Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli

The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined. Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon. Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence. Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites. Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified. Probable terminator sequences were not found in any of these three regulatory regions. Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein. Computer techniques were used to analyse the mtlD gene and its product.     

Corrected sequence of the mannitol (mtl) operon in Escherichia coli

The previously published sequences of the operator-promoter region of the mannitol operon of Escherichia coli and of the mtlD gene have been found to contain a number of errors. The major conclusions reported previously were correct, but additionally it is now clear that a C-terminal portion of mannitol-1-phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino-terminal region of human liver fructose-6-phosphate-2-kinase:fructose-2,6-bisphosphatase.     

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.     

Purification and characterization of the recombinant Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.     

Role of cell-associated N-acetyl-d-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro

Abstract Previously a N -acetyl- d -glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.     

Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII^Mtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [³H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII^Mtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。     

Conversion of D-mannitol to D-ribose: a newly discovered pathway in Escherichia coli.

A mutant (mtlD) strain of Escherichia coli unable to oxidize mannitol-1-phosphate to fructose-6-phosphate was used to study the fate of mannitol-1-phosphate. D-[1-14C]mannitol entered the cells via the phosphotransferase system and was phosphorylated equally at carbon 1 or 6. The label disappeared gradually from the mannitol-1-phosphate pool, and some 60% of the 14C was recovered in nucleic acids. Ribose was isolated from the purified RNA. The 14C label distribution in the isolated ribose precluded a simple hexose-to-pentose conversion by elimination of one terminal carbon from mannitol-1-phosphate. The 14C from mannitol-1-phosphate that did not enter macromolecules was found in CO2 and in some organic, non-phosphorylated compounds that were not identified. We suggest that the de novo synthesis of mannitol-1-phosphate in E. coli may be a reaction specifically dedicated to the biosynthesis of ribose.     

Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata

The mtl operon of Klebsiella pneumoniae KAY2026 (formerly Aerobacter aerogenes 1033-5P14) was shown to contain as the promoter-proximal gene mtlA, encoding a D-mannitol-specific enzyme II transporter (IICBA(Mtl)). This gene is followed by mtlD, coding for a mannitol-1-phosphate dehydrogenase (MtlD, 382 amino acid residues), and mtlR (MtlR, 195 amino acid residues) coding for a putative repressor, gene mtlR overlaps the termination codon of mtlD. The DNA and protein sequences are highly similar to the corresponding genes (81% identical bp) and proteins (79-85% identical amino acids) of Escherichia coli K-12. A truncated form of MtlD lacking the 162 C-terminal amino acid residues still shows 10% dehydrogenase activity which may explain the controversy in the literature concerning the properties of mannitol-phosphate and other medium-length dehydrogenases.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.     

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus.

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.     

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.     

Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K(m) and V(max) values for Man-6P of purified recombinant PMI1 were 41.3+/-4.2 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn(2+), Cd(2+), and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.     

Overproduction of heterologous mannitol 1-phosphatase: a key factor for engineering mannitol production by Lactococcus lactis

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.     

高度耐盐双价转基因烟草的研究

随着全球性人口的增长和土地退化的加剧,开发利用广阔盐碱地和干旱土地的需要日益迫切。植物生物技术的日臻完善,为培育高效耐盐植物迎来了一丝曙光。在高渗条件下,耐盐的微生物或植物细胞通过增加胞内一些相溶性溶质的浓度来维持渗透压的平衡。这些可溶性溶质包括无机离子、糖类、多元醇、氨基酸和生物碱等。通过基因工程手段,使细胞内积累脯氮酸⑴、甜菜碱⑵、甘露醇⑶、海藻糖⑷,能够不同程度地提高转基因烟草的耐盐性。多元醇含有多个羟基,亲水性能强,能有效维持细胞内水活度。山梨醇、甘露醇等己糖分子结构、理化性质和生理功能相近。故此．我们认为：不同糖醇在转基因烟草中的积累．可能具有协同(或累加)效应,有希望更大地提高植物耐盐性。我们在获得大肠杆菌mtlD基因(编码l-磷酸甘露醇脱氢酶)和gutD基因(编码6-磷酸山梨醇脱氢酶)克隆⑸的基础上,获得了分别表达mtlD和gutD基因的单价转基因烟草,并首次证实了gucD基因的表达,能显著地提高转基因烟草的耐盐性⑹。本文工作进一步报道同时表达大肠杆菌mtlD和gutD基因双价转基因烟草的高效高度耐盐性。     

Engineering of the metabolism of Saccharomyces cerevisiae for anaerobic production of mannitol

Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells.