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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, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of [Cys.sup.162] by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA.lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

Fructooligosaccharides (FOSs)--fructose-based oligosaccharides--are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcanemolasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose. [ABSTRACT FROM AUTHOR]

Leucocroton havanensis Borhidi is an endemic plant species of Cuba able to hyperaccumulate nickel. In order to sustain the conservation of this species, an efficient protocol for its micropropagation, via axillary bud proliferation, is described. We placed apical segments from aseptic seedlings on basal medium supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP) or thidiazuron (TDZ), individually or in combination. On a medium containing 0.5 mg L-1 IAA and 1.0 mg L-1 BAP explants (65.5%) developed axillary buds. Nevertheless, combinations of 0.5 mg L-1 IAA with 0.1 mg L-1 TDZ was the most effective treatment producing the highest number of buds per explant (30.3); while the control treatment, without growth regulators, produced no buds at all. Transfer of buds to medium supplemented with indole-3-butyric acid, indicated that 0.25 mg L-1 is the amount of hormone required to generate roots on young buds (100%). In order to assess DNA variations in micropropagated plants, an Amplified Fragment Length Polymorphism analysis was performed and no genetic variation was detected. This study demonstrates that a high multiplication rate can be obtained by means of the reported protocol, and that plantlets can be readily hardened (96% survival) in a greenhouse by transplanting them on serpentine soil. [ABSTRACT FROM AUTHOR]

The article focuses on a study on the identification and recombinant expression of a bacterial exolevanase useful for the production of high fructose syrups. The sugarcane endophyte Gluconacetobacter diazotrophicus is producing levan from sucrose by a secreted levansucrase (LsdA). Recombinant expression of the levanase-encoding gene (lsdB) in Escherichia coli and Pichia pastoris resulted in a functional protein that is able to hydrolyze levan and inulin to free fructose without the formation of oligofructans.