Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

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

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.

The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, Gluconacetobacter (formerly Acetobacter) diazotrophicus is a nonpathogenic, nitrogen-fixing endophyte of sugarcane and other predominantly sucrose-rich crops (11,20,47). This gramnegative bacterium lacks a sucrose transport system (3) and depends on the secretion of a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose (16,17). The levansucrase gene (lsdA) and an exolevanase gene (lsdB) downstream form a chromosomal operon with a high level of conservation among G. diazotrophicus isolates (17, 26).-All known levansucrases are extracellular proteins, although they follow different secretion routes. Levansucrase secretion in gram-positive bacteria involves cleavage of signal-peptidecontaining precursors in Bacillus subtilis (43), Bacillus amyloliquefaciens (46), Geobacillus stearothermophilus (24), Paenibacillus polymyxa (7), Streptococcus salivarius (34), Actinomyces naeslundii (6), Arthrobacter nicotinovorans (36), and Lactobacillus reuteri (49). However, with the exception of LsdA, the other gram-negative levansucrases characterized so far are secreted by a signal-peptide-independent pathway; this occurs in Zymomonas mobilis (41), Erwinia amylovora (14), Rahnella aquatilis (42), Pseudomonas syringae (18, 23), and Gluconacetobacter xylinus (45). LsdA is synthesized as a precursor with a 30-residue N-terminal signal peptide, which is cleaved off during transport to the periplasm, where the enzyme adopts its final conformation. Then, in a rate-limiting step, the mature LsdA is transferred across the outer membrane and released into the extracellular medium without further proteolytic cleavage (15).A wide spectrum of gram-negative bacteria, mainly plantand animal-interacting species, utilize the type II secretion pathway for extracellular release of particular proteins, such as hydrolytic enzymes and toxins. Following translocation to the periplasm by the signal-peptide-dependent Sec or TAT system (32, 52), the folded substrate protein is transported across the outer membrane by the type II secretion apparatus, consisting of a multiprotein complex of at least 12 components (reviewed in reference 38).Here we report that the endophytic bacterium G. diazotrophicus possesses a type II secretion operon required for LsdA transport across the outer membrane. The locus was identified downstream of the levansucrase-levanase (lsdA-lsdB) operon on the chromosome of the 14 strains tested, recovered from different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II secretion pathway in the Acetobacteraceae. MATERIALS AND METHODSBacterial strains, media, and plasmids....