Effect of TraN key residues involved in DNA binding on pIP501 transfer rates in Enterococcus faecalis

Conjugation is a major mechanism that facilitates the exchange of antibiotic resistance genes among bacteria. The broad-host-range Inc18 plasmid pIP501 harbors 15 genes that encode for a type IV secretion system (T4SS). It is a membrane-spanning multiprotein complex formed between conjugating donor and recipient cells. The penultimate gene of the pIP501 operon encodes for the cytosolic monomeric protein TraN. This acts as a transcriptional regulator by binding upstream of the operon promotor, partially overlapping with the origin of transfer. Additionally, TraN regulates traN and traO expression by binding upstream of the P traNO promoter. This study investigates the impact of nine TraN amino acids involved in binding to pIP501 DNA through site-directed mutagenesis by exchanging one to three residues by alanine. For three traN variants, complementation of the pIP501∆traN knockout resulted in an increase of the transfer rate by more than 1.5 orders of magnitude compared to complementation of the mutant with native traN. Microscale thermophoresis (MST) was used to assess the binding affinities of three TraN double-substituted variants and one triple-substituted variant to its cognate pIP501 double-stranded DNA. The MST data strongly correlated with the transfer rates obtained by biparental mating assays in Enterococcus faecalis. The TraN variants TraN_R23A-N24A-Q28A, TraN_H82A-R86A, and TraN_G100A-K101A not only exhibited significantly lower DNA binding affinities but also, upon complementation of the pIP501∆traN knockout, resulted in the highest pIP501 transfer rates. This confirms the important role of the TraN residues R23, N24, Q28, H82, R86, G100, and K101 in downregulating pIP501 transfer. Although TraN is not part of the mating pair formation complex, TraE, TraF, TraH, TraJ, TraK, and TraM were coeluted with TraN in a pull-down. Moreover, TraN homologs are present not only in Inc18 plasmids but also in RepA_N and Rep_3 family plasmids, which are frequently found in enterococci, streptococci, and staphylococci. This points to a widespread role of this repressor in conjugative plasmid transfer among Firmicutes.

Supplementary Table S9 TraEpIP501 and TraJpIP501 homologs in putative non-conjugative plasmids encoding traN-like sequences.All plasmids lack a TraA-like relaxase and four out of five plasmids an oriT-like sequence.

Supplementary Table S2 Oligonucleotides used in this work. name sequence (5'-3') nucleotide position/reference traN expression cloning
All primers without reference in the table were designed in this study.Supplementary TableS3Final concentration of double-stranded DNA oligos in the dilution series utilized for MST measurements.Supplementary TableS4Transfer rates obtained in biparental mating assays.
mating A:

testing single point mutations in putative TraN key residues
Supplementary TableS5Alignment of predicted structures of TraN variants with TraN showing the number of aligned atoms, RMSD values and the color code of Supplementary FigureS3.In silico digest of Tra proteins showing the resulting peptides for MS analysis.Putative TraNpIP501 homologs encoded on plasmids or transposons.TraN homologs were detected via a tBLASTn search using the NCBI database.Search criteria are described in detail in 2.12 in the main text.Percentage amino acid identities were calculated with the BLAST algorithm.
*mean values are given with standard deviation (±SD).Transfer rates are given as the number of transconjugants per recipient cell.n= 3. Mating A corresponds to Figure1B, mating B to Figure1C.c Rep_3 family (repUS35) and d for Rep_trans family (rep7a).Unlabeled plasmids contain a predicted oriT and a complete pIP501-like T4SS (TraA