Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4

Abstract

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) ( M A ) was isolated from a genomic library. The nucleotide sequence of a 2-3 kb DNA fragment sufficient for complementation of a levansucrase-def icient mutant (obtained by EMS treatment) was determined. The M A gene (1751 bp) coded for a polypeptide of molecular mass 649 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 lsdA 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 M A gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymornonas mobilis and Ennrinia amylowora, respectively, but only 28-31 YO identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gramnegative 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 synthesized product. Disruption of the lsdA 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. 1

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

“…Broad-host-range plasmids were used to transform E. coli strain S17.1 and were mobilized to G. diazotrophicus by conjugative mating as previously described (4). The transconjugants were selected on LGIE agar medium supplemented with tetracycline and chloramphenicol; the latter antibiotic was used to counterselect E. coli donors.…”

“…Plasmid pALS130 was constructed by joining in a quadruple-ligation reaction the 3,991-bp BamHI-HindIII fragment from pALS32, the 1.6-kbp HindIII nptII-ble cassette from pUC4KIXX, and the 3,560-bp HindIII-KpnI fragment from pALS119 in BamHI-KpnI-digested pBluescript SK(ϩ). The three plasmids were separately introduced into G. diazotrophicus SRT4 by electroporation, and the recombinant colonies were selected on kanamycin-containing LGIE medium plates as described previously (4). Insertion of the nptII marker into the lsd type II secretion genes in the chromosomes of nonmucous colonies was confirmed by Southern hybridization.…”

“…The sequence of the identified type II lsd genes is available in the DDBJ/EMBL/GenBank databases under accession number L41732. zotrophicus mutants that had active LsdA in the periplasm but were unable to transfer the enzyme across the outer membrane were previously isolated by EMS treatment of strain SRT4 (4). Introduction of the library cosmid clone p21R1 restored the Sec ϩ , levan-forming (mucous colony) phenotype in one of the seven mutants (Fig.…”

Characterization and Mutational Analysis of Three Allelic lsc Genes Encoding Levansucrase in Pseudomonas syringae

“…Levan is a high-molecular-weight ␤-(2,6)-polyfructan with extensive branching through ␤-(2,1) linkages. Lsc produces both linkage types.Levansucrases have been isolated from various bacteria, such as Bacillus subtilis, Streptococcus mutans, Zymomonas mobilis, Acetobacter diazotrophicus, Erwinia amylovora, and Pseudomonas syringae (2,9,18,22,34,41). In contrast to levansucrases from gram-positive bacteria, which differ widely in their biochemical characteristics, levansucrases from gramnegative bacteria are similar in their molecular mass and substrate-independent expression (16,22,23,43).…”

Molecular Characterization of the Growth Phase-Dependent Expression of the lsrA Gene, Encoding Levansucrase of Rahnella aquatilis

“…Disruption of the gene encoding levansucrase of the fireblight pathogen Erwinia amylovora retards the development of necrotic symptoms in inoculated pear seedlings, suggesting that the enzyme participates in phytopathogenesis (14). This enzyme is also involved in the sucrose metabolism of Acetobacter diazotrophicus, in which disruption of the gene resulted in a mutant lacking the ability to utilize sucrose as a carbon source (1).…”