IncI shufflons: Assembly issues in the next-generation sequencing era

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• The shufflon is a site-specific recombination system in IncI1 plasmids, notably R64.
• It generates diversity in the PilV protein's C-terminus via rearrangement and inversion of its segments by the Rci recombinase.
• PilV is crucial for bacterial conjugation, potentially influencing recipient cell specificity.

Purpose of the Study:

• To address challenges in assembling IncI1 shufflon sequences, particularly with short-read sequencing technologies.
• To investigate the conservation and variability of shufflon segments across different IncI1 plasmids.
• To evaluate the biological relevance of reported shufflon segment arrangements.

Main Methods:

• Analysis of IncI1 sequence data from Roche-454 and Illumina platforms.
• Comparative analysis of shufflon segments from plasmids in The Netherlands, Australia, and GenBank.
• Utilizing plasmid multi-locus sequencing typing (PMLST) for plasmid grouping.

Main Results:

• Difficulties in assembling the shufflon region using standard short-read sequencing methods were observed.
• The number of shufflon segments appears conserved within plasmid groups defined by PMLST.
• Shufflon segment sequences are highly conserved, with minimal nucleotide variation.
• Reported variations in shufflon segment arrangements may not be biologically significant.

Conclusions:

• The shufflon's repetitive nature complicates plasmid assembly, necessitating specialized approaches.
• Despite potential arrangement variability, the core shufflon segment count and sequences are conserved within IncI1 plasmid lineages.
• The biological significance of observed shufflon arrangement differences requires further investigation.