Genome-Scale Metabolic Network Validation of Shewanella oneidensis Using Transposon Insertion Frequency Analysis

Abstract

Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

“…This estimate is larger than the 3,355 non-essential genes reported by Deutschbauer et al 13. due to a larger progenitor collection, and understandably larger than the 2,216 reported by Yang et al 28. and the 3,030 gene fitness values provided by Brutinel et al 27.…”

“…There will likely be some non-essential genes into which transposon insertion is a low to zero probability event due to the scarcity or absence of the insertion site (NE2) (an AT or TA dinucleotide for the mariner system used here52). Conversely, there will be a small number of essential genes that can accept transposon insertions, for instance at the end of the coding region28 (E1), whereas insertion at any point in most will be fatal (E2). Finally, there will likely be intergenic regions in which the transposon insertion site is scarce or absent or in which insertion will be fatal (I2).…”

“…Recently developed techniques that use next-generation sequencing (NGS) of pooled transposon mutant libraries232425 have made dramatic advances in the characterization of genes that can be selected for fitness contributions26 to species including S. oneidensis 132728. Nevertheless, clonally isolated collections of mutants remain critically important for the characterization of phenotypes such as virulence factors2930, secondary and cryptic metabolite production31 and behaviours synonymous with Shewanellae, such as biofilm formation3233 and EET34.…”

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

“…For example, the relative lack of mutants in a particular genetic element in a high-density transposon library can indicate the essentiality of that element. Previous studies have used several criteria to determine the immutable regions of a bacterial genome from Tn-seq data in the absence of control conditions, including the prevalence of transposon insertions detected per genetic element or the probability of encountering a DNA segment with no insertions given that segment's length (15)(16)(17). However, these methods do not always account for two types of information available in Tn-seq data: (i) the abundance of each transposon mutant and (ii) the variability observed in Tn-seq biological replicates.…”

Sequence-based Network Completion Reveals the Integrality of Missing Reactions in Metabolic Networks

“…The four organisms were selected because the associated gene knock-out libraries were generated through full-length gene deletion methods rather than transposon insertion methods. Transposon knockouts can display complex gene knockdown behavior that complicates the interpretation of gene essentiality predictions (44). The gap-filling reactions added by the Model SEED were stripped from the downloaded models.…”