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

Abstract

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. mobiis. 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.The ethanologenic gram-negative bacterium Zymomonas mobilis can grow only on glucose, fructose, or sucrose and metabolizes these sugars with the production of ethanol and carbon dioxide as main fermentation products (1,22,41). Carbohydrate metabolism in Z. mobilis has been reviewed recently (43). The monosaccharides glucose and fructose are transported inside the cell by a facilitated diffusion system mediated by a carrier (12), phosphorylated by a specific kinase, and metabolized through the Entner-Doudoroff pathway. The disaccharide, sucrose, is first hydrolyzed to liberate glucose and fructose in the culture medium, and these sugars enter the cell by using the transport system described above. The number and nature of the enzymes involved in sucrose catabolism are not clearly known in Z. mobilis.Sucrose metabolism has been intensively studied in Bacillus subtilis (15). Three saccharolytic enzymes are present: an intracellular sucrase (sacA gene), an extracellular levansucrase (sacB gene), and a levanase (sacC gene). All enzymes act as 13-D-fructofuranosidases; in addition, levansucrase catalyzes the formation of levan, a high-molecular-weight polymer of fructose. The nucleotide sequences of sacA, sacB, and sacC genes have been determined (13,21,39). A strong homology of the N-terminal protein sequences of sucrase, levanase, and yeast invertase (SUC2 gene) was observed, while no similarity with levansucrase could be detected (21).Levan formation during growth of Z. mobilis on sucrose is well known and the presence of levansucrase is generally well accepted (22, 41). Furthermore, it has been demonstrated that levan formation is cell linked (25), while a high saccharolytic activity was detected in culture medium (28,33). These results raised the question of the existence, in addition to levansucrase, of a second enzyme, a sucrase, which may be liberated in culture medium during cell growth (41). More recently, two other polymers has been characterized: a cell-linked, high-...

Characterization of a sucrase gene from Staphylococcus xylosus

“…The amino acid sequence of the ScrB protein shows strong homology with other bacterial sucrases. Homology is found throughout the S. xylosus enzyme, and six highly conserved regions in sucrases, as pointed out by Gunasekaran et al (16), are clearly detectable (Fig. 2).…”

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis

“…E. coli RR1 cells were transformed with plasmids obtained from the genomic library of Z. mobilis ZM1 constructed in pUC19 as previously described (11). Transformants were screened for DnaKz production by colony blotting since the DnaKz antiserum was shown not to cross-react with E. coli proteins.…”

Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

“…The SacA enzyme is a monomer with a molecular weight of 58 kDa. The sacA gene from Z. mobilis has been cloned, sequenced and characterized (Gunasekaran et al, 1990a). The deduced amino acid sequence of sacA gene product showed strong homology with the intracellular sucrase of Bacillus subtilis and yeast invertases.…”

“…The differential expression of gap and pgk appears to be controlled at the mRNA level. The pattern of codon usage in sacB gene was compared with two classes of genes known in Z. mobilis -highly expressed genes like pyruvate decarboxylase (pdc), glucose-6-phosphate dehydrogenase (zwf) and poorly expressed genes like intracellular sucrase (SacA), acid phosphatase (PhoC) (Pond et al, 1989) and ligase (lig) (Gunasekaran et al, 1990a). The codon bias was calculated according to Shark and Conway (1992).…”