PIRT-Seq: a high-resolution whole-genome assay to identify protein-coding genes

Area of Science:

• Genomics
• Molecular Biology
• Microbial Genetics

Background:

• Bacterial genomes possess undiscovered coding potential, particularly in small or overlapping genes.
• Existing protein detection methods struggle to identify these overlooked coding sequences.
• High-density mutagenesis and data-mining hint at a vast, unannotated proteome.

Purpose of the Study:

• To develop and apply a novel genetics-based method for large-scale identification of translated open reading frames.
• To overcome limitations of current protein detection and genome annotation techniques.
• To discover novel protein-coding sequences (CDS) in Escherichia coli, independent of genome annotation.

Main Methods:

• Combined transposon insertion sequencing with a dual-selection transposon and a translation reporter.
• Applied the method to Escherichia coli to identify translated open reading frames genome-wide.
• Protein Identification through Reporter Transposon-Sequencing (PIRT-Seq) was developed and utilized.

Main Results:

• Identified over 200 putative novel protein-coding sequences (CDS) in E. coli.
• The majority of newly identified CDS are short (less than 50 amino acids).
• Found conserved CDS located near functionally important genes, with validated expression via chromosomal tags.

Conclusions:

• PIRT-Seq is a powerful, high-throughput method for discovering unannotated bacterial genes and conditional gene expression.
• This technique complements existing methods like proteomics and ribosome trapping for identifying condition-dependent protein CDS.
• PIRT-Seq provides a foundation for future high-throughput genetic studies to explore unannotated genes across bacterial species.