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Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
Published on: January 4, 2017
TraN variants mediate conjugation species specificity of IncA/C, IncH, and Acinetobacter baumannii plasmids
Shan He1, Sophia David2, Jaie Rattle1
1Department of Life Sciences, Imperial College London, London, United Kingdom.
Abstract:
IncA/C and IncH plasmids commonly carry antimicrobial resistance genes, notably blaNDM-1. Although these plasmids disseminate among Gram-negative pathogens via conjugation, the mechanisms underlying mating pair stabilization (MPS) and conjugation species specificity remain poorly understood. In IncF plasmids, MPS is mediated by interactions between outer membrane proteins (OMP) encoded by the plasmids in the donor (TraN) and by the chromosome in the recipient. Using the Plascad database, we extracted 1,436 TraN sequences from 1,517 plasmids: 62.5% (898/1,436), mainly in IncF plasmids, are 550-660 amino acids (aa) (we renamed TraN short, TraNS); 15% (216/1,436), in IncA/C plasmids, are 880-950 aa (TraN medium, TraNM); and 11% (160/1,436), in IncH plasmids, are 1,050-1,070 aa (TraN long, TraNL). One TraN, found in six plasmids from Acinetobacter baumannii (891 aa), was designated TraN V-shaped (TraNV). Like TraNS, TraNM and TraNL contain a base and one distal tip domain essential for conjugation, whereas TraNV has a base and two distinct tip domains forming a V-shaped structure. TraNM, TraNL, and TraNV determine conjugation species specificity, with TraNL cooperating with OmpA. Tip swapping reverses conjugation specificity, revealing how TraNM and TraNL diversity influence plasmid host range and AMR dissemination. Our new data reveal the molecular basis of plasmid host specificity and broaden our understanding of how conjugation drives the dissemination of antimicrobial resistance genes among clinically relevant bacteria.
Importance:
Plasmid conjugation drives the spread of antimicrobial resistance genes between different bacterial species. In IncF plasmids, this process relies on tight interactions between an outer-membrane protein in the recipient and the plasmid-encoded TraN, which consists of conserved base and variable tip domains. So far, TraN was only studied in IncF plasmids. We show that IncA/C and IncH plasmids encode a larger TraN with distinct isoforms that shape host range and species specificity. We also identify a novel TraN variant in Acinetobacter baumannii plasmids containing a base and two tips. These findings broaden our understanding of conjugation specificity and the mechanisms that influence the dissemination of resistance genes across diverse bacterial communities and highlight the evolutionary flexibility of plasmid transfer systems.
Insights
Diverse plasmid TraN proteins mediate antimicrobial resistance gene spread. Larger TraN variants in IncA/C and IncH plasmids, including a novel V-shaped TraN in Acinetobacter baumannii, dictate host range and conjugation specificity.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Plasmid conjugation is a primary mechanism for the dissemination of antimicrobial resistance (AMR) genes among Gram-negative pathogens.
- Mating pair stabilization (MPS) and conjugation species specificity are crucial for efficient plasmid transfer but remain incompletely understood.
- In IncF plasmids, MPS involves interactions between recipient outer membrane proteins (OMPs) and donor plasmid-encoded TraN proteins.
Purpose of the Study:
- To investigate the diversity of TraN proteins beyond IncF plasmids, particularly in IncA/C and IncH plasmids.
- To elucidate the structural variations in TraN proteins and their impact on conjugation species specificity and host range.
- To identify novel TraN variants and understand their role in AMR gene dissemination.
Main Methods:
- Utilized the Plascad database to extract and analyze 1,436 TraN sequences from 1,517 plasmids.
- Classified TraN sequences into distinct size categories (TraNS, TraNM, TraNL, TraNV) based on amino acid length and plasmid type.
- Investigated the structural features of different TraN isoforms, including tip domain variations and their functional implications.
- Examined the role of TraNL in cooperating with OmpA and assessed the effect of tip swapping on conjugation specificity.
Main Results:
- Identified three main TraN size classes: TraNS (550-660 aa) in IncF plasmids, TraNM (880-950 aa) in IncA/C plasmids, and TraNL (1,050-1,070 aa) in IncH plasmids.
- Discovered a novel TraN variant, TraNV (891 aa), in *Acinetobacter baumannii* plasmids, characterized by a V-shaped structure with two distinct tip domains.
- Demonstrated that TraNM, TraNL, and TraNV are key determinants of conjugation species specificity, with TraNL interacting with OmpA.
- Showed that tip swapping experiments can reverse conjugation specificity, highlighting the plasticity of TraN in influencing plasmid host range.
Conclusions:
- The diversity in TraN protein structure, particularly in larger isoforms found in IncA/C, IncH, and novel variants like TraNV, significantly shapes plasmid host specificity.
- Variations in TraN tip domains are critical for determining conjugation species specificity and influence the dissemination of AMR genes.
- Understanding these TraN-mediated mechanisms provides crucial insights into the evolution of plasmid transfer systems and the spread of antibiotic resistance.
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