Molecular Description and Industrial Potential of Tn 6098 Conjugative Transfer Conferring Alpha-Galactoside Metabolism in Lactococcus lactis

Abstract

A novel 51-kb conjugative transposon of Lactococcus lactis, designated Tn6098, encoding the capacity to utilize ␣-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use ␣-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in ␣-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates.Lactic acid bacteria (LAB) are widely applied in food fermentations, where they contribute to the organoleptic, preservation, and health properties of the fermented food stuffs. Because of their industrial relevance, numerous LAB genome sequencing initiatives have been conducted (13,17,32). Comparative genomics of LAB has revealed the existence of a large inter-and intraspecies genomic diversity (4,5,9,18,26,27,35,39). Horizontal gene transfer (HGT) appears to be a primary driver in LAB evolution and the creation of diversity. HGT is facilitated by the widespread occurrence of plasmids, phages, and mobile genetic elements such as insertion sequence (IS) elements and conjugative transposons, which serve as effective vectors for the spread of genes and traits within and over the species barrier (20,40).Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, especially cheese and butter. Because of its tremendous industrial importance, it has long been the primary model organism for LAB research revealing molecular insight into many traits relevant for industrial applications, including the production of flavor compounds, vitamins and other nutraceuticals, and exopolysaccharides relevant for texture development (3, 8, 39).L. lactis occupies two distinct niches. The occurrence of the species in dairy substrates has long been described, and L. lactis strains are readily isolated from artisanal and spontaneo...

Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution

“…With the exception of one deletion in a stretch of three adenine residues, all INDELs identified were located in mononucleotide stretches of at least 8 nt, which is consistent with the majority of mutations found in dairy-adapted streptococci (Table 2; Bolotin et al 2004). Furthermore, strains NZ5522 and NZ5523 had lost an identical 51.3-kb genomic fragment that is similar to a lactococcal sucrose transposon, designated Tn6098 (Kelly et al 2000;Machielsen et al 2011). Since Tn6098 contains the only predicted a-galactosidase of the parental KF147 strain, the relative fraction of a-galactosidase-negative strains throughout the evolution experiment could be determined by selective plating.…”

The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin

“…4). The location of the raffinose gene cluster differs for strain A12: it is located on a plasmid in strain A12 but on the chromosome in strains KF147 (30,45,46) and ATCC 43920 (47), close to the galKT genes cluster involved in the galactose metabolism. This was confirmed by hybridization of the PFGE fingerprint using LL2138 as a probe (data not shown).…”

Improving Oenococcus oeni to overcome challenges of wine malolactic fermentation

“…Conjugative transposons are mobile genetic elements capable of independent replication and insertion of a copy within the genome. An example is the conjugative transposon Tn6098, which encodes the capacity to utilize a-galactosides in Lactococcus lactis strains isolated from plants [13]. The transposon was characterized and transferred into a strain of L. lactis derived from milk, enabling the recipient strain to grow well in soy milk (a substrate rich in a-galactosides) but retaining the flavor-forming capabilities important in dairy L. lactis [13].…”

“…An example is the conjugative transposon Tn6098, which encodes the capacity to utilize a-galactosides in Lactococcus lactis strains isolated from plants [13]. The transposon was characterized and transferred into a strain of L. lactis derived from milk, enabling the recipient strain to grow well in soy milk (a substrate rich in a-galactosides) but retaining the flavor-forming capabilities important in dairy L. lactis [13]. Problematically, the current methods of conjugation for O. oeni do not allow gene replacement, as the transfer frequency is lower than the recombination frequency [14].…”