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.     

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.     

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.     

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.     

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.     

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.     

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.     

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.     

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.     

Characterization,expression, and mutation of the Lactococcus lactis galPMKTE genes,involved in galactose utilization via the Leloir pathway

A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.     

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.