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Figure 5 The lac operon in lactococci. The first four structural genes (lacABCD) code for enzymes of the tagatose pathway, lacFE code for lactose-specific PTS proteins, and lacG codes for phospho-P-galactosidase. The divergently transcribed lacR codes for a repressor; the function of lacX is not known. Promoter sites and directions are shown by arrows, and potential transcriptional terminators are shown as hairpin loops. The number of amino acids for each protein is given. (Adapted from de Vos et al., 1990.)

cluster (lacTEGF) encodes, respectively, for a regulatory protein, two PTS proteins, and phospho-P-galactosidase. Genes coding for galactose metabolism (lacABCD in Lc. lactis) are absent in the Lb. casei lac cluster. Although expression of lac genes is repressed by a CcpA-mediated process, as in Lc. lactis, an additional regulatory mechanism dependent on an antiterminator also exists in Lb. casei (Gosalbes et al., 1999).

D. Lactose Transport and Hydrolysis by S. thermophilus

Although the PTS is widely distributed among lactic acid bacteria, several important dairy species rely on a lactose permease for transport and a P-galactosidase for hydrolysis. Some species have both pathways for lactose, and some have a PTS for one sugar and a permease for another. The best example of the lactose permease/P-galactosidase system is that which occurs in S. thermophilus, Lb. helveticus, and Lb. delbruecki subsp. bulgaricus (Fig. 6). In these bacteria, lactose accumulates in an unmodified form via the LacS permease. A similar system also exists in some strains of Lc. lactis, but clearly it is not the primary system. The lactose permease in S. thermophilus is dramatically different from other, well-studied lactose permeases, such as the LacY system in Escherichia coli. In

E. coli, lactose transport is fueled by a proton motive force (PMF), and the perme-ase binds and transports its substrate lactose in symport with a proton. In S. thermophilus, lactose transport can also be fueled by a PMF, but that is not the main way the permease can function. Instead, the transporter has exchange or lactose galactose lactose galactose lactose lactose glucose

EM pathway glucose galactose

EM pathway lactic acid

Figure 6 Lactose transport and hydrolysis by S. thermophilus. Lactose uptake is driven by galactose efflux; both solutes may be transported in symport with a proton.

antiporter activity, so that lactose uptake can be driven by efflux of galactose. That is, ''uphill'' lactose transport (uptake against a concentration gradient) occurs as a result of ''downhill'' galactose efflux (Hutkins and Ponne, 1991). Since generation of a PMF requires ATP (or its equivalent), not having to use the PMF for lactose uptake conserves energy. The lactose:galactose exchange reaction is actually quite remarkable, in that, as discussed below, galactose efflux, rather than galactose utilization, appears to be the preferred pathway for most strains of S. thermophilus. Why this phenomenon occurs and the important practical implications for this will be discussed later.

Detailed analysis of the S. thermophilus LacS system has revealed that the permease protein itself is a hybrid consisting of two distinct regions or domains (Poolman et al., 1989). The deduced amino acid sequence of the amino-terminal region is very similar to the melibiose permease of E. coli. However, the carboxy-terminal region is structurally similar to an E. coli PTS enzyme IIA domain. In fact, this enzyme IIA-like domain can be phosphorylated by HPr, reducing transport activity of LacS. It now appears that the permease region functions as the lactose carrier and the enzyme IIA-like domain functions as a regulatory unit.

Hydrolysis of lactose in S. thermophilus occurs via a P-galactosidase that has modest amino acid homology to other LacZ-like enzymes (30-50%). After hydrolysis, S. thermophilus rapidly ferments glucose to lactic acid by the Embden-Meyerhoff pathway, yet most strains, especially those used as dairy starter cultures, do not ferment the galactose moiety of lactose. Rather, galactose is effluxed and accumulates in the extracellular medium. In the manufacture of dairy products made with an S. thermophilus-containing culture, such as yogurt or mozzarella cheese, galactose may appear in the finished product. With yogurt, accumulated galactose is of little consequence, but for mozzarella cheese, even a small amount of galactose can present problems. This is because of the nonenzy-matic browning reaction that occurs when galactose, a reducing sugar, is heated in the presence of free amino acids. Since most mozzarella cheese is used for pizzas, high-temperature baking accelerates nonenzymatic browning reactions. Cheese containing galactose can brown excessively, a phenomenon considered as a defect by many pizza manufacturers. Therefore, mozzarella producers may be asked by their customers to satisfy specifications for ''low-browning'' or low-galactose cheese. Although some cheese manufacturers can rely on their cheese-making know-how and simply modify the production procedures to remove un-fermented galactose, other manufacturers have chosen to use cultures that have low-browning potential, as described below.

Why are most strains of S. thermophilus phenotypically galactose negative (Gal") and unable to ferment either free or lactose-derived galactose? Evidence from several laboratories indicates that S. thermophilus does contain genes necessary for galactose metabolism (see below), but that these genes are not ordinarily expressed even under inducing conditions. Mutants have been isolated, however, that ferment free galactose, but when these strains are grown on lactose, galactose utilization is still repressed (Thomas and Crow, 1984, Benateya et al., 1991). Thus, it has been suggested that of the two routes that galactose can take, the efflux reaction is favored over the catabolic pathway.

E. Lactose Metabolism by Lactobacillus and Other Lactic Acid Bacteria

Most other lactic acid bacteria rely on one or the other of the two pathways described earlier. Table 1 lists the pathways used by species that have been studied in sufficient detail. With the exception of Lc. lactis and Lb. casei, however, most dairy lactic acid bacteria do not have a lactose PTS, and instead use a lactose permease/p-galactosidase system for metabolism of lactose. Some strains have more than one system; for example, Lc. lactis and Lb. casei have both a lactose PTS and a lactose permease/p-galactosidase. It is important to note that not all strains or species that use non-PTS pathways for lactose metabolism excrete galactose into the medium, as described for S. thermophilus. Many of the lactoba-cilli and Leuconostoc spp. that transport and hydrolyze lactose by a permease and a p-galactosidase, respectively, also ferment glucose and galactose simultaneously. This is important, because in almost all fermented dairy products made with a culture containing S. thermophilus, a galactose-fermenting Lactobacillus sp. is also present (see Chap. 11). For some products, such as Swiss-style cheeses, the galactose that is effluxed into the curd by S. thermophilus is subsequently fermented by Lb. helveticus. Otherwise, the free galactose could be fermented by other members of the microflora, resulting in heterofermentative endproducts that could contribute to off-flavors and other product defects.

F. Galactose Metabolism

During growth in milk, lactic acid bacteria ordinarily encounter free galactose only after intracellular hydrolysis of lactose. For lactococci and those lactobacilli

Table 1 Lactose Transport and Metabolic Systems in Dairy Lactic Acid Bacteria

Organism

Lactose transport system

Galactose pathway

Streptococcus thermophilus

Lac permease

Leloir

Lactococcus lactis

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