Phosphorylation state of HPr determines the level of expression and the extent of phosphorylation of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus is composed of a translocator domain and a regulatory domain that is phosphorylated by HPr(His approximately P), the general energy coupling protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Lactose transport is affected by the phosphorylation state of HPr through changes in the activity of the LacS protein as well as expression of the lacS gene. To address whether or not CcpA-HPr(Ser-P)-mediated catabolite control is involved, the levels of LacS were determined under conditions in which the cellular phosphorylation state of HPr greatly differed. It appears that HPr(Ser-P) is mainly present in the exponential phase of growth, whereas HPr(His approximately P) dominates in the stationary phase. The transition from HPr(Ser-P) to HPr(His approximately P) parallels an increase in LacS level, a drop in lactose and an increase in galactose concentration in the growth medium. Because the K(m)(out) for lactose is higher than that for galactose, the lactose transport capacity decreases as lactose concentration decreases and galactose accumulates in the medium. Our data indicate that S. thermophilus compensates for the diminished transport capacity by synthesizing more LacS and phosphorylating the protein, which results in increased transport activity. The link between transport capacity and lacS expression levels and LacS phosphorylation are discussed.     

Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems.

The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two HindIII fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; greater than 50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of Lactobacillus bulgaricus (lacL; greater than 60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the beta-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the beta-galactosidase appear to be separated by a 3-base-pair intercistronic region.     

Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.     

Lactose transport system of Streptococcus thermophilus. The role of histidine residues.

The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain. The histidine residues of LacS were changed individually into glutamine or arginine residues. Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport. The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport. The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside. Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein. Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein. The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides. The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs. P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E. coli in which the lactose transport gene was expressed. The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E. coli.     

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.     

Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.     

Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus catalyzes the uptake of lactose in an exchange reaction with intracellularly formed galactose. The interactions between the substrate and the cytoplasmic and extracellular binding site of LacS have been characterized by assaying binding and transport of a range of sugars in proteoliposomes, in which the purified protein was reconstituted with a unidirectional orientation. Specificity for galactoside binding is given by the spatial configuration of the C-2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety. Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier groups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair transport. In fact, the hydrophobic groups increased the binding affinity but decreased transport rates compared with galactose. Binding and transport characteristics of deoxygalactosides from either side of the membrane showed that the cytoplasmic and extracellular binding site interact differently with galactose. Compared with galactose, the IC(50) values for 2-deoxy- and 6-deoxygalactose at the cytoplasmic binding site were increased 150- and 20-fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis of the galactose moiety and are restricted along the C-2 to C-6 axis. The differences in affinity at the cytoplasmic and extracellular binding site ensure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.     

HPr(His approximately P)-mediated phosphorylation differently affects counterflow and proton motive force-driven uptake via the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The IIA domain of LacS is phosphorylated on His-552 by the general energy coupling proteins of the PTS, which are Enzyme I and HPr. To study the effect of phosphorylation on transport, the LacS protein was purified and incorporated into liposomes with the IIA domain facing outwards. This allowed the phosphorylation of the membrane-reconstituted protein by purified HPr(His approximately P) of S. thermophilus. Phosphorylation of LacS increased the V(max) of counterflow transport, whereas the V(max) of the proton motive force (delta p)-driven lactose uptake was not affected. In line with a range of kinetic studies, we propose that phosphorylation affects the rate constants for the reorientation of the ternary complex (LacS with bound lactose plus proton), which is rate-determining for counterflow but not for delta p-driven transport.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.     

Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.