Genetics of lactose utilization in lactic acid bacteria

Abstract: Lactose utilization is the primary function of lactic acid bacteria used in industrial dairy fermentations. The mechanism by which lactose is transported determines largely the pathway for the hydrolysis of the internalized disaccharide and the fate of the glucose and galactose moieties. Biochemical and genetic studies have indicated that lactose can be transported via phosphotransferase systems, transport systems dependent on ATP binding cassette proteins, or secondary transport systems including proton symport and lactose-galactose antiport systems. The genetic determinants for the group translocation and secondary transport systems have been identified in lactic acid bacteria and are reviewed here. In many cases the lactose genes are organized into operons or operon-like structures with a modular organization, in which the genes encoding lactose transport are tightly linked to those for lactose hydrolysis. In addition, in some cases the genes involved in the galactose metabolism are linked to or co-transcribed with the lactose genes, suggesting a common evolutionary pathway. The lactose genes show characteristic configurations and very high sequence identity in some phylogenetically distant lactic acid bacteria such as Leuconostoc and Lactobacillus or Lactococcus and Lactobacillus . The significance of these results for the adaptation of lactic acid bacteria to the industrial milk environment in which lactose is the sole energy source is discussed.     

Energetics of glucose uptake in Salmonella typhimurium

We have studied the energetics of glucose uptake in Salmonella typhimurium. Strain PP418 transprots glucose via the phosphoenolpyruvate: glucose phosphotransferase system, while strain PP1705 lacks this system and can only use the galactose permease for glucose uptake. These two strains were cultured anaerobically in glucose-limited chemostats. Both strains produced ethanol and acetate in equimolar amounts but a significant difference was observed in the molar growth yield on glucose (Y _Glc). It is suggested that this difference is due to a difference in the energetics of the glucose uptake systems in the two strains.Assuming an equal Y _ATP for both strains, we could calculate that uptake of 1 mole of glucose via the galactose permease consumes the equivalent of 0.5 mole of ATP. With the additional assumption that one proton is transported in symport with one glucose molecule, these results imply a stoichiometry of two protons per ATP hydrolysed.Abbreviations PTS Phosphoenolpyruvate: carbohydrate phosphotransferase system - D dilution rate (h^-1 - DW dry weight - GalP galactose permease - EtOH ethanol - HAc acetate - Lact lactate - Suc succinate - HFo formate - Glc Glucose - Y _Glc, Y _ATP yield of cells per glucose or ATP - q specific production rate     

Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556.

Regulation of transport of D-glucose and D-fructose was studied in Kluyveromyces marxianus grown in continuous culture. Both substrates could be transported by at least two different transport systems, low-affinity transport and high-affinity proton-sugar symport. The low-affinity transporter, specific for both glucose and fructose, was constitutively present and was apparently not regulated by carbon catabolite repression. Regulation of the activity of the glucose- and fructose-specific proton symport systems appeared to proceed mainly through catabolite repression. Activation of symport did not need the presence of specific inductor molecules in the medium. Nevertheless, the capacities of the proton-sugar symporters varied in cells grown on a wide variety of carbon sources. The possibility that the control of proton symport activity is related to the presence of specific intracellular metabolites is discussed.     

Roles of extracellular lactose hydrolysis in cellulase production by Trichoderma reesei Rut C30 using lactose as inducing substrate

Lactose, an inexpensive, soluble substrate, offers reasonably good induction for cellulase production by Trichoderma reesei. The fungus does not uptake lactose directly. Lactose is hydrolyzed to extracellular glucose and galactose for subsequent ingestion. The roles of this extracellular hydrolysis step were investigated in this study. Batch and continuous cultures were grown on the following substrates: lactose, lactose–glycerol mixtures, glucose, galactose, and glucose–galactose mixtures. Cell growth, substrate consumption, lactose hydrolysis, and lactase and cellulase production were followed and modeled. Cells grew much faster on glucose than on galactose, but with comparable cell yields. Glucose (at >0.3 g/L) repressed the galactose consumption. Cellulase synthesis was growth-independent while lactase synthesis was growth-dependent, except at D < ∼0.065 h⁻¹ where a basal level lactase production was observed. For cellulase production the optimal D was 0.055–0.065 h⁻¹ where the enzyme activity and productivity were both near maxima. The model suggested that lactase synthesis was subject to weak galactose repression. As the galactose concentration increased at high D (>0.1 h⁻¹), lactase synthesis became repressed. The insufficient lactase synthesis limited the lactose hydrolysis rate. Extracellular lactose hydrolysis was concluded to be the rate-limiting step for growth of T. reesei Rut C30 on lactose.     

Characterization of the Lactose Transport System in the Strain Bifidobacterium bifidum DSM 20082

Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose ¹⁴C. K _m and V _max values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the β-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-β-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na⁺, Li⁺, or K⁺ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the strain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.     

Metabolic control of lactose entry in Escherichia coli.

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain beta-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by beta-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of omicron-nitrophenyl-beta-D-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of omicron-nitrophenyl-beta-D-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG.     

Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.     

Lactose permease H+-lactose symporter: mechanical switch or Brownian ratchet?

Lactose permease structure is deemed consistent with a mechanical switch device for H(+)-coupled symport. Because the crystallography-assigned docking position of thiodigalactoside (TDG) does not make close contact with several amino acids essential for symport; the switch model requires allosteric interactions between the proton and sugar binding sites. The docking program, Autodock 3 reveals other lactose-docking sites. An alternative cotransport mechanism is proposed where His-322 imidazolium, positioned in the central pore equidistant (5-7 A) between six charged amino acids, Arg-302 and Lys-319 opposing Glu-269, Glu-325, Asp-237, and Asp-240, transfers a proton transiently to an H-bonded lactose hydroxyl group. Protonated lactose and its dissociation product H(3)O+ are repelled by reprotonated His-322 and drift in the electrostatic field toward the cytosol. This Brownian ratchet model, unlike the conventional carrier model, accounts for diminished symport by H322N mutant; how H322 mutants become uniporters; why exchanging Lys-319 with Asp-240 paradoxically inactivates symport; how some multiple mutants become revertant transporters; the raised export rate and affinity toward lactose of uncoupled mutants; the altered specificity toward lactose, melibiose, and galactose of some mutants, and the proton dissociation rate of H322 being 100-fold faster than the symport turnover rate.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Occurrence and taxonomic aspects of proton movements coupled to sugar transport in the yeast genusKluyveromyces

The occurrence of proton symport mechanisms for the transport of glucose, galactose, fructose, raffinose and sucrose in 21 yeast strains representing the species of the genusKluyveromyces was surveyed. Proton symport of one or more sugars occurred in 57% of the strains. Similarly, all the sugars investigated were transported by symports by several strains. Symport systems for non-utilisable sugars were rare. Starvation of cells frequently resulted in the appearance of a symport absent in non-starved glucose-grown cells, indicating that repression of proton symports by glucose and subsequent derepression by starvation is a general phenomenon in members ofKluyveromyces. The addition of a sugar to cell suspensions resulted in acidification in 80% of cases, indicating the activity of a membrane-bound ATPase. Acidification was also observed with a number of sugars that cannot be utilised by the particular species. Interesting correlations between the number of proton symports and the abundance of other phenotypic characteristics in members of the genus emerged. Most members of the infertile group of species showing an increase in the number of small chromosomes, inability to produce well-developed pseudomycelium, linoleic and linolenic acid, a decrease in the number of carbon compounds utilised and inability to utilise ethylamine also had no proton symports, whereas most members of the interfertile species produced one or more proton symports. It was concluded that the distribution of the number of proton symports amongstKluyveromyces species coincided with that of other positive characteristics and may therefore be of taxonomic value.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate

Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK _m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V _max) are dependent on the dilution rate (D). At D=0.12 h^–1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h^–1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.     

Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism

In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified. In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active. In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively. These symporters appeared to be sensitive to catabolite inactivation. This process is induced by incubating cells in the presence of glucose, fructose or mannose. Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin. Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose. Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium. The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.     

Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis

The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.