Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB

Abstract

The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens1,2,3. Although aspects of IncC plasmid conjugation have been well studied4,5,6,7,8,9, many roles of conjugation genes have been assigned based solely on sequence similarity. We applied hypersaturated transposon mutagenesis and transposon-directed insertion-site sequencing to determine the set of genes required for IncC conjugation. We identified 27 conjugation genes, comprising 19 that were previously identified (including two regulatory genes, acaDC) and eight not previously associated with conjugation. We show that one previously unknown gene, acaB, encodes a transcriptional regulator that has a crucial role in the regulation of IncC conjugation. AcaB binds upstream of the acaDC promoter to increase acaDC transcription; in turn, AcaDC activates the transcription of IncC conjugation genes. We solved the crystal structure of AcaB at 2.9-Å resolution and used this to guide functional analyses that reveal how AcaB binds to DNA. This improved understanding of IncC conjugation provides a basis for the development of new approaches to reduce the spread of these multi-drug-resistance plasmids.

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Fig. 1: TraDIS analysis of IncC plasmid conjugation genes.
Fig. 2: AcaB activates conjugation in an AcaDC-dependent manner.
Fig. 3: AcaB is a DNA-binding protein.
Fig. 4: Structural and functional analysis of AcaB.

Data availability

TraDIS read data have been deposited in the Sequence Read Archive: pre-conjugation-1 (SRR3990757), pre-conjugation-2 (SRR3990758), post-conjugation-1 (SRR8271039) and post-conjugation-2 (SRR8271040). The complete sequence of plasmid pMS6198A is available from Genbank (accession no. CP015835.1). Genbank accession numbers for other IncC plasmids are as follows: pSTY1-2010K-1587 (CP016864.1), pKAZ5 (KR827394.1) and pVCR94deltaX (KF551948.1). Sanger sequencing data confirming the identity of all plasmid constructs are available from the authors upon reasonable request. The structures have been deposited in the Protein Data Bank (PDB ID 6N8B for native AcaB and PDB ID 6N8A for the SeMet-containing protein). Source data are provided with this paper.

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Acknowledgements

We acknowledge the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland, the Institute for Molecular Bioscience Sequencing Facility and the Australian Synchrotron MX beamlines, part of ANSTO, and we made use of the Australian Cancer Research Foundation detector. This work was supported by Project (nos. GNT1106590, GNT1033799 and GNT1067455) and Program (no. GNT1071659) grants from the Australian National Health and Medical Research Council (NHMRC). S.J.H. is supported by an Australian Government Research Training Program Scholarship. M.A.S. is supported by an NHMRC Senior Research Fellowship (no. GNT1106930), S.A.B. is supported by a NHMRC Career Development Fellowship (no. GNT1090456) and B.K. is supported by a NHMRC Principal Research Fellowship (no. GNT1110971) and an Australian Research Council Laureate Fellowship (no. FL180100109). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Contributions

S.J.H. and Z.L. performed experiments, with assistance from M.-D.P., A.W.L., K.M.P., N.T.K.N., B.M.F., J.W. and J.Y. S.J.H., Z.L., M.-D.P., B.K. and M.A.S. designed the study. S.J.H., Z.L., M.-D.P., B.M.F., J.W., J.Y., R.A.S., S.A.B., B.K. and M.A.S. analysed the data. M.-D.P., R.A.S., D.L.P., T.R.W., B.K., S.A.B. and M.A.S. supervised aspects of the project and provided essential expert analysis. All authors contributed to the interpretation of the results. S.J.H., Z.L., M.-D.P., B.K. and M.A.S. wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Minh-Duy Phan or Bostjan Kobe or Mark A. Schembri.

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Extended data

Extended Data Fig. 1 Schematic representation of the pMS6198A backbone.

Rings from inside-out represent: GC-content, GC-skew, notable DNA elements (green, AcaDC binding site7; red, origin of replication (oriV); blue, origin of transfer (oriT)), coding sequences (CDS) on reverse strand, and CDS on forward strand. CDS are colour-coded by function; red, replication; yellow, stability; blue, conjugation; green, conjugation regulation. Blue brackets show defined conjugation regions.

Extended Data Fig. 2 AcaB is activated by AcaDC.

a, qRT-PCR targeting acaB, standardised to repA. b, pQF50-PacaB in the presence/absence of AcaDC. Individual values of biological replicates are shown as dot points with the grey bar indicating the mean. All strains were assayed in biological triplicates. Statistical tests were performed on log10-transformed values using (a) One-way ANOVA and Sidak’s multiple comparisons and (b) two-sided unpaired t-test comparisons.

Extended Data Fig. 3 Electrophoretic mobility shift assay with AcaB showing binding specificity for Pacr1-567.

Competitive (comp) DNA was amplified from other regions of pMS6198A (409bp: 7975..8383 and 685bp: 7907..8591), see Supplementary Table 4. Experiment was repeated three times with similar results. No molecular weight ladder is present. Full unprocessed image provided as Source Data Fig. 2.

Extended Data Fig. 4 DNaseI footprinting analysis of AcaB bound to Pacr1, targeting the region -689 to -412 bp relative to acr1 ATG.

End-labelled primers were used to map the protected region in the presence of increasing concentrations of AcaB. Nucleotide sequencing (GA ladder) was performed using the same primers. The protected region is indicated, along with the corresponding DNA sequence. The experiment was performed once.

Extended Data Fig. 5 Superimposition of the two β-α-α units in AcaB.

Yellow: residues 23-97; cyan: residues 109-183. Residues are superimposed using Cα atoms.

Extended Data Fig. 6 Comparison of the AcaB structure to characterised RHH proteins.

The β-sheet region responsible for DNA-binding is shown in red.

Extended Data Fig. 7 Analyses of AcaB oligomerisation in solution.

a, SEC-MALS. Red and blue curves indicate the normalised UV traces of the WT protein and the E69A mutant, respectively. The dotted lines under the peaks are calculated average molecular mass using the same colour scheme. b, Glutaraldehyde and SDS-PAGE. Lane 1: AcaB with 0 % (v/v) glutaraldehyde; Lane 2: AcaB with 0.005 % (v/v) glutaraldehyde; Lane 3: AcaBE69A with 0 % (v/v) glutaraldehyde; Lane 4: AcaBE69A with 0.005 % (v/v) glutaraldehyde. Each experiment was repeated three times with similar results.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 1–5.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed immunolot.

Source Data Fig. 2

Unprocessed EMSA.

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Hancock, S.J., Phan, M., Luo, Z. et al. Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB. Nat Microbiol 5, 1340–1348 (2020). https://doi.org/10.1038/s41564-020-0775-0

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