Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.     

Galactokinase activity in Streptococcus thermophilus

ATP-dependent phosphorylation of [14C]galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains. Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal+ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal+ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.     

Galactokinase activity in Streptococcus thermophilus.

ATP-dependent phosphorylation of [14C]galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains. Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal+ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal+ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.     

The gal locus from Haemophilus influenzae: cloning, sequencing and the use of gal mutants to study lipopolysaccharide

The gal locus from Haemophilus influenzae was cloned and sequenced. Four genes were identified by amino acid homology: galT, galK, galM and galR. The coding direction of galT, galK and galM is divergent from that of galR. There are non-coding intergenic regions between galR and galT, galT nd galK, and galK and galM. Deletion-insertion mutations constructed in galK and galE, which is in lic3, were moved into the H. influenzae chromosome generating each of the single mutants as well as the double gal mutant. Even when grown on complex media, the double mutant failed to react with an anti-lipopolysaccharide monoclonal antibody known to react with a digalactoside epitope. Both the galE single and the galE galK double mutants were serum-sensitive and relatively avirulent in infant rats, indicating a critical role for galactose metabolism, and providing evidence to support a central role for lipopolysaccharide, in H. influenzae virulence.     

Multiple regulator gene control of the galactose operon in Escherichia coli K-12

Previous studies showed that nonsense mutations in either of two genes (capR or capS) or an undefined mutation in a third gene (capT) led to pleiotropic effects: (i) increased capsular polysaccharide synthesis (mucoid phenotype); (ii) increased synthesis of enzymes specified by at least four spatially separated operons involved in synthesis of capsular polysaccharide including the product of the galE gene, UDP-galactose-4-epimerase (EC 5.1.3.2) in capR mutants. The present study demonstrated that the entire galactose (gal) operon (galE, galT, and galK) is derepressed by mutations in either the capR or the capT genes, but not by mutation in capS. Double mutants (capR9 capT) were no more derepressed than the capR9 mutant, indicating that capR9 and capT regulate the gal operon via a common pathway. Isogenic double mutants containing either galR(+), galR(-), galR(s), or galO(c) in combination with either capR(+) or capR9 were prepared and analyzed for enzymes of the gal operon. The results demonstrated that capR9 caused derepression as compared to capR(+) in all of the combinations. Strains with a galR(s) mutation are not induced, for the gal operon, by any galactose compound including d-fucose, and this was confirmed in the present study using d-fucose. Nevertheless, the derepression of galR(s) capR9 compared to galR(s) capR(+) was four- to sixfold. The same derepression was observed when galR(+)capR9 was compared to galR(+)capR(+). The data eliminate the explanation that internal induction of the gal operon by a galactose derivative was causing increased gal operon enzyme synthesis in capR or capT mutants. Furthermore, the same data suggest that the galR and capR genes are acting independently to derepress the gal operon. A modified model for the structure of the gal operon is proposed to explain these results. The new feature of the model is that two operator sites are suggested, one to combine with the galR repressor and one to combine with the capR repressor.     

Molecular and biochemical analysis of the galactose phenotype of dairy Streptococcus thermophilus strains reveals four different fermentation profiles

Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.     

Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector

The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.     

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.     

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.