Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4.
The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthetized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.
Arrieta
Juan
J
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Hernández
Lázaro
L
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Coego
Alberto
A
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Suárez
Vivian
V
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Balmori
Ezequiel
E
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Menéndez
Carmen
C
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
Petit-Glatron
Marie-Françoise
MF
Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris 7-Denis Diderot, Laboratoire Génétique et Membranes, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France.
Chambert
Régis
R
Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris 7-Denis Diderot, Laboratoire Génétique et Membranes, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France.
Selman-Housein
Guillermo
G
Center for Genetic Engineering and Biotechnology, Agriculture Division, PO Box 6162, Havana, Cuba.
D000091:Acetobacter / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase