Nucleotide sequences of thegalE gene and thegalT gene ofE.coli

Lemaire, H. G.; Müller‐Hill, Benno

doi:10.1093/nar/14.19.7705

Cited by 102 publications

(58 citation statements)

References 16 publications

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“…3A and Fig. 5) (25) (38% amino acid identity), S. lividans (1) (42% amino acid identity), and the amino-terminal domain of K. lactis UDPglucose 4-epimerase (43) (40% amino acid identity) (Fig. 3A and 5).…”

Section: Resultsmentioning

confidence: 99%

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

Poolman¹,

Royer²,

Mainzer³

et al. 1990

J Bacteriol

108

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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-gahM-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 Il-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose-and glucose-grown S. thermophilus cells matched the mRNA levels.Streptococcus thermophilus transports lactose by means of a proton motive force-linked mechanism (33). Lactose enters the cell as a free sugar, and the disaccharide is hydrolyzed into glucose and galactose by P-galactosidase (20,33). Glucose enters the glycolytic pathway, whereas in the presence of excess lactose, the galactose moiety of lactose is excreted into the medium (39).The lac genes of S. thermophilus have recently been cloned, sequenced, and partially characterized (20, 33; C. J. Schroeder, C. Robert, G. Lenzen, L. L. McKay, and A.Mercenier, submitted for publication). The lactose transport gene (lacS) encodes a 69,454-dalton (Da) protein consisting of an amino-terminal domain with homology to the melibiose carrier of Escherichia coli and a carboxy-terminal domain with homology to enzyme III or enzyme III domains of various phosphoenolpyruvate-dependent phosphotransferase systemns from gram-positive and gram-negative organisms. A similar transport protein has been found in Lactobacillus bulgaricus (33,38), and the function(s) of the different domains of the transport...

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Section: Resultsmentioning

confidence: 99%

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

Poolman¹,

Royer²,

Mainzer³

et al. 1990

J Bacteriol

108

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“…The galK and galT genes were translated into amino acid sequences and compared by dot plot analysis with the corresponding E. coli genes (11,26). The results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

Mollet

Pilloud

1991

J Bacteriol

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By complementing appropriate gal lesions in Escherichia coli K802, we were able to isolate the galactokinase (gaLK) and galactose-1-phosphate uridyl transferase (gall) genes of Lactobacillus helveticus. Tn1O transposon mutagenesis, together with in vivo complementation analysis and in vitro enzyme activity measurements, allowed us to map these two genes. The DNA sequences of the genes and the flanking regions were determined. These revealed that the two genes are organized in the order galK-galT in an operonlike structure. In an in vitro transcription-translation assay, the galK and galf gene products were identified as 44-and 53-kDa proteins, respectively, data which corresponded well with the DNA sequencing data. The deduced amino acid sequence of the galK gene product showed significant homologies to other prokaryotic and eukaryotic galactokinase sequences, whereas galactose-1-phosphate uridyl transferase did not show any sequence similarities to other known proteins. This observation, together with a comparison of known gal operon structures, suggested that the L. helveticus operon developed independently to a translational expression unit having a different gene order than that in E. coli, Streptococcus lividans, or Saccharomyces cerevisiae. DNA sequencing of the flanking regions revealed an open reading frame downstream of the galKT operon. It was tentatively identified as galM (mutarotase) on the basis of the significant amino acid sequence homology with the corresponding Streptococcus thermophilus gene.Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus are used together with Streptococcus thermophilus in the dairy industry as starter cultures for cheese, yogurt, and other dairy product fermentations. As a primary carbon and energy source, they utilize lactose, the only sugar present in milk, and ferment it to lactate. The uptake of lactose by these organisms occurs via a permeasetype system and is followed by a ,3-galactosidase-catalyzed cleavage of the sugar into its monosaccharide glucose and galactose moieties (18,40).In L. delbrueckii subsp. bulgaricus and S. thermophilus, the glucose moiety is metabolized, while the galactose moiety is excreted stoichiometrically into the medium (18). In fact, it has been observed that galactose efflux from these cells is coupled to uptake of lactose or methyl-p-D-thiogalactopyranoside from the external medium and that the transport system involved acts as a lactose-galactose antiporter (21, 39, 39a). However, L. helveticus, which is related to L. delbrueckii subsp. bulgaricus and difficult to distinguish taxonomically from it (22, 38), is able to ferment the glucose and galactose moieties of lactose and does not accumulate free galactose in the external medium (47). It can utilize galactose as an energy source. Hickey et al. (18) observed that there is no lactose or galactose phosphotransferase system in L. helveticus and suggested that the galactose moiety of the lactose is metabolized via the Leloir rather than the tagatose 6-phosphate pat...

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“…The CgtB glycosyltransferase from C. jejuni is a (35), and E. coli O55:H7 (36); GalEEco is the UDP-glucose 4-epimerase from E. coli (37). This alignment was made using ClustalX (38) and was reformatted in GeneDoc (39).…”

Section: Sequence Analysis Of Gale From C Jejuni Strain Nctcmentioning

confidence: 99%

“…This epimerization of UDPGlcNAc and UDP-GalNAc by the C. jejuni GalE has not been demonstrated before. Because GalE from C. jejuni possesses UDP-N-acetylglucosaminyl 4-epimerase activity, it will henceforth be designated Gne (UDP-GlcNAc 4-epimerase), after the enzymes with the same function in B. subtilis, E. coli, and Y. enterocolitica (20,37,43).…”

Section: Sequence Analysis Of Gale From C Jejuni Strain Nctcmentioning

confidence: 99%

A Single Bifunctional UDP-GlcNAc/Glc 4-Epimerase Supports the Synthesis of Three Cell Surface Glycoconjugates in Campylobacter jejuni

Bernatchez

Szymanski

Ishiyama

et al. 2005

Journal of Biological Chemistry

111

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The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The K m(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 M, whereas those for UDP-Glc and UDP-Gal are 780 and 784 M. The k cat and k cat /K m(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDPGlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugarbinding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.

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Nucleotide sequences of thegalE gene and thegalT gene ofE.coli

Cited by 102 publications

References 16 publications

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

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

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes

A Single Bifunctional UDP-GlcNAc/Glc 4-Epimerase Supports the Synthesis of Three Cell Surface Glycoconjugates in Campylobacter jejuni

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