Identification of putative essential protein domains from high-density transposon insertion sequencing

Flyn Gallardo,Silvia T Cardona,Lukas Timmerman,A S M Zisanur Rahman

doi:10.1038/s41598-022-05028-x

Flyn Gallardo, Silvia T Cardona + Show 2 more

Open Access

https://doi.org/10.1038/s41598-022-05028-x

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Journal: Scientific Reports	Publication Date: Jan 19, 2022
Citations: 3	License type: open-access

Affiliation: University of Manitoba

Abstract

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term “essential” to whole genes rather than the protein domain sequences that encode the essential functions. However, genes can code for multiple protein domains that evolve their functions independently. Therefore, when essential genes code for more than one protein domain, only one of them could be essential. In this study, we defined this subset of genes as “essential domain-containing” (EDC) genes. Using a Tn-seq data set built-in Burkholderia cenocepacia K56-2, we developed an in silico pipeline to identify EDC genes and the essential protein domains they encode. We found forty candidate EDC genes and demonstrated growth defect phenotypes using CRISPR interference (CRISPRi). This analysis included two knockdowns of genes encoding the protein domains of unknown function DUF2213 and DUF4148. These putative essential domains are conserved in more than two hundred bacterial species, including human and plant pathogens. Together, our study suggests that essentiality should be assigned to individual protein domains rather than genes, contributing to a first functional characterization of protein domains of unknown function.

Highlights

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential
We operationally defined this subclass of essential genes as “essential domain-containing” (EDC) genes (Fig. 1c, d) and present a computational pipeline to identify them in a Tn-seq dataset built-in Burkholderia cenocepacia K56-217
Using a CRISPR Interference (CRISPRi)[18] platform we developed for Burkholderia[19], we experimentally confirmed growth defects, representing the loss of a putative essential function, in 27 EDC gene knockdowns

Summary

Introduction

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. Essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq) These studies assign the term “essential” to whole genes rather than the protein domain sequences that encode the essential functions. Tn-seq analysis may classify a gene as “non-essential” due to the presence of transposon insertions in a non-essential coding region, despite the gene coding for a second domain not spanning through the whole gene length that might be e ssential[3,15,16] We operationally defined this subclass of essential genes as “essential domain-containing” (EDC) genes (Fig. 1c, d) and present a computational pipeline to identify them in a Tn-seq dataset built-in Burkholderia cenocepacia K56-217. This study highlights that gene essentiality depends on the function of individual protein domains rather than entire proteins

Methods

Results

Conclusion