
in L. pentosus (Mahr et al., 2000) and of ptsHI in S. thermophilus (Cochu et al., 2003) did not vary in the presence of different carbohydrates.
Carbon catabolite repression is likely present in L. acidophilus, since all the necessary regulatory proteins are encoded within its genome, cre-like sequences are present in the promoter-operator regions of several carbohydrate loci (Barrangou et al., 2003), and transcription of operons involved in utilization of non-preferred carbohydrates is repressed by glucose (Barrangou et al., 2003).
Carbon catabolite repression illustrates how lactic acid bacteria adapt dynamically to the diverse carbohydrate sources available in their various habitats.