Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB)

The nucleotide sequence of a 2.119-kb DNA fragment containing the Vibrio alginolyticus sucrase gene (scrB) was determined. The complete sequence (484 aa residues) of the sucrase was deduced and homology was detected between the sucrase enzymes from V. alginolyticus and the Gram-positive bacteria Bacillus subtilis and Streptococcus mutans. In Escherichia coli cells the cloned V. alginolyticus sucrase is translocated to the periplasm. Transposon phoA mutagenesis experiments strongly suggested that V. alginolyticus sucrase in E. coli is not exported across the cytoplasmic membrane by means of a typical signal sequence.     

Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli.

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.     

Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides

Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids. Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L. mesenteroides DNA by Escherichia coli host cells. The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced. To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19. The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E. coli. pLmz was sequenced showing that the enzyme consists of 485 amino acids. L. mesenteroides Glc6PD is 31% identical to the human enzyme.     

Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.     

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli.

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1

The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).     

Dextran synthesis by immobilized dextran sucrase

Dextran sucrase has been produced by fermentation of Leuconostoc mesenteroides NRRL B-512, with and without continuous sucrose addition to improve enzyme production. The enzyme preparation has been concentrated from the fermentation broth by ultrafiltration and purified by gel permeation chromatography on Ultrogel. The specific activity of the dextran sucrase was greatly enhanced by calcium chloride addition to the purified enzyme. This enzyme preparation has been immobilized by covalent coupling onto an amino porous silica support (Spherosil) activated with glutaraldehyde. Immobilized dextran sucrase derivatives with an activity up to 830 dextran sucrase units per g. support could thus be obtained. The effect of the support specific area on coupling efficiency and reaction kinetics has been investigated, and the effect of intraparticular diffusion underlined. The molecular weight distribution of the dextran has been determined when varying several parameters.     

Molecular characterization and functional analysis of a secA gene homolog in Actinobacillus actinomycetemcomitans

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram-negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross-reacted with anti-SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azide mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera-specific Sec proteins to complement some Sec mutations in E. coli.     

Characteristic of saccharose fermentation by pathogenic Escherichia coli strains--phenotypic and genotypic elements

The purpose of the study was to characterize fermentation of sucrose by Escherichia coli strains and to answer why some of these strains doesn't utilize this disaccharide. Investigations included 16 E. coli strains. Only 5 of these strains utilized sucrose. Genotypic analysis demonstrated the presence of cscB gene (encoding the sucrase permease which catalyzes transport of sucrose through the plasma membrane of the cell) in 5 strains of E. coli and cscA gene (encoding an enzyme sucrase that catalyzes the utilization of sucrose) in 6 strains of E. coli. These 5 of E. coli strains which possessed a chromosomally encoded sucrose metabolic pathway utilized sucrose with a different time. 3 of them destroyed this disaccharide after 24 h and 2 of them destroyed it after 48 h. Ten of E. coli strains hadn't cscA gene and 11 of them had not cscB genes. The lack of these genes can be the prove that it is not possible for 11 of E. coli strains to synthesize sucrose permease and for 10 of them to synthesize sucrase and it may be the reason of not utilize disaccharide sucrose by these bacteria.     

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.     

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus

The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.