TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance

Yasir, Muhammad; Turner, A. Keith; Bastkowski, Sarah; Baker, David; Page, Andrew J.; Telatin, Andrea; Phan, Minh-Duy; Monahan, Leigh G.; Savva, George M.; Darling, Aaron E.; Webber, Mark A.; Charles, Ian G.

doi:10.1101/gr.254391.119

Cited by 41 publications

(80 citation statements)

References 38 publications

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“…We recently described the construction of a high-density (insert approximately every 6 bp) TraDIS-Xpress library in E. coli BW25113, which was used in this work. 24 BW25113 is a commonly used reference strain, a full and well-annotated genome sequence is available, and it was the parent strain for the KEIO collection of defined insertion mutants, thus enabling easier validation of the roles of candidate genes. 24 The transposon used {a mini-Tn 5 transposon coding for kanamycin resistance [ aph(3′)-Ia ]} incorporates an outward-transcribing tac promoter 3′ of the kanamycin cassette that is inducible by IPTG, allowing over-expression or repression of genes adjacent to insertion sites (depending on insert orientation) as well as gene inactivation.…”

Section: Methodsmentioning

confidence: 99%

“… 24 BW25113 is a commonly used reference strain, a full and well-annotated genome sequence is available, and it was the parent strain for the KEIO collection of defined insertion mutants, thus enabling easier validation of the roles of candidate genes. 24 The transposon used {a mini-Tn 5 transposon coding for kanamycin resistance [ aph(3′)-Ia ]} incorporates an outward-transcribing tac promoter 3′ of the kanamycin cassette that is inducible by IPTG, allowing over-expression or repression of genes adjacent to insertion sites (depending on insert orientation) as well as gene inactivation. This allows the roles of essential genes in response to a stress to be analysed based on expression changes; traditionally these loci have been cryptic in TraDIS experiments as insertions within them are lethal.…”

Section: Methodsmentioning

confidence: 99%

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A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems

Turner

Yasir

Bastkowski

et al. 2020

Journal of Antimicrobial Chemotherapy

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Background Fosfomycin is an antibiotic that has seen a revival in use due to its unique mechanism of action and efficacy against isolates resistant to many other antibiotics. In Escherichia coli, fosfomycin often selects for loss-of-function mutations within the genes encoding the sugar importers, GlpT and UhpT. There has, however, not been a genome-wide analysis of the basis for fosfomycin susceptibility reported to date. Methods Here we used TraDIS-Xpress, a high-density transposon mutagenesis approach, to assay the role of all genes in E. coli involved in fosfomycin susceptibility. Results The data confirmed known fosfomycin susceptibility mechanisms and identified new ones. The assay was able to identify domains within proteins of importance and revealed essential genes with roles in fosfomycin susceptibility based on expression changes. Novel mechanisms of fosfomycin susceptibility that were identified included those involved in glucose metabolism and phosphonate catabolism (phnC-M), and the phosphate importer, PstSACB. The impact of these genes on fosfomycin susceptibility was validated by measuring the susceptibility of defined inactivation mutants. Conclusions This work reveals a wider set of genes that contribute to fosfomycin susceptibility, including core sugar metabolism genes and two systems involved in phosphate uptake and metabolism previously unrecognized as having a role in fosfomycin susceptibility.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems

Turner

Yasir

Bastkowski

et al. 2020

Journal of Antimicrobial Chemotherapy

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“…The construction of the TraDIS library in the ST131 clone EO499, using a mini-Tn5 transposon encoding chloramphenicol resistance, has been described [ 37 ]. The library has approximately 450,000 unique insertion sites across the genome (5.4 Mba), an average density of 1 insertion every twelve bases.…”

Section: Methodsmentioning

confidence: 99%

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions

Bushell

Herbert

Sannasiddappa

et al. 2020

Genes

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Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism’s ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

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“…A customised sequencing library was prepared to identify transposon insertions. DNA was tagmented using a MuSeek DNA fragment library preparation kit (ThermoFisher) and customised Tn5-i5 and i7 primers were used in PCR for 28 cycles 33 . DNA fragments of 300-500 bp were size selected using AMPure beads (Beckman Coulter) and nucleotide sequences were generated using a NextSeq 500 and a NextSeq 500/550 High Output v2 kit (75 cycles) (Illumina).…”

Section: Tradis-xpress Sequencingmentioning

confidence: 99%

Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation inEscherichia coli

Holden

Yasir

Turner

et al. 2020

Preprint

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Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to seed biofilms in new environments. Progression through this life cycle requires controlled temporal gene expression to maximise fitness at each stage. Previous studies have focused on the essential genome for the formation of a mature biofilm, but here we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress; a massively parallel transposon mutagenesis approach using transposon-located promoters to assay expression of all genes in the genome. We determined how gene essentiality and expression affects the fitness of E. coli growing as a biofilm on glass beads after 12, 24 and 48 hours. A selection of genes identified as important were then validated independently by assaying biofilm biomass, aggregation, curli biosynthesis and adhesion ability of defined mutants. We identified 48 genes that affected biofilm fitness including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points. Adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes including some which were both beneficial and detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included ychM and truA involved in cell division, crfC and maoP in DNA housekeeping and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.

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TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance

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Cited by 41 publications

References 38 publications

A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems

A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions

Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation inEscherichia coli

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