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

Abstract

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.

Keywords: DNA binding proteins; Enterococcus faecalis; antibiotic resistance; conjugative transfer; pIP501; transfer regulator; type IV secretion system.

FIGURE 1

Biparental mating assays of pIP501, pIP501∆traN, and pIP501∆traN substituted with traN and traN carrying point mutations in the putative key residues listed below. E. faecalis JH2-2 (pIP501 isogenic wild type (obtained during generation of the traN knockout)), E. faecalis JH2-2 (pIP501∆traN) and E. faecalis JH2-2 (pIP501∆traN and pEU327-RBS-traN) were used as reference. (A) Crystal structure of TraN in complex (PDB: 6G1T) with original binding site (oBS). Mutated residues labeled and shown as sticks. (B) Complementation strains of traN knockout expressing single-substituted traN variants in trans (pEU327-RBS-traN_Q28A, pEU327-RBS-traN_H82A, pEU327-RBS-traN_K101A) and (C) complementation strains of traN knockout expressing multiple-substituted traN variants in trans (pEU327-RBS-traN_R23A-N24A-Q28A, pEU327-RBS-traN_G47A-G48A, pEU327-RBS-traN_H82A-R86A, and pEU327-RBS-traN_G100A-K101A) were applied as donors, E. faecalis OG1X as recipient. Transfer frequencies are presented as number of transconjugants per recipient cell. n = 3. Mean values are depicted with standard deviation. **p < 0.01, *p < 0.1 as determined by the Mann–Whitney U test.

FIGURE 2

Coomassie-stained 12% SDS polyacrylamide gels of expression and purification of TraN and its variants. kDa: Pierce™ Unstained Protein molecular weight marker (Thermo Fisher). The expected molecular weight of all constructs ranges from 17.10 kDa (TraN_R23A-N24A-Q28A) to 17.32 kDa (TraN_G47A-G48A).

FIGURE 3

TraN fold seems unaffected in TraN variants, TraN_R23A-N24A-Q28A, TraN_G47A-G48A, TraN_H82A-R86A, and TraN_G100A-K101A. (A) Circular dichroism (CD) spectra of His-TraN and its variants. (B) Structural alignment of the crystal structure of TraN and the predicted structures of its variants showing that the substitutions do not have an impact on the protein fold. TraN is depicted in purple. The residues mutated in the variants are shown as green sticks. C- and N-termini are labeled. The color code for the TraN variants is shown in Supplementary Table S5. (C) Close-up of the wing motif of the C-terminal wHTH motif (TraN_G100A-K101A). The alanines likely protrude into the minor groove upon DNA binding. (D) Close-up of motif 1. The mutated residues of TraN_R23A-N24A-Q28A are shown as light pink sticks. (E) Close-up of the wing motif of the N-terminal wHTH motif (TraN_G47A-G48A). Introduced alanine residues likely protrude into the minor groove upon DNA binding. (F) Close-up of motif 2 (TraN_H82A-R86A).

FIGURE 4

Graphical representation of TraN and its variants interacting with original binding site (oBS) and random ds-oligonucleotide (random DNA). (A) Cartoon representation of class (I) interactions between amino acid side chains and corresponding bases of oBS. The images were taken from the structure of TraN bound to oBS pIP501 DNA (PDB: 6G1T). Interacting residues are highlighted in cyan and respective bases of the oBS are highlighted in green. Water molecules are depicted in dark blue, hydrogen bonds in light blue. Normalized dose-response curves of TraN and its variants for interactions with oBS (red) and unspecific random DNA (green) acquired from MST binding assays. Wherever the fit was successful, the resulting curves exhibit a sigmoidal shape from which a dissociation constant (K_d) was calculated. (B) MST-derived dissociation constants (K_d) of TraN and its variants to oBS and random DNA.

FIGURE 5

Western blots of the elution fractions of the pull-down with TraN-Strep. The following antibodies were used: (A) anti-TraB, (B) anti-TraE, (C) anti-TraF, (D) anti-TraG, (E) anti-TraH, (F) anti-TraJ, (G) anti-TraK, (H) anti-TraM, (I) anti-TraN, and (J) anti-TraO, targeting the respective Tra proteins. The MW of the respective protein is indicated above each blot. MW marker (Blue Prestained Protein Standard, New England Biolabs) was included to assess protein sizes (kDa). Three fractions of the TraN-based pull-down were analyzed: lysate (Lys) in lane 1, flow-through (FT) in lane 2, and elution fraction (EF) in lane 3. *14.4 kDa is the MW of native TraN. The fused Strep-tag adds 1 kDa; therefore, the protein appears slightly larger than in the immunoblots performed with native TraN.

FIGURE 6

Western blots of the pull-down with GFP-Strep. The cell lysate (Lys) in lane 1, the flow-through (FT) in lane 2, and the elution fraction (EF) in lane 3 were analyzed. (A) Anti-TraN and (B) anti-TraF antibodies were used as primary antibodies. The used antibody and the MW of the respective Tra protein are given above each blot. MW marker (kDa) (Blue Prestained Protein Standard, New England Biolabs) was applied.

FIGURE 7

Alignments of TraN_pIP501 and its homologs. (A) Multiple sequence alignment using Expresso algorithm implemented in T-Coffee. Homologs were identified by tBLASTn search. As these homologs are hypothetical, the names of the plasmids encoding the TraN_pIP501 homolog are used instead of protein annotation. Asterisks (*) represent conserved amino acids in all aligned sequences, (:) indicates amino acids of high similarity, dots (.) indicate partial similarities. The top panel shows the evaluation scores from the T-Coffee Server (TCS) evaluation. The closer the score to 100, the better the alignment. The quality of the alignment is also represented in red. (B) Backbone alignment of TraN_pIP501 (purple) with the homolog from p3-38 (cyan) using theoretical structures predicted with coLAB AlphaFold2 server. Residues (Arg23, Asn24, Gln28, Gly47, Gly48, His82, Arg86, Gly100, and Lys101) are shown as green sticks.

FIGURE 8

Structural alignment of TraN_pIP501 (PDB: 4P0Z) with (A) Tra20_pNP40, (B) TrsR_pUC11B, (C) TrsR_pAF22, (D) TrsR_p001F, and (E) Orf5_pMRC01. TrsR_pUC08B is 100% identical to TrsR_pUC11B, and TrsR_pIBB477a to Orf5_pMRC01. (A–E) Homologs are shown in rainbow, colored by pLDDT (per-residue local distance difference test) score (red = high pLDDT = high confidence; blue = low pLDDT = low confidence), TraN_pIP501 in purple blue. All aligned proteins resemble the C-terminal half of TraN_pIP501.