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Tomofusa Tsuchiya - One of the best experts on this subject based on the ideXlab platform.
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A cryptic Melibiose transporter gene possessing a frameshift from Citrobacter freundii.
Journal of biochemistry, 2001Co-Authors: Toshi Shimamoto, Noriko Okazaki, Tadashi Shimamoto, Hideyuki Kawakami, Tomofusa TsuchiyaAbstract:Wild-type Citrobacter freundii cannot grow on Melibiose as a sole source of carbon. The Melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize Melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active Melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to Melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the Melibiose transporters.
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Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999Co-Authors: Shigenobu Matsuzaki, Tomofusa Tsuchiya, Audrey C. Weissborn, Eiji Tamai, T. Hastings WilsonAbstract:AbstractThe Melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of Melibiose. Starting with the cysteine-less Melibiose carrier, we have individually substituted cysteine for amino acids 39–66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of Melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of Melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of Melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an α-helical structure and thus the rows are tilted at an angle of 25° to the helical axis. We suggest that these residues line the path of Melibiose and its associated cation through the carrier
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Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the Melibiose operon in Escherichia coli
The Journal of biological chemistry, 1998Co-Authors: Eiji Tamai, Tadashi Shimamoto, Masaaki Tsuda, Tohru Mizushima, Tomofusa TsuchiyaAbstract:The Melibiose utilization system ofEscherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 °C. The reason for this temperature sensitivity was thought to be that the Melibiose transporter (MelB) of W3133 cells was temperature-sensitive. A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize Melibiose between 37 and 42 °C. However, we found that the Melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the Melibiose transporter at 37 °C (or 40 °C) in both E. coli W3133 and W3133-2 were exactly the same. Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the Melibiose operon) of W3133-2 was exactly the same as that of W3133. Activity of α-galactosidase (product of the first gene, melA, of the Melibiose operon) of W3133 cells grown at 40 °C was very low, although that of W3133-2 cells grown at 40 °C was high. These observations suggested that expression of the Melibiose operon in W3133 is also temperature-sensitive. In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method. Furthermore, we identified mutations in the promoter region of the Melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the −35 region. This may stabilize a possible stem structure due to the inverted repeat at 37–42 °C.
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Mutants of Citrobacter freundii That Transport and Utilize Melibiose
Journal of bacteriology, 1998Co-Authors: Noriko Okazaki, Masayuki Kuroda, Toshi Shimamoto, T H Wilson, Tomofusa TsuchiyaAbstract:The Melibiose transporter of Escherichia coli is a secondary transporter which mediates symport of monovalent cations and Melibiose or its analogs (16). This transporter is a valuable system for the investigation of structure-function relationships in a cation-coupled symporter. Either Na+, H+, or Li+ is utilized as a coupling cation for transport of Melibiose or other galactosides (or galactose). The coupling cation utilized varies depending on the substrate transported (16). Na+ is the most effective coupling cation for Melibiose transport, followed by H+ and Li+ (Li+ is a poor coupling cation). With methyl-β-d-thiogalactoside (TMG) as the substrate, both Na+ and Li+, but not H+, are utilized (5, 16). We cloned the gene (melB) encoding the Melibiose transporter and sequenced it (3, 18). Thus, the primary structure of the Melibiose transporter (MelB) was deduced. Mutational analysis revealed many amino acid residues that are important for the function of the Melibiose transporter, especially for cation recognition (11, 17). Analyses of functionally and structurally related proteins are valuable for the understanding of structure-function relationships in the proteins. Several microorganisms possess Melibiose transporters. The Melibiose transporters from Salmonella typhimurium (6), Klebsiella pneumoniae (2), Enterobacter aerogenes (9), and Enterobacter cloacae (8), in addition to E. coli, have been characterized and sequenced (13). Such analyses are also useful for understanding the evolutionary relationships of the transporters (and microorganisms). Citrobacter freundii is a member of the Enterobacteriaceae and is often found in clinical specimens as an opportunistic or secondary pathogen (12). Although cells of C. freundii are able to utilize lactose as a carbon source (10), they are unable to utilize Melibiose. Here we report the isolation of C. freundii mutants able to grow on Melibiose. We also describe the properties of the Melibiose transporter in the mutants. Isolation of mutants. Cells of C. freundii ATCC 8090 grown in L medium (4) were densely streaked on agar plates containing a minimal medium (14) supplemented with 10 mM Melibiose. Na+ salts in the minimal medium were replaced with K+ salts. After incubation at 37°C for 2 days, colonies appeared on the plates. Since these mutant cells utilized Melibiose as a carbon source, they must have expressed a transporter for Melibiose and an enzyme for the degradation of Melibiose. We isolated the colonies and purified them on agar plates containing minimal medium and Melibiose. Thereafter, we measured the growth of two of the mutants, M4 and M7, on Melibiose. The mutant cells grew well on Melibiose, although the wild-type cells did not (data not shown). Cells of M4 showed better growth than cells of M7. The generation time for M7 was about 1.5 times longer than that for M4. α-Galactosidase activity in the mutants. Wild-type and mutant cells of C. freundii were grown in minimal medium supplemented with 1% tryptone either in the absence or presence of 10 mM Melibiose at 37°C under aerobic conditions, and α-galactosidase activity was measured as described previously (15). As shown in Table Table1,1, cells of the wild type and M7 grown in the absence of Melibiose had no α-galactosidase activity. Cells of M4 grown in the absence of Melibiose, however, showed some α-galactosidase activity. When grown in the presence of Melibiose, cells of M4 showed very high α-galactosidase activity, cells of M7 showed moderate activity, and wild-type cells showed no activity. Thus, cells of M4 and M7 possessed inducible α-galactosidase activities, although the activity was partially constitutive in M4 cells (Table (Table1).1). TABLE 1 α-Galactosidase activity in wild-type and mutant cells of C. freundii Melibiose transporter in the mutants. For transport experiments, cells were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. Transport of [3H]Melibiose (Rotem) and [14C]TMG (DuPont, NEN, Boston, Mass.) was measured as reported previously (5). Wild-type cells showed no Melibiose transport activity, M7 cells showed some activity, and M4 cells showed higher activity than M7 cells (Fig. (Fig.1A).1A). When TMG was used as the substrate, M4 cells showed very high activity and M7 cells showed moderate activity (Fig. (Fig.1B).1B). M4 cells showed a little TMG transport activity when cells were grown in the absence of Melibiose (data not shown). FIG. 1 Melibiose and TMG transport activities in wild-type and mutant cells of C. freundii. Cells of the wild type (○), mutant M4 (•), or mutant M7 (▴) were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose ... We tested the effects of several sugars (50-fold excess) on TMG (0.1 mM) transport in M4 cells induced with Melibiose. Among the sugars tested, galactosyl-β-d-thiogalactoside(thiodigalactoside) showed the strongest inhibition (82%), followed by Melibiose (76%), galactose (48%), and lactose (48%). Cation coupling in the mutants. Cation coupling to Melibiose and TMG transport in the wild-type and mutant cells were investigated. For this experiment, cells of the wild type, M4, and M7 were grown as described above. Since transport of Melibiose or TMG in cells of E. coli and S. typhimurium is stimulated by Na+ or Li+ (5, 7, 16), we tested the effects of Na+ or Li+ on Melibiose transport and on TMG transport in the C. freundii mutants. However, no significant effect was observed (data not shown). Thus, it seems that neither Na+ nor Li+ is a coupling cation for Melibiose transport or TMG transport in the C. freundii mutants. We then investigated whether H+ and/or Na+ uptake was observed when the transport substrate was added to the cell suspension by using ion-selective electrodes (H+ electrode and Na+ electrode), as described previously (16). We observed uptake of H+ elicited by the addition of Melibiose or TMG in M4 cells (Fig. (Fig.2),2), indicating that Melibiose or TMG is taken up by cells by a mechanism of symport with H+. TMG gave a larger H+ uptake than did Melibiose. Cells of M7 showed some H+ uptake elicited by the addition of Melibiose or TMG. The wild-type cells showed no H+ uptake. Uptake of Na+ was not detected when Melibiose or TMG was added to the cell suspension of M4, M7, or the wild type (data not shown). Thus, we concluded that the coupling cation for Melibiose transport or TMG transport in the C. freundii mutants is H+. FIG. 2 Uptake of H+ driven by downhill sugar entry into cells of C. freundii. Cells of the wild type, mutant M4, or mutant M7 were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. ... melB homolog in C. freundii. Since both α-galactosidase activity and Melibiose transport activity were detected in the mutant cells but not in the wild-type cells, it seemed that wild-type C. freundii possesses a cryptic Melibiose operon. We tested this possibility by Southern blot analysis with a DNA fragment derived from the E. coli melB gene used as a probe. Chromosomal DNA was prepared from cells grown in minimal medium supplemented with 1% tryptone, as described previously (1). Chromosomal DNA prepared from cells of E. coli, S. typhimurium, C. freundii, Citrobacter amalonaticus, or Citrobacter diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% agarose gel, and blotted onto a nitrocellulose membrane. The melB probe used was a BamHI-BamHI fragment (1.1 kbp) derived from the melB gene of E. coli (18). The probes were labeled with [32P]dCTP by using a Multiprime DNA Labelling Kit (Amersham), as suggested by the manufacturer. The 32P-labeled melB probe hybridized with the DNA blot on the nitrocellulose. As shown in Fig. Fig.3,3, we detected a band which hybridized with the probe in a DNA digest from C. freundii. In a control experiment, we detected a hybridized band in a DNA digest from E. coli and S. typhimurium (Fig. (Fig.3).3). No hybridized band was detected with a DNA digest from C. amalonaticus (ATCC 25405) or C. diversus (ATCC 25408). Thus, we conclude that wild-type C. freundii possesses a cryptic melB homolog in the chromosomal DNA but that the other species of Citrobacter, C. amalonaticus and C. diversus, do not possess such a gene. It should be pointed out that we were unable to obtain mutants from C. amalonaticus and C. diversus that utilize Melibiose (data not shown). FIG. 3 Southern hybridization analysis. Chromosomal DNA prepared from E. coli, S. typhimurium, C. freundii, C. amalonaticus, or C. diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% ... Cloning of the melB-like gene from C. freundii is now under way. In all of the mutants tested, we detected both α-galactosidase activity and Melibiose transport activity. Thus, it seems that a gene for α-galactosidase and a gene for the Melibiose transporter are organized into an operon.
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The construction of a cysteine-less Melibiose carrier from E. coli
Biochimica et biophysica acta, 1997Co-Authors: Audrey C. Weissborn, Tomofusa Tsuchiya, Masayuki Kuroda, Martyn C. Botfield, T. Hastings WilsonAbstract:The Melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four Melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a Melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less Melibiose carrier had no significant decrease in ability to accumulate Melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the Melibiose carrier.
T H Wilson - One of the best experts on this subject based on the ideXlab platform.
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Cysteine substitutions for individual residues in helix VI of the Melibiose carrier of Escherichia coli.
The Journal of membrane biology, 2001Co-Authors: Ping Z. Ding, A.c. Weissborn, T H WilsonAbstract:The Melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H+, Na+, or Li+. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the Melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting Melibiose carriers retained 22–100% of their ability to transport Melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of Melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which Melibiose passes.
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Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.
Biochimica et biophysica acta, 1999Co-Authors: Shigenobu Matsuzaki, A.c. Weissborn, E Tamai, T Tsuchiya, T H WilsonAbstract:The Melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of Melibiose. Starting with the cysteine-less Melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of Melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of Melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of Melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of Melibiose and its associated cation through the carrier.
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Mutants of Citrobacter freundii That Transport and Utilize Melibiose
Journal of bacteriology, 1998Co-Authors: Noriko Okazaki, Masayuki Kuroda, Toshi Shimamoto, T H Wilson, Tomofusa TsuchiyaAbstract:The Melibiose transporter of Escherichia coli is a secondary transporter which mediates symport of monovalent cations and Melibiose or its analogs (16). This transporter is a valuable system for the investigation of structure-function relationships in a cation-coupled symporter. Either Na+, H+, or Li+ is utilized as a coupling cation for transport of Melibiose or other galactosides (or galactose). The coupling cation utilized varies depending on the substrate transported (16). Na+ is the most effective coupling cation for Melibiose transport, followed by H+ and Li+ (Li+ is a poor coupling cation). With methyl-β-d-thiogalactoside (TMG) as the substrate, both Na+ and Li+, but not H+, are utilized (5, 16). We cloned the gene (melB) encoding the Melibiose transporter and sequenced it (3, 18). Thus, the primary structure of the Melibiose transporter (MelB) was deduced. Mutational analysis revealed many amino acid residues that are important for the function of the Melibiose transporter, especially for cation recognition (11, 17). Analyses of functionally and structurally related proteins are valuable for the understanding of structure-function relationships in the proteins. Several microorganisms possess Melibiose transporters. The Melibiose transporters from Salmonella typhimurium (6), Klebsiella pneumoniae (2), Enterobacter aerogenes (9), and Enterobacter cloacae (8), in addition to E. coli, have been characterized and sequenced (13). Such analyses are also useful for understanding the evolutionary relationships of the transporters (and microorganisms). Citrobacter freundii is a member of the Enterobacteriaceae and is often found in clinical specimens as an opportunistic or secondary pathogen (12). Although cells of C. freundii are able to utilize lactose as a carbon source (10), they are unable to utilize Melibiose. Here we report the isolation of C. freundii mutants able to grow on Melibiose. We also describe the properties of the Melibiose transporter in the mutants. Isolation of mutants. Cells of C. freundii ATCC 8090 grown in L medium (4) were densely streaked on agar plates containing a minimal medium (14) supplemented with 10 mM Melibiose. Na+ salts in the minimal medium were replaced with K+ salts. After incubation at 37°C for 2 days, colonies appeared on the plates. Since these mutant cells utilized Melibiose as a carbon source, they must have expressed a transporter for Melibiose and an enzyme for the degradation of Melibiose. We isolated the colonies and purified them on agar plates containing minimal medium and Melibiose. Thereafter, we measured the growth of two of the mutants, M4 and M7, on Melibiose. The mutant cells grew well on Melibiose, although the wild-type cells did not (data not shown). Cells of M4 showed better growth than cells of M7. The generation time for M7 was about 1.5 times longer than that for M4. α-Galactosidase activity in the mutants. Wild-type and mutant cells of C. freundii were grown in minimal medium supplemented with 1% tryptone either in the absence or presence of 10 mM Melibiose at 37°C under aerobic conditions, and α-galactosidase activity was measured as described previously (15). As shown in Table Table1,1, cells of the wild type and M7 grown in the absence of Melibiose had no α-galactosidase activity. Cells of M4 grown in the absence of Melibiose, however, showed some α-galactosidase activity. When grown in the presence of Melibiose, cells of M4 showed very high α-galactosidase activity, cells of M7 showed moderate activity, and wild-type cells showed no activity. Thus, cells of M4 and M7 possessed inducible α-galactosidase activities, although the activity was partially constitutive in M4 cells (Table (Table1).1). TABLE 1 α-Galactosidase activity in wild-type and mutant cells of C. freundii Melibiose transporter in the mutants. For transport experiments, cells were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. Transport of [3H]Melibiose (Rotem) and [14C]TMG (DuPont, NEN, Boston, Mass.) was measured as reported previously (5). Wild-type cells showed no Melibiose transport activity, M7 cells showed some activity, and M4 cells showed higher activity than M7 cells (Fig. (Fig.1A).1A). When TMG was used as the substrate, M4 cells showed very high activity and M7 cells showed moderate activity (Fig. (Fig.1B).1B). M4 cells showed a little TMG transport activity when cells were grown in the absence of Melibiose (data not shown). FIG. 1 Melibiose and TMG transport activities in wild-type and mutant cells of C. freundii. Cells of the wild type (○), mutant M4 (•), or mutant M7 (▴) were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose ... We tested the effects of several sugars (50-fold excess) on TMG (0.1 mM) transport in M4 cells induced with Melibiose. Among the sugars tested, galactosyl-β-d-thiogalactoside(thiodigalactoside) showed the strongest inhibition (82%), followed by Melibiose (76%), galactose (48%), and lactose (48%). Cation coupling in the mutants. Cation coupling to Melibiose and TMG transport in the wild-type and mutant cells were investigated. For this experiment, cells of the wild type, M4, and M7 were grown as described above. Since transport of Melibiose or TMG in cells of E. coli and S. typhimurium is stimulated by Na+ or Li+ (5, 7, 16), we tested the effects of Na+ or Li+ on Melibiose transport and on TMG transport in the C. freundii mutants. However, no significant effect was observed (data not shown). Thus, it seems that neither Na+ nor Li+ is a coupling cation for Melibiose transport or TMG transport in the C. freundii mutants. We then investigated whether H+ and/or Na+ uptake was observed when the transport substrate was added to the cell suspension by using ion-selective electrodes (H+ electrode and Na+ electrode), as described previously (16). We observed uptake of H+ elicited by the addition of Melibiose or TMG in M4 cells (Fig. (Fig.2),2), indicating that Melibiose or TMG is taken up by cells by a mechanism of symport with H+. TMG gave a larger H+ uptake than did Melibiose. Cells of M7 showed some H+ uptake elicited by the addition of Melibiose or TMG. The wild-type cells showed no H+ uptake. Uptake of Na+ was not detected when Melibiose or TMG was added to the cell suspension of M4, M7, or the wild type (data not shown). Thus, we concluded that the coupling cation for Melibiose transport or TMG transport in the C. freundii mutants is H+. FIG. 2 Uptake of H+ driven by downhill sugar entry into cells of C. freundii. Cells of the wild type, mutant M4, or mutant M7 were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. ... melB homolog in C. freundii. Since both α-galactosidase activity and Melibiose transport activity were detected in the mutant cells but not in the wild-type cells, it seemed that wild-type C. freundii possesses a cryptic Melibiose operon. We tested this possibility by Southern blot analysis with a DNA fragment derived from the E. coli melB gene used as a probe. Chromosomal DNA was prepared from cells grown in minimal medium supplemented with 1% tryptone, as described previously (1). Chromosomal DNA prepared from cells of E. coli, S. typhimurium, C. freundii, Citrobacter amalonaticus, or Citrobacter diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% agarose gel, and blotted onto a nitrocellulose membrane. The melB probe used was a BamHI-BamHI fragment (1.1 kbp) derived from the melB gene of E. coli (18). The probes were labeled with [32P]dCTP by using a Multiprime DNA Labelling Kit (Amersham), as suggested by the manufacturer. The 32P-labeled melB probe hybridized with the DNA blot on the nitrocellulose. As shown in Fig. Fig.3,3, we detected a band which hybridized with the probe in a DNA digest from C. freundii. In a control experiment, we detected a hybridized band in a DNA digest from E. coli and S. typhimurium (Fig. (Fig.3).3). No hybridized band was detected with a DNA digest from C. amalonaticus (ATCC 25405) or C. diversus (ATCC 25408). Thus, we conclude that wild-type C. freundii possesses a cryptic melB homolog in the chromosomal DNA but that the other species of Citrobacter, C. amalonaticus and C. diversus, do not possess such a gene. It should be pointed out that we were unable to obtain mutants from C. amalonaticus and C. diversus that utilize Melibiose (data not shown). FIG. 3 Southern hybridization analysis. Chromosomal DNA prepared from E. coli, S. typhimurium, C. freundii, C. amalonaticus, or C. diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% ... Cloning of the melB-like gene from C. freundii is now under way. In all of the mutants tested, we detected both α-galactosidase activity and Melibiose transport activity. Thus, it seems that a gene for α-galactosidase and a gene for the Melibiose transporter are organized into an operon.
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Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus Melibiose).
Journal of bacteriology, 1997Co-Authors: Manuel F. Varela, Robert J. Brooker, T H WilsonAbstract:The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar Melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on Melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of Melibiose downhill transport. Accumulation (uphill) of Melibiose was completely defective in all of the mutants. Kinetic analysis of Melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for Melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.
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GLY113-->ASP can restore activity to the ASP51-->SER mutant in the Melibiose carrier of Escherichia coli.
Biochemical and biophysical research communications, 1995Co-Authors: D.m. Wilson, H Hama, T H WilsonAbstract:ASP51 in the putative membrane-spanning helix 2 of the Melibiose carrier of Escherichia coli was replaced by SER. This mutation caused failure of the cell to transport Melibiose and failure to ferment Melibiose on indicator plates. A Melibiose-positive revertant was isolated from these plates and was found to have two additional mutations, GLY113-->ASP (in helix 4) and PHE16-->LEU (in helix 1). The double mutant ASP51-->SER/GLY113-->ASP was constructed and showed accumulation of Melibiose. On the other hand ASP51-->SER/PHE16-->LEU showed no activity. It is concluded that the new carboxyl group at position 113 compensates for the loss of the carboxyl group at position 51.
Lan Guan - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium Melibiose transporter MelB.
BMC biology, 2018Co-Authors: Parameswaran Hariharan, Elena B. Tikhonova, João Medeiros-silva, Aike Jeucken, Mikhail V Bogdanov, William Dowhan, Jos F. Brouwers, Markus Weingarth, Lan GuanAbstract:Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling Melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the Melibiose transport catalyzed by this protein. Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled Melibiose active transport to different extents. Interestingly, neither the co-substrate binding (Melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in Melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.
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Structural and functional characterization of protein–lipid interactions of the Salmonella typhimurium Melibiose transporter MelB
BMC Biology, 2018Co-Authors: Parameswaran Hariharan, João Medeiros-silva, Aike Jeucken, Mikhail V Bogdanov, William Dowhan, Jos F. Brouwers, Markus Weingarth, Elena Tikhonova, Lan GuanAbstract:Background Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelB_St) is a symporter coupling Melibiose translocation with a cation (Na^+, Li^+, or H^+). We present an extensive study on the effects of specific phospholipids on the structure of MelB_St and the Melibiose transport catalyzed by this protein. Results Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelB_St at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelB_St; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H^+- and Na^+-coupled Melibiose active transport to different extents. Interestingly, neither the co-substrate binding (Melibiose or Na^+) nor MelB_St folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelB_St with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. Conclusions (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelB_St. (2) The headgroups of PE and PG, but not of CL, play important roles in Melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelB_St.
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insights into the inhibitory mechanisms of the regulatory protein iiaglc on Melibiose permease activity
Journal of Biological Chemistry, 2014Co-Authors: Parameswaran Hariharan, Lan GuanAbstract:The phosphotransfer protein IIAGlc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIAGlc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIAGlc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIAGlc directly binds to MelB of Salmonella typhimurium (MelBSt) and Escherichia coli MelB (MelBEc) at a stoichiometry of unity in the absence or presence of Melibiose. The dissociation constant values are 3–10 μm for MelBSt and 25 μm for MelBEc. All of the binding is solely driven by favorable enthalpy forces. IIAGlc binding to MelBSt in the absence or presence of Melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIAGlc-bound MelBSt exhibits a decreased binding affinity for Melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIAGlc inhibits the induced fit process and restricts the conformational dynamics of MelBSt.
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role of gly117 in the cation Melibiose symport of melb of salmonella typhimurium
Biochemistry, 2012Co-Authors: Lan Guan, Vivek S Jakkula, Alexey A. HodkoffAbstract:The Melibiose permease of Salmonella typhimurium (MelBSt) catalyzes symport of Melibiose with Na+, Li+, or H+, and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of th...
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Role of Gly117 in the cation/Melibiose symport of MelB of Salmonella typhimurium
Biochemistry, 2012Co-Authors: Lan Guan, S. Vivek Jakkula, Alexey A. HodkoffAbstract:The Melibiose permease of Salmonella typhimurium (MelBSt) catalyzes symport of Melibiose with Na+, Li+, or H+, and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of th...
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Comparison of Melibiose and trehalose as stabilising excipients for spray-dried β-galactosidase formulations.
International journal of pharmaceutics, 2018Co-Authors: Tiina Lipiäinen, Marikki Peltoniemi, Heikki Räikkönen, Anna-maija Kolu, Anne Mari JuppoAbstract:Abstract Spray-dried protein formulations commonly require stabilising excipients to prevent protein degradation during processing and storage, and trehalose has been commonly used. The purpose of this work was to evaluate Melibiose in spray-dried protein formulations in comparison to trehalose. The protein-activity-preserving efficacy, process behaviour and storage stability were studied. Spray drying of β-galactosidase was carried out using different process temperature, drying air flow and feed liquid atomisation settings. Both Melibiose and trehalose reduced protein activity loss during drying. A decrease in activities was observed when the process temperature exceeded a threshold temperature. During storage (30 days at 18% RH and 20 or 40 °C), the formulations dried below this threshold temperature showed no further activity loss, and the stabilising efficacy of the two disaccharides was equal. With higher process temperatures, the remaining protein activities after storage trended higher with Melibiose formulations. All formulations remained amorphous. The powder yields of Melibiose formulations were similar to trehalose. There was a difference in residual moisture contents, with Melibiose formulations giving drier products. In conclusion, protein formulations with Melibiose could be spray dried into amorphous powders that were physically stable, contained lower moisture contents and protected protein activity at least as well as trehalose formulations.
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Spray-dried amorphous isomalt and Melibiose, two potential protein-stabilizing excipients.
International Journal of Pharmaceutics, 2016Co-Authors: Tiina Lipiäinen, Marikki Peltoniemi, Heikki Räikkönen, Anne Mari JuppoAbstract:Abstract The possibility of producing amorphous isomalt and Melibiose by spray drying was studied. The impact of process parameters on yield and solid-state stability was compared to sucrose and trehalose. All powders remained amorphous during 2–3 weeks. Processing was challenging due to powder stickiness. Low-temperature and low-humidity drying processes generally performed best. Most isomalt and sucrose powder was retrieved when using 60 °C inlet temperature, 800 L/h atomizing rate, 1.4 ml/min feed rate, 15% concentration and 100% aspirator rate, giving 42–43 °C outlet temperature. Isomalt was the most problematic, because it had the lowest T g and became sticky very easily, therefore process parameters needed to be precisely balanced. There was more freedom in designing processes for Melibiose but best yields were obtained with low-temperature (50 °C inlet temperature, 800 L/h atomizing rate, 4.9 ml/min feed rate, 10% concentration and 100% aspirator, 39 °C outlet temperature). Trehalose was different in that higher temperatures resulted in better yields. Yet, trehalose generally contained the highest moisture contents. The possibility to produce amorphous isomalt and Melibiose at low-temperature process conditions makes them promising considering spray drying applications for heat-sensitive proteins. Melibiose is a better candidate than isomalt because of easier processability and superior solid-state stability.
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The tableting properties of Melibiose monohydrate
International journal of pharmaceutics, 2013Co-Authors: Satu Lakio, Janne Sainio, Petteri Heljo, Tuomas Ervasti, Niina Kivikero, Anne Mari JuppoAbstract:In this research, the tableting properties of α-Melibiose monohydrate were studied. Melibiose is a disaccharide which bears structural resemblance to lactose, because they both consist of galactose and glucose monosaccharide subunits. Compactibility and deformation behavior of two Melibiose batches from different suppliers were studied and compared with α-lactose monohydrate and some other typical tableting excipients. Differences in the deformation behavior were determined comparing the shape of the Heckel plots, the yield pressure values and the strain rate sensitivity (SRS) indexes. In addition, the effect of moisture on the tabletability was studied. According to the yield pressures and SRS indexes Melibiose was concluded to be fragmenting, even at higher degree than lactose monohydrate. However, the overall deformation behavior of Melibiose was found to be similar to that of lactose monohydrate. Increase in moisture content resulted in higher tensile strengths of tablets for both Melibiose batches, but it seemed to have more effect on compactibility of the other batch. In conclusion, Melibiose has potential to be used as an excipient in tablet formulations.
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the effect of relative humidity on the physical properties of two Melibiose monohydrate batches with differing particle size distributions and surface properties
Journal of Pharmaceutical Sciences, 2013Co-Authors: Ville P Heljo, Satu Lakio, Janne Sainio, Niina Kivikero, Anna Shevchenko, Anne Mari JuppoAbstract:Melibiose monohydrate has shown promise when employed as a pharmaceutical excipient, but its physical properties have not been adequately characterized. Therefore, two different Melibiose monohydrate batches were analyzed as received or after storage under different relative humidity (RH) atmospheres. The particle size distributions and specific surface areas of the two batches were shown to differ considerably, which also had an effect on their water sorption tendencies and on the intermolecular structure of Melibiose after storage. The relatively large primary particles that were more abundant in one of the batches were shown to possess a porous surface structure, and water evaporation from them occurred in two phases when heated. Furthermore, storing the batch with smaller mean particle size under dry conditions affected the crystal structure and molecular vibrations of the sample more than in the case of the batch with larger mean particle size. It was concluded that the physical properties of Melibiose monohydrate after storage at different RH atmospheres is largely governed by the primary particle size and porosity.
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Stability of rituximab in freeze-dried formulations containing trehalose or Melibiose under different relative humidity atmospheres.
Journal of pharmaceutical sciences, 2012Co-Authors: Ville P Heljo, Vasco Filipe, Stefan Romeijn, Wim Jiskoot, Anne Mari JuppoAbstract:The objective of the study was to compare the effectiveness of trehalose with that of Melibiose in protecting a monoclonal antibody (rituximab) from aggregation, fragmentation, and secondary structure alterations during processing and subsequent storage. Because reducing disaccharides such as Melibiose participate in Maillard reaction with proteins, especially in the presence of water, the lyophilizates were stored under different relative humidity (RH 5%, 11%, and 23%) atmospheres. Freeze drying was shown to cause clear alterations in rituximab secondary structure, an increase in noncovalent protein aggregation, and in some cases fragmentation. However, these changes were less pronounced in the formulation containing Melibiose. Storing the lyophilizates under low RH (5%) proved to be most harmful to the stability of rituximab, intensifying secondary structure alterations and increasing protein aggregate content. Again, these changes were less aggravated in the formulation containing Melibiose. Surprisingly, the concentration of aggregates larger than 1 μm decreased in some cases during storage at RH 11% and 23%. There was no indication that storage even under the highest RH (23%) would have caused significant amounts of Maillard reaction end products to be formed during 3 months of storage.
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Mutants of Citrobacter freundii That Transport and Utilize Melibiose
Journal of bacteriology, 1998Co-Authors: Noriko Okazaki, Masayuki Kuroda, Toshi Shimamoto, T H Wilson, Tomofusa TsuchiyaAbstract:The Melibiose transporter of Escherichia coli is a secondary transporter which mediates symport of monovalent cations and Melibiose or its analogs (16). This transporter is a valuable system for the investigation of structure-function relationships in a cation-coupled symporter. Either Na+, H+, or Li+ is utilized as a coupling cation for transport of Melibiose or other galactosides (or galactose). The coupling cation utilized varies depending on the substrate transported (16). Na+ is the most effective coupling cation for Melibiose transport, followed by H+ and Li+ (Li+ is a poor coupling cation). With methyl-β-d-thiogalactoside (TMG) as the substrate, both Na+ and Li+, but not H+, are utilized (5, 16). We cloned the gene (melB) encoding the Melibiose transporter and sequenced it (3, 18). Thus, the primary structure of the Melibiose transporter (MelB) was deduced. Mutational analysis revealed many amino acid residues that are important for the function of the Melibiose transporter, especially for cation recognition (11, 17). Analyses of functionally and structurally related proteins are valuable for the understanding of structure-function relationships in the proteins. Several microorganisms possess Melibiose transporters. The Melibiose transporters from Salmonella typhimurium (6), Klebsiella pneumoniae (2), Enterobacter aerogenes (9), and Enterobacter cloacae (8), in addition to E. coli, have been characterized and sequenced (13). Such analyses are also useful for understanding the evolutionary relationships of the transporters (and microorganisms). Citrobacter freundii is a member of the Enterobacteriaceae and is often found in clinical specimens as an opportunistic or secondary pathogen (12). Although cells of C. freundii are able to utilize lactose as a carbon source (10), they are unable to utilize Melibiose. Here we report the isolation of C. freundii mutants able to grow on Melibiose. We also describe the properties of the Melibiose transporter in the mutants. Isolation of mutants. Cells of C. freundii ATCC 8090 grown in L medium (4) were densely streaked on agar plates containing a minimal medium (14) supplemented with 10 mM Melibiose. Na+ salts in the minimal medium were replaced with K+ salts. After incubation at 37°C for 2 days, colonies appeared on the plates. Since these mutant cells utilized Melibiose as a carbon source, they must have expressed a transporter for Melibiose and an enzyme for the degradation of Melibiose. We isolated the colonies and purified them on agar plates containing minimal medium and Melibiose. Thereafter, we measured the growth of two of the mutants, M4 and M7, on Melibiose. The mutant cells grew well on Melibiose, although the wild-type cells did not (data not shown). Cells of M4 showed better growth than cells of M7. The generation time for M7 was about 1.5 times longer than that for M4. α-Galactosidase activity in the mutants. Wild-type and mutant cells of C. freundii were grown in minimal medium supplemented with 1% tryptone either in the absence or presence of 10 mM Melibiose at 37°C under aerobic conditions, and α-galactosidase activity was measured as described previously (15). As shown in Table Table1,1, cells of the wild type and M7 grown in the absence of Melibiose had no α-galactosidase activity. Cells of M4 grown in the absence of Melibiose, however, showed some α-galactosidase activity. When grown in the presence of Melibiose, cells of M4 showed very high α-galactosidase activity, cells of M7 showed moderate activity, and wild-type cells showed no activity. Thus, cells of M4 and M7 possessed inducible α-galactosidase activities, although the activity was partially constitutive in M4 cells (Table (Table1).1). TABLE 1 α-Galactosidase activity in wild-type and mutant cells of C. freundii Melibiose transporter in the mutants. For transport experiments, cells were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. Transport of [3H]Melibiose (Rotem) and [14C]TMG (DuPont, NEN, Boston, Mass.) was measured as reported previously (5). Wild-type cells showed no Melibiose transport activity, M7 cells showed some activity, and M4 cells showed higher activity than M7 cells (Fig. (Fig.1A).1A). When TMG was used as the substrate, M4 cells showed very high activity and M7 cells showed moderate activity (Fig. (Fig.1B).1B). M4 cells showed a little TMG transport activity when cells were grown in the absence of Melibiose (data not shown). FIG. 1 Melibiose and TMG transport activities in wild-type and mutant cells of C. freundii. Cells of the wild type (○), mutant M4 (•), or mutant M7 (▴) were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose ... We tested the effects of several sugars (50-fold excess) on TMG (0.1 mM) transport in M4 cells induced with Melibiose. Among the sugars tested, galactosyl-β-d-thiogalactoside(thiodigalactoside) showed the strongest inhibition (82%), followed by Melibiose (76%), galactose (48%), and lactose (48%). Cation coupling in the mutants. Cation coupling to Melibiose and TMG transport in the wild-type and mutant cells were investigated. For this experiment, cells of the wild type, M4, and M7 were grown as described above. Since transport of Melibiose or TMG in cells of E. coli and S. typhimurium is stimulated by Na+ or Li+ (5, 7, 16), we tested the effects of Na+ or Li+ on Melibiose transport and on TMG transport in the C. freundii mutants. However, no significant effect was observed (data not shown). Thus, it seems that neither Na+ nor Li+ is a coupling cation for Melibiose transport or TMG transport in the C. freundii mutants. We then investigated whether H+ and/or Na+ uptake was observed when the transport substrate was added to the cell suspension by using ion-selective electrodes (H+ electrode and Na+ electrode), as described previously (16). We observed uptake of H+ elicited by the addition of Melibiose or TMG in M4 cells (Fig. (Fig.2),2), indicating that Melibiose or TMG is taken up by cells by a mechanism of symport with H+. TMG gave a larger H+ uptake than did Melibiose. Cells of M7 showed some H+ uptake elicited by the addition of Melibiose or TMG. The wild-type cells showed no H+ uptake. Uptake of Na+ was not detected when Melibiose or TMG was added to the cell suspension of M4, M7, or the wild type (data not shown). Thus, we concluded that the coupling cation for Melibiose transport or TMG transport in the C. freundii mutants is H+. FIG. 2 Uptake of H+ driven by downhill sugar entry into cells of C. freundii. Cells of the wild type, mutant M4, or mutant M7 were grown in minimal medium supplemented with 1% tryptone and 10 mM Melibiose at 37°C under aerobic conditions. ... melB homolog in C. freundii. Since both α-galactosidase activity and Melibiose transport activity were detected in the mutant cells but not in the wild-type cells, it seemed that wild-type C. freundii possesses a cryptic Melibiose operon. We tested this possibility by Southern blot analysis with a DNA fragment derived from the E. coli melB gene used as a probe. Chromosomal DNA was prepared from cells grown in minimal medium supplemented with 1% tryptone, as described previously (1). Chromosomal DNA prepared from cells of E. coli, S. typhimurium, C. freundii, Citrobacter amalonaticus, or Citrobacter diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% agarose gel, and blotted onto a nitrocellulose membrane. The melB probe used was a BamHI-BamHI fragment (1.1 kbp) derived from the melB gene of E. coli (18). The probes were labeled with [32P]dCTP by using a Multiprime DNA Labelling Kit (Amersham), as suggested by the manufacturer. The 32P-labeled melB probe hybridized with the DNA blot on the nitrocellulose. As shown in Fig. Fig.3,3, we detected a band which hybridized with the probe in a DNA digest from C. freundii. In a control experiment, we detected a hybridized band in a DNA digest from E. coli and S. typhimurium (Fig. (Fig.3).3). No hybridized band was detected with a DNA digest from C. amalonaticus (ATCC 25405) or C. diversus (ATCC 25408). Thus, we conclude that wild-type C. freundii possesses a cryptic melB homolog in the chromosomal DNA but that the other species of Citrobacter, C. amalonaticus and C. diversus, do not possess such a gene. It should be pointed out that we were unable to obtain mutants from C. amalonaticus and C. diversus that utilize Melibiose (data not shown). FIG. 3 Southern hybridization analysis. Chromosomal DNA prepared from E. coli, S. typhimurium, C. freundii, C. amalonaticus, or C. diversus was digested with BamHI (except S. typhimurium DNA) or EcoRV (S. typhimurium DNA), separated by electrophoresis in a 1% ... Cloning of the melB-like gene from C. freundii is now under way. In all of the mutants tested, we detected both α-galactosidase activity and Melibiose transport activity. Thus, it seems that a gene for α-galactosidase and a gene for the Melibiose transporter are organized into an operon.
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The construction of a cysteine-less Melibiose carrier from E. coli
Biochimica et biophysica acta, 1997Co-Authors: Audrey C. Weissborn, Tomofusa Tsuchiya, Masayuki Kuroda, Martyn C. Botfield, T. Hastings WilsonAbstract:The Melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four Melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a Melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less Melibiose carrier had no significant decrease in ability to accumulate Melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the Melibiose carrier.
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Sequence of a Melibiose transporter gene of Enterobacter cloacae
Biochimica et biophysica acta, 1997Co-Authors: Noriko Okazaki, Tadashi Shimamoto, Masayuki Kuroda, Xu Xing Jue, Hideki Miyake, Tomofusa TsuchiyaAbstract:Abstract We cloned a fragment of the chromosomal DNA of Enterobacter cloacae , which enabled a Melibiose-negative Escherichia coli mutant lacking melB to grow on Melibiose as the sole source of carbon. Transformed cells harboring the hybrid plasmid carrying the cloned DNA showed Melibiose transport activity. The nucleotide sequence of the DNA region was determined. One complete open reading frame (ORF) and a part of another ORF were found in the region, and the amino acid sequences were deduced. The complete ORF was found to encode a Melibiose transporter which consisted of 425 amino acid residues. Hydropathy analysis revealed that there are about 12 hydrophobic domains in this transporter. The incomplete ORF which exists in the upstream region of the transporter gene seemed to encode an α -galactosidase.
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Characteristics of the Melibiose transporter and its primary structure in Enterobacter aerogenes.
Biochimica et biophysica acta, 1997Co-Authors: Noriko Okazaki, Tadashi Shimamoto, Masayuki Kuroda, Toshi Shimamoto, Tomofusa TsuchiyaAbstract:Abstract Cells of Enterobacter aerogenes can grow on Melibiose as a sole source of carbon. This suggests the presence of Melibiose operon in this organism. We found that E. aerogenes cells possess both α -galactosidase activity and Melibiose transport activity, which were induced by Melibiose. Neither Na + nor Li + stimulated the Melibiose transport. However, transport of methyl- β -thiogalactoside (TMG) was stimulated by Li + but not by Na + . These findings suggest that the major coupling cation for the Melibiose transporter in E. aerogenes is H + . In fact, we observed H + entry into cells caused by an influx of Melibiose and some of its analogs. We cloned the melB gene which encodes the Melibiose transporter, and sequenced it. Deduced amino acid sequence of the transporter revealed that the Melibiose transporter consists of 471 amino acid residues and the molecular weight was calculated to be 52 214 Da. The sequence showed high homology with the sequences of the Melibiose transporters of Escherichia coli , Salmonella typhimurium and Klebsiella pneumoniae . Higher homology was found with the Melibiose transporter of K. pneumoniae than with that of E. coli and S. typhimurium .
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A Melibiose transporter and an operon containing its gene in Enterobacter cloacae.
Journal of bacteriology, 1997Co-Authors: Noriko Okazaki, Tadashi Shimamoto, Masayuki Kuroda, Xu Xing Jue, Hideki Miyake, Tomofusa TsuchiyaAbstract:We detected inducible Melibiose transport activity in cells of Enterobacter cloacae IID977. H+, but not Na+, was found to be the coupling cation for this transporter. We cloned and sequenced the gene encoding the Melibiose transporter. A homology search of a protein sequence database revealed that this Melibiose transporter has high sequence similarity with the lactose transporter (LacY) and the raffinose transporter (RafB) and has some similarity with the Melibiose transporter (MelB) of Escherichia coli.