Inventory of the GH70 enzymes encoded by Leuconostoccitreum NRRL B-1299-identification of three novel -transglucosylases

Leuconostoccitreum NRRL B-1299 has long been known to produce -glucans containing both -(16) and -(12) linkages, which are synthesized by -transglucosylases of the GH70 family. We sequenced the genome of Leuconostoccitreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucraseA (BRS-A) grafts linear -(16) dextran with -(12)-linked glucosyl units, and is probably involved in the -(12) branching of L.citreum NRRL B-1299 dextran. This is the first report of a naturally occurring -(12) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for -(16) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27000gmol(-1). In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L.citreum genomes further revealed that dsrDP is unique to L.citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L.citreum strains.