A Noise Trimming and Positional Significance of Transposon Insertion System to Identify Essential Genes in Yersinia pestis

Abstract

Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.

High-throughput analysis of Yersinia pseudotuberculosis gene essentiality in optimised in vitro conditions, and implications for the speciation of Yersinia pestis

“…Interestingly, we detected five LysR/DeoR transcriptional regulators ( yptb1518 , yptb1927 , yptb2301, yptb2533 and yptb3075 ) uniquely essential to Y. pseudotuberculosis , suggesting a non-redundant role in metabolism that is of diminished importance in Y. pestis . Similar regulators have been reported in Y. pestis as being conditionally upregulated in the flea, including rovM [ 63 ] and yfbA , which is required for biofilm formation and colonisation of the insect vector [ 64 ], but no such regulators were required for in vitro growth according to the published gene set [ 26 ].…”

“…We identified a similar number of essential in vitro growth genes in Y. pseudotuberculosis as those found in Y. pestis by Yang et al [ 26 ], and most of these genes were orthologous. These represent conserved genes whose importance has remained vital despite substantial genomic divergence by Y. pestis ; they include genes encoding ribosomal proteins, DNA polymerase and topoisomerase.…”

“…By comparing our findings with the published Y. pestis in vitro essential genes [ 26 ], cultured using identical methods, we were able to identify shared and contrasting essential genes between the species (Additional file 2 : Table S2). A Venn diagram showing selected shared and unique essential genes between Y. pseudotuberculosis and Y. pestis highlights some prominent trends, such differences relating to iron acquisition and oxidative phosphorylation (Fig.…”

“…Some artefact genes were identified such as those encoding flagellar proteins, mutation of which may lower competitive fitness in liquid broth conditions without being true essential genes. Similarly, some genes have been reported to tolerate insertions at the three-prime region without complete loss of function [ 26 ]. While the risk of naming false essential genes can be reduced through adequate library complexity, biological replicates and the use of insertion-index scores, we acknowledge a margin of error inherent in any TraDIS/Tnseq study.…”

“…In the present work, we demonstrate the efficacy of actinonin, a deformylase inhibitor, against Y. pseudotuberculosis based on the discovery that peptide deformylase, yptb3665 , is required for growth in vitro. Furthermore, we exploit the recently published Y. pestis essential gene data [ 26 ] and compare this to our Y. pseudotuberculosis TraDIS. This allowed us to contrast the differing importance of genes required for growth of either Yersinia spp.…”