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. 2013 Nov;195(22):4999-5006.
doi: 10.1128/JB.00367-13. Epub 2013 Aug 30.

A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

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Free PMC article

A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

Anabel Alperi et al. J Bacteriol. .
Free PMC article

Abstract

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.

Figures

Fig 1
Fig 1
Known translocation signals in T4SS substrates. The T4SS is indicated on the left. Substrate features determined as relevant for secretion are indicated as follows. Gray boxes, C-terminal TSs, including a positively charged secretion motif (R-X7-R-X-R-X-R-X-Xn) (4, 29, 30); vertically hatched boxes, BID domains (13); horizontally hatched boxes, N-terminal and C-terminal regions of H. pylori CagA (12); diagonally hatched boxes, TS-containing regions in relaxases, as determined by CRAfT assays (9, 10); black boxes, consensus TraI/F sequences (G[E/D]R[L/M]R[V/F]T) (10).
Fig 2
Fig 2
(A) ClustalO alignment of TSs determined for R1/F-TraI and R1162-MobA against the whole R388-TrwC and pKM101-TraI sequences. The asterisks indicate positions that have a single, fully conserved residue; the colons indicate conservation between groups of strongly similar properties. (B) The protein secondary-structure prediction was obtained using Phyre2 for RecD2; wild-type TrwC; and TrwC variants TS1*, TS2*, and TS1* plus TS2*. To simplify the results, the consensus secondary structures are represented by showing the amino acid sequences and the β-strands (arrows) or α-helices (cylinders).
Fig 3
Fig 3
Conjugative DNA transfer mediated by TrwC derivatives. Donor bacteria contained the plasmid coding for the Dtr region of R388 (oriT-trwABC) and a helper plasmid (pSU4058) to provide R388 T4SS. The TrwC variant in each construct is indicated at the bottom: TrwC*, a variant with different C-terminal 32 residues; double colons indicate domains fused to the C terminus; variants in TS motifs are indicated with asterisks (see the text for details). DNA transfer is expressed as the number of transconjugants per donor. The bars represent means of 5 independent experiments. The error bars indicate standard errors of the mean. Student's t test was used to analyze the data referred to the positive control, TrwC. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001.
Fig 4
Fig 4
Percentages of eGFP-positive EA.hy926 cells infected by B. henselae carrying the indicated TrwC variant compared with its own positive control. The TrwC derivative in each construct is indicated at the bottom, as in Fig. 3. The data shown come from the subset of experiments in which the particular construct was assayed in parallel with its positive control. The bars represent means from at least 3 independent experiments done in triplicate. The error bars indicate standard errors of the mean. Student's t test was used to analyze the data referred to the positive control, TrwC. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001.

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