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.     

Gluconate kinase from Zymomonas mobilis: isolation and characteristics

The enzyme gluconate kinase EC 2.7.1.12 has been found at high levels in glucose-grown Zymomonas mobilis cells. A simple procedure, based on differential dye-ligand chromatography, has been used to isolate the enzyme, purifying it some 600-fold. The purified enzyme is a monomer of molecular weight 18,000 Da, which is much smaller than other gluconate kinases reported. It has a relatively low affinity for ATP. (Km = 1.5 mM), but high for gluconate (Km = 0.33 mM), and has little activity with any other potential substrates.     

Cloning,sequencing and characterization of the alkaline phosphatase gene (phoD) from Zymomonas mobilis

The phoD gene encoding the membrane-bound alkaline phosphatase (ALPI) from Zymomonas mobilis CP4 was cloned and sequenced. Both the translated sequence and the properties of the recombinant enzyme were unusual. Z. mobilis ALPI was monomeric (_r 62926) and hydrolysed nucleotides more effectively than sugar phosphates. The translated sequence contained a single hydrophobic segment near the N-terminus which may serve as a membrane-anchor in Z. mobilis, although the recombinant enzyme was recovered in the cytoplasmic fraction of Escherichia coli. The predicted amino acid sequence for ALPI did not align well with other ALPs or other known genes. However, some similarity to E. coli ALP was noted in the metal-binding and phosphate-binding regions. Two other regions were identified with similarity to the active sites of pyruvate kinase and mammalian 5′-nucleotide phosphodiesterase (also membrane-bound), respectively. It is likely that Z. mobilis phoD represents a new class of alkaline phosphatase genes which has not been described previously.     

Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis.

The gene encoding the enzyme gluconolactonase (D-glucono-delta-lactone lactonohydrolase, EC 3.1.1.17) has been isolated from a recombinant library of genomic Zymomonas mobilis DNA, by detection of enzyme activity in recombinant clones. The gene encoded a protein of 320 amino acids, which is processed to the mature enzyme of 285 amino acids (31079 Da) by cleavage at an Ala-Ala bond, as determined from N-terminal sequencing of the purified enzyme. A minor sequence commencing at amino acid 6 is suggestive of an alternative start of translation at the ATG codon of amino acid 5; in this case the expressed enzyme would remain cytoplasmic, whereas it is presumed that the main portion is directed to the membrane of periplasm by the leader sequence.     

Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis.

Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.     

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis.

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.     

Cloning, sequencing, and characterization of the principal acid phosphatase, the phoC+ product, from Zymomonas mobilis.

The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an Mr 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active with magnesium ions than with zinc ions. Its promoter region was similar to the -35 "pho box" region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomyces cerevisiae acid phosphatase (PHO5). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5' transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.     

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%).     

Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis.

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.