Delineation of a lactococcal conjugation system reveals a restriction‐modification evasion system

Abstract

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Plasmid‐mediated horizontal gene mobilisation: Insights from two lactococcal conjugative plasmids

“… Schematic representation of the pNP40 and pUC11B conjugation gene clusters, displaying a colour‐coded representation of the shared protein functionalities between the clusters, based on mutational and predictive functional analysis based on sequence and structural similarities, adapted from Ortiz Charneco et al. ( 2021 , 2023 ). The oriT regions from both plasmids is highlighted, with the IRs highlighted in green and the presumed nick region in yellow.…”

“…Bacterial strains used in this study are listed in Table S1 . Overnight cultures were prepared by inoculating cells from frozen stocks into 10 mL of M17 supplemented with 0.5% (v/v) glucose (GM17) containing either nisin (2.5 μg/mL, for selection of pNP40), streptomycin (500 μg/mL, to select for the recipient L. cremoris MG1614) (Gasson, 1983 ), tetracycline (10 μg/mL, to select strains harbouring pUC11B or pPTPi) or erythromycin (5 μg/mL, to select for strains containing the mobilisable plasmids, pNZ8048E or pPEPi, or derivatives thereof) (Ortiz Charneco et al., 2023 ) and incubated at 30°C for 16 h. All plasmids and constructs are summarised in Table S3 . Electrocompetent cells of L. cremoris were prepared as previously described (Holo & Nes, 1989 ).…”

“…mobC and mobA together, or mobC or mobA on their own) into the low‐copy number vector pPTPi (O'Driscoll et al., 2004 ) using the oligonucleotides listed in Table S2 . Furthermore, the oriT ‐containing sequences from either pNP40 or pUC11B were individually cloned into the erythromycin‐resistant derivative pPEPi (Ortiz Charneco et al., 2023 ), into which the aforementioned mob genes from pDRC3A and pUC11C were cloned. All constructs were sequenced by Sanger sequencing (Eurofins, Ebersberg, Germany) to verify sequence integrity.…”

“…Several lactococcal plasmids encode conjugation functions which facilitate plasmid transfer from a donor to a recipient cell via a presumed Type IV Secretion System (T4SS) process (Cabezón et al., 2015 ; Ortiz Charneco et al., 2023 ). The conjugation‐associated T4SS represents a multiprotein complex that spans the bacterial cell envelope in both Gram‐positive and Gram‐negative bacteria (Grohmann et al., 2018 ).…”

“…pMRC01‐like plasmids, among which is the conjugative plasmid pUC11B, form another prevalent conjugation system encoded by certain lactococcal plasmids (Ortiz Charneco et al., 2021 ). A previous study (Ortiz Charneco et al., 2023 ) determined that plasmids pMRC01, pAF22 and pUC11B were part of the ‘pMRC01‐like’ group of plasmid‐encoded conjugation systems in lactococci, representing the second most prevalent type of plasmid‐encoded conjugation systems among lactococcal strains. Among these three plasmids, the conjugative plasmid pUC11B remains the least characterised, with pUC11B‐mediated mobilisation not being reported to date.…”