UDP galactose epimerase glucose-1 -P
Figure 7 Leloir pathway in lactic acid bacteria. Phosphorylation of galactose may require isomerization by mutarotase (not shown). The subsequent steps convert galactose-1-phosphate into glucose-6-phosphate, which feeds into the EM pathway (homofermentative bacteria) or phosphoketolase (PK) pathway (heterofermentative bacteria).
permease/p-galactosidase system and therefore generate free intracellular galactose. In some instances, they will also encounter free extracellular galactose, especially if they are grown in the presence of galactose-nonfermenting strains, as described earlier. Subsequent galactose fermentation by Lb. helveticus and Leuco-nostoc lactis occurs via the Leloir pathway. Transport is mediated by a permease, apparently driven by a PMF. A mutarotase (the product of the galM gene) may also be necessary to convert P-D-galactose (the product of lactose hydrolysis) to its anomeric isomer, a-D-galactose, before it can be efficiently phosphorylated by galactokinase.
Despite the inability of most strains of S. thermophilus to ferment galactose, genes coding for enzymes of the Leloir pathway appear to be present and functional (Grossiord et al., 1998; Poolman et al., 1990; Mustapha et al., 1995). The S. thermophilus gal operon consists of four structural genes (galKTEM) and one divergently transcribed regulatory gene (galR). Transcription of these genes, however, does not occur in most wild-type strains, accounting for the galactose nonfermenting phenotype. Mutations in the gal promoter/regulatory region led to isolation of galactose-fermenting mutants that expressed gal genes and fermented galactose. Such efforts suggest that genetic modification of S. thermophilus may provide the basis for obtaining stable galactose-fermenting derivatives that would be of considerable value to the dairy industry (de Vos, 1996).
Although the gal genes in S. thermophilus, Leuc. lactis, Lc. lactis, Lb. casei, and Lb. helveticus share significant amino acid sequence homology and are chromosomally encoded, they are organized in a somewhat different order (Grossiord et al., 1998). All contain galK (galactokinase), galT(galactose-1-phos-phate uridyl transferase), and galE (UDP-galactose-4-epimerase), and some clusters also contain the galM gene coding for mutarotase. In S. thermophilus, the gal genes are located immediately upstream of the lacS-lacZ cluster. There is also rather significant variation with respect to genetic structure even between strains of the same species. For example, a galA gene, thought to encode for a permease, is the first gene in the Lc. lactis MG1363 gal cluster, but this gene does not appear in gal clusters from other organisms.
The ability of these strains, especially lactobacilli, to ferment galactose can be quite variable, and strain selection is important. Galactose fermentation by lactobacilli has also been used as a basis for distinguishing between Lb. helveticus (Gal+) and Lb. delbrueckii subsp. bulgaricus (Gal"). As noted earlier, some culture suppliers promote ''nonbrowning'' cultures for use in mozzarella cheese production; invariably, these cultures contain galactose-fermenting lactobacilli.
As described earlier, lactic acid bacteria are ordinarily considered as being either homofermentative or heterofermentative, with some species being able to metab olize sugars by both pathways. However, sugar metabolism, even by obligate homofermentative strains, can result in formation of endproducts other than lactic acid by a variety of pathways (Fig. 8). In general, these alternative fermentation products are formed as a consequence of accumulation of excess pyruvate and the requirement of cells to maintain a balanced NADH/NAD+ ratio. That is, when the intracellular pyruvate concentration exceeds the rate at which lactate can be formed via lactate dehydrogenase, other pathways must be recruited not only to remove pyruvate but also to provide a means for oxidizing NADH. These alternative pathways may also provide cells with the means to make additional ATP. Under what conditions or environments would pyruvate accumulate? As noted earlier, when fermentation substrates are limiting, and the glycolytic activator, fructose-1,6-diphosphate, is in short supply, activity of the allosteric enzyme, lactate dehydrogenase, is reduced and pyruvate accumulates. Low carbon flux may also occur during growth on galactose or other less preferred carbon sources, resulting in excess pyruvate. When the environment is highly aerobic, NADH that would normally reduce pyruvate is instead oxidized directly by molecular oxygen and is unavailable for the lactate dehydrogenase reaction.
Several enzymes and pathways have been identified in lactococci and other lactic acid bacteria that are responsible for diverting pyruvate away from lactic acid and toward other products (Cocaign-Bousquet et al., 1996; Garrigues et al., 1997). In anaerobic conditions, and when carbohydrates are limiting and growth rates are low, a mixed-acid fermentation occurs, and ethanol, acetate, and formate are formed. Under these conditions, pyruvate-formate lyase is activated, and pyr-uvate is split to form formate and acetyl-CoA. Acetyl-CoA can be converted to ethanol and/or acetate. The latter also results in formation of an ATP via acetate acetate a-acetolactate acetate a-acetolactate
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