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Figure 2 The phosphoketolase pathway used by heterofermentative lactic acid bacteria.

of alternative transport systems. Finally, the metabolic behavior of a particular strain and how that strain functions in fermented dairy foods may be influenced by the actual operation of the transport system itself.

B. Lactose Phosphotransferase System of Lc. lactis

There are, in general, two different systems used by lactic acid bacteria to transport carbohydrates, and it is convenient to group lactic acid bacteria according to the system used to transport their primary substrate, lactose. The phosphoenol-pyruvate (PEP)-dependent phosphotransferase system (PTS) is used by most mesophilic, homofermentative lactic acid bacteria, especially lactococci used as starter cultures for cottage, Cheddar, Gouda, and other common cheese varieties. In contrast, other starter culture bacteria, such as S. thermophilus and Lactobacillus spp. that are used for yogurt, Swiss, and mozzarella cheese production, transport lactose via a lactose permease. Dairy Leuconostoc bacteria also rely on a lactose permease for uptake of lactose. Some lactococci and lactobacilli have the ability to use both systems. Not only do these two systems differ in biochemical characteristics, but energy sources used to drive transport and accumulated intra-cellular products differ as well. These differences have practical implications.

The Lactococcus lactose PTS, first described by McKay et al. (1969), consists of a cascade of cytoplasmic and membrane-associated proteins that transfer a high-energy phosphate group from its initial donor, PEP, to the final acceptor molecule, lactose. Phosphorylation of lactose occurs concurrent with the vectorial movement of lactose across the cytoplasmic membrane (from out to in) and results in intracellular accumulation of lactose phosphate. As shown in Figure 3, there are two cytoplasmic proteins, enzyme I and histidine-containing protein (HPr), that are nonspecific and function as the initial phosphorylating proteins for all PTS substrates. The substrate-specific PTS components comprise the enzyme II complex, which for the lactose PTS in Lc. lactis, represents three protein domains (Enz IIAlac and Enz IIBClac). The phosphoryl group is transferred first from PEP to enzyme I, then to HPr, then to the cytoplasmic protein, Enz IIAlac, which then transfers it to the cytoplasmic domain of Enz IIBClac. As lactose is translocated across the membrane by the integral membrane domain of Enz IIBClac, it becomes phosphorylated.

The product of the lactose PTS, thus, is lactose-phosphate, or more specifically, glucose-P-1,4-galactosyl-6-phosphate. Hydrolysis of this compound occurs via phospho-P-galactosidase, yielding glucose and galactose-6-phosphate. Glucose is phosphorylated by hexokinase (via an ATP) to glucose-6-phosphate, which then feeds directly into the Embden-Meyerhoff pathway, as described earlier. Galactose-6-phosphate, in contrast, takes a different route altogether, as it is first isomerized to tagatose-6-phosphate and then phosphorylated to form taga-tose-1,6-diphosphate (Fig. 4). The latter is then split by tagatose-1,6-diphosphate

Figure 3 Signal transduction and the phosphotransferase system in gram-positive bacteria. HPr can be phosphorylated at His-15 (by Enz I) or at Ser-46 by an HPr kinase. The latter, along with CcpA and fructose diphosphate (FDP), form a complex that recognizes CRE sites and prevents transcription of catabolic genes. (Adapted from Saier et al., 1995.)

Figure 3 Signal transduction and the phosphotransferase system in gram-positive bacteria. HPr can be phosphorylated at His-15 (by Enz I) or at Ser-46 by an HPr kinase. The latter, along with CcpA and fructose diphosphate (FDP), form a complex that recognizes CRE sites and prevents transcription of catabolic genes. (Adapted from Saier et al., 1995.)

Figure 4 Tagatose pathway in lactococci. Galactose-6-phosphate is formed from hydrolysis of lactose-phosphate, the product of the lactose PTS. Isomerization and phosphoryla-tion form tagatose-1, 6-diphosphate, which is split by an aldolase, yielding the triose phosphates that feed into the EM pathway.

Figure 4 Tagatose pathway in lactococci. Galactose-6-phosphate is formed from hydrolysis of lactose-phosphate, the product of the lactose PTS. Isomerization and phosphoryla-tion form tagatose-1, 6-diphosphate, which is split by an aldolase, yielding the triose phosphates that feed into the EM pathway.

aldolase to form the triose phosphates, glyceraldehyde-3-phosphate and dihy-droxyacetone phosphate, in a reaction analogous to the aldolase of the Embden-Meyerhoff pathway. It is important to note that in Lc. lactis, glucose and galactose moieties of lactose, despite taking parallel pathways, are fermented simultaneously.

C. Regulation of the Phosphotransferase System

In Lc. lactis, lactose fermentation is regulated at several levels. First, several glycolytic enzymes are allosteric, and their activities are therefore influenced by the intracellular concentration of specific glycolytic metabolites via feedback inhibition. During active lactose metabolism (i.e., when lactose is plentiful), the high intracellular concentration of fructose-1,6-diphosphate (FDP) and low level of inorganic phosphate stimulate pyruvate kinase. Thus, much of the PEP made via glycolysis is used to drive ATP synthesis, which is consistent with a period of active cell growth. The activity of the NADH-dependent lactate dehydrogenase is also stimulated, which is important because reduced NAD+, formed via the glyceraldehyde-3-phosphate dehydrogenase reaction, must be reoxidized to maintain the NAD+/NADH balance. In contrast, when lactose is limiting, pyruvate kinase activity decreases causing PEP to accumulate, which forms a ''bottleneck'' in glycolysis. The concentration of triose phosphates subsequently increases, forming a pool of PEP equivalents. Thus, during a period when lactose is unavailable, a PEP ''potential'' exists, poising the cell for when lactose is available (Thompson, 1987).

A second and more effectual mechanism for controlling or regulating lactose metabolism is exerted at the level of the transport machinery itself. In particular, the phosphorylation state of HPr, the cytoplasmic PTS phosphocarrier protein, plays a major role in sugar metabolism. As noted earlier, HPr is phos-phorylated by enzyme 1. This phosphorylation occurs specifically at the histidine-15 (His-15) residue of HPr. However, HPr can also be phosphorylated at a serine residue (Ser-46) by an ATP-dependent HPr kinase, which is activated by fructose-1,6-diphosphate (as would occur during active sugar metabolism). When HPr is in this state, that is, HPr (Ser-46-P), phosphorylation at His-15 is inhibited; thus, PTS activity is also inhibited and entry of other potential PTS substrates is prevented. Additional experimental evidence that HPr (Ser-46-P) can directly inhibit transport of sugars was provided by Saier and coworkers (Ye and Saier, 1995a, 1995b; Ye et al., 1994), who showed that HPr (Ser-46-P) can bind to or otherwise inactivate sugar permeases, a process known as inducer exclusion. Yet another means by which HPr (Ser-46-P) regulates sugar flux is via inducer expulsion. Presumably, this occurs when sugar phosphates have accumulated intracellularly beyond the rate at which metabolism can occur or when nonmetabolizable sugars have been taken up. Since these sugar phosphates could inhibit metabolism, they must first be dephosphorylated and then effluxed. In inducer expulsion, therefore, HPr (Ser-46-P) activates a sugar-specific phosphatase that dephosphorylates the sugar phosphates so that efflux of the free sugar can then occur (Thompson and Chassy, 1983).

HPr not only exerts biochemical control on transport, but HPr (Ser-46-P) also plays an important role at the gene level through its interaction with the DNA-binding protein, CcpA, or catabolite control protein A. As illustrated in Figure 3, HPr (Ser-46-P) and CcpA (with the participation of fructose-1,6-diphos-phate) affect metabolism by blocking transcription of catabolic genes, including other PTS genes, a process called catabolite repression. CcpA or CcpA-like proteins appear to be widely distributed among gram-positive bacteria, including several species of lactic acid bacteria (Luesink et al., 1998a), and this mechanism of gene regulation, therefore, may be common. According to this model of carbon source-mediated gene regulation, HPr exists in one of two phosphorylation states, HPr (His-15-P) or HPr (Ser-46-P). The former accumulates when lactose (or another PTS sugar, such as glucose) is unavailable, since the enzyme II complex is without its substrate. In contrast, when lactose is available and the energy state of the cell is high, intracellular FDP levels increase and HPr kinase is activated, causing HPr (Ser-46-P) to accumulate. A complex is then formed between HPr (Ser-46-P) and CcpA. This complex, along with a glycolytic activator (fructose-1,6-diphosphate or glucose-6-phosphate), binds to 14-base pair DNA regions called catabolite responsive elements (CREs) located near the transcription start sites of catabolic genes. When these CRE regions are occupied by the HPr (Ser-46-P)-CcpA complex, transcription by RNA polymerase is effectively blocked or reduced. In contrast, mutations in ccpA or deletions of cre regions eliminate catabolite repression. Since CRE regions are found in the promoter regions of several catabolic genes, the phosphorylation status of HPr can have a profound effect on whether these genes are expressed. Identified gene clusters preceded by CRE regions in lactococci include genes coding for galactose (and thus lactose) and sucrose metabolism. For example, when Lc. lactis is grown on glucose, a PTS substrate, transcription of genes coding for galactose metabolism is repressed (Luesink et al., 1998b). Even the presence of galactose fails to induce expression of gal genes as long as glucose, the repressing sugar, is present.

Not only does HPr have a negative regulatory role, but it was recently shown that HPr (Ser-46-P) and CcpA can also activate gene expression (Luesink et al., 1998b, 1999). Specifically, expression of the las operon, coding for lactate dehydrogenase, phosphofructokinase, and pyruvate kinase, is activated at high sugar conditions. The net effect, therefore, is that the phosphorylation state of HPr serves as a signal for activating expression of genes coding for glycolytic enzymes when the cell is actively metabolizing sugars. Recent genetic evidence (Luesink et al., 1999) indicates that HPr is also important in influencing sugar uptake by establishing a hierarchy for different sugars preferentially fermented by Lc. lactis.

Finally, lactose metabolism is also genetically regulated via expression and repression of the lactose PTS genes (Fig. 5). The lactose metabolism genes in Lc. lactis, like the genes coding for other important metabolic pathways, are often located on plasmids of varying size. Strains cured of the lactose plasmid, which encodes lactose metabolism genes, are unable to ferment lactose. In Lc. lactis MG1820, the lac genes are organized as an 8-kb operon, consisting of eight genes in the order lacABCDFEGX (de Vos et al., 1990). The first four genes, lacABCD, actually code for enzymes of the tagatose pathway and are necessary for galactose utilization (see below). The lactose-specific genes, lacFEG, code for PTS proteins and phospho-P-galactosidase. The operon is negatively regulated by LacR, a repressor protein encoded by the lacR gene, which is located upstream of the lac promoter and which is divergently transcribed (van Rooijen and de Vos, 1990). In the presence of lactose, lacR expression is repressed, and transcription of the lac operon is induced. During growth on glucose or when lactose is unavailable (and cells are uninduced), LacR is expressed and transcription of the lac genes is repressed. A CRE site is also located near the transcriptional start site of the lac operon. However, when lacR is inactivated, expression of lac genes becomes constitutive regardless of carbon source (i.e., under conditions that presumably would activate CcpA-mediated repression). This implies that LacR, along with inducer expulsion-exclusion, have primary responsibility for regulating sugar metabolism, rather than CcpA, and that catabolite repression in lactococci is mediated mainly via the concentration of inducer (Luesink et al., 1998).

The lactose PTS, as described earlier for Lc. lactis, also exists in other dairy lactic acid bacteria, including Lb. casei. However, in Lb. casei, lac genes are chromosomally encoded and the nucleotide sequence and genetic organization are different from those in Lc. lactis (Gosalbes et al., 1997). The Lb. casei lac lacR

lacA

tacB

lacC

/acD

lacF

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