The design and analysis of transposon insertion sequencing experiments

Chao, Michael C.; Abel, Sören; Davis, Brigid M.; Waldor, Matthew K.

doi:10.1038/nrmicro.2015.7

Cited by 188 publications

(213 citation statements)

References 62 publications

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“…The combined read counts per gene were further normalized per million mapped reads to account for the differences in sizes of mapped reads among sequenced libraries. Then a genomic location bias correction was performed in each library as described by (16). During bacterial growth and replication, the amount of DNA close to the origin of replication (Ori) increases, which results in more DNA around the Ori becoming available for sequencing.…”

Section: Methodsmentioning

confidence: 99%

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum

Sanchez-Larrayoz

Elhosseiny

Chevrette

et al. 2017

The Journal of Immunology

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Acinetobacter baumannii is a bacterial pathogen with increasing impact in healthcare settings, due in part to this organism’s resistance to many antimicrobial agents, with pneumonia and bacteremia as the most common manifestations of disease. A significant proportion of clinically-relevant A. baumannii strains are resistant to killing by normal human serum (NHS), an observation supported here by showing that 12 out of 15 genetically diverse strains of A. baumannii are resistant to NHS killing. To expand our understanding of the genetic basis of A. baumannii serum resistance, a transposon-sequencing (Tn-seq) approach was used to identify genes contributing to this trait. An ordered Tn-library in strain AB5075 with insertions in every non-essential gene was subjected to selection in NHS. We identified 50 genes essential for the survival of A. baumannii in NHS, including already known serum resistance factors, and many novel genes not previously associated with serum resistance. This latter group included the maintenance of lipid asymmetry (mla) genetic pathway as a key determinant in protecting A. baumannii from the bactericidal activity of NHS via the alternative complement pathway. Follow up studies validated the role of 8 additional genes identified by Tn-seq in A. baumannii resistance to killing by NHS but not by normal mouse serum, highlighting the human-species specificity of A. baumannii serum resistance. The identification of a large number of genes essential for serum resistance in A. baumannii indicates the degree of complexity needed for this phenotype, which might reflect a general pattern pathogens rely on to cause serious infections.

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Section: Methodsmentioning

confidence: 99%

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum

Sanchez-Larrayoz

Elhosseiny

Chevrette

et al. 2017

The Journal of Immunology

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“…However, some mutants may behave differently in a pool where there can be both competition as well trans complementation than when grown individually. Secondly, technically, transposon libraries are often constructed with an initial isolation on a rich medium and then subjected to a selection for growth on the condition of interest (often a more minimal media or media that models the host [13]. Because the isolation step is in fact a selection on rich media, genes that are essential in rich media cannot be evaluated in this way.…”

Section: Discussionmentioning

confidence: 99%

“…The different tools can vary dramatically both in the assumptions built into the analysis and how conservatively each model calls essentiality i.e., whether one is more willing to tolerate false positives or false negatives. For example, a Hidden Markov Model (HMM) and sliding window approach rely on a stretch of TA sites that have zero to very low level insertions to denote an essential gene [13,31]. An advantage of these methods is that intergenic regions and essential domains within a larger gene can be queried.…”

Section: Discussionmentioning

confidence: 99%

“…We next sought to perform a comprehensive and quantitative analysis of our 90 Tn-Seq datasets. However, while many methods exist for analyzing Tn-Seq data [13,[30][31][32], significant variation exists in the complexity of these methods and how conservative they are in calling a gene essential. Additionally, because of their complexity, many of them require implicit assumptions that may have contributed to their widely varying predictions of which genes are essential when applied to our datasets (data not shown).…”

Section: Poulsen Be Et Almentioning

confidence: 99%

“…Taking advantage of massively parallel sequencing, methods have emerged (Tn-Seq; also known as TIS, INseq, HITS, TraDIS, [13][14][15][16][17]) for performing genome-wide negative selections studies to quantitatively measure the relative fitness of a pool of mutants. Using transposon insertion sequencing, genes which are required for optimal growth under a specific growth condition can be identified by mapping the gene location of the disrupting transposon in fitness impaired mutants; at an extreme, mutants that cannot be detected at all in the pool correspond to genes that are essential under that condition.…”

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confidence: 99%

See 2 more Smart Citations

Defining the core essential genome ofPseudomonas aeruginosa

Poulsen

Yang

Clatworthy

et al. 2018

Preprint

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Pseudomonas aeruginosa is a clinically significant pathogen that has alarming antibiotic resistance rates and very few candidate drugs in development. The challenges of novel drug discovery are exacerbated by incomplete knowledge of the essential genes across the species required for survival under infection conditions. We thus sought to define the core essential genome of P. aeruginosa by performing transposon insertion sequencing (Tn-Seq) on nine strains of P. aeruginosa isolated from different infection sites including wound, eye, lung, urinary tract, and blood, with an environmentally isolated strain for comparison, in five different media conditions: three were infection relevant media (serum, sputum, urine) and two were lab-based media (LB and M9 minimal). We developed a novel statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain-media combinations. FiTnEss required minimal assumptions and had good predictive power in a limited set of validation studies with mutant strains of PA14 containing clean gene deletions. A core set of 321 essential genes emerged that are the highest probability targets for successful novel drug discovery against this important pathogen.

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Biochemical and structural characterization reveals Rv3400 codes for β‐phosphoglucomutase in Mycobacterium tuberculosis

Singh,

Karthikeyan,

Thakur

2024

Protein Science

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Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β‐phosphoglucomutase (β‐PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β‐PGM activity and hence, Rv3400 encodes for β‐PGM in Mtb. Our data also confirm that Mtb β‐PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β‐PGM converts β‐D‐glucose‐1‐phosphate to β‐D‐glucose‐6‐phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β‐PGM in the physiology of Mtb.

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The design and analysis of transposon insertion sequencing experiments

Cited by 188 publications

References 62 publications

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum

Defining the core essential genome ofPseudomonas aeruginosa

Biochemical and structural characterization reveals Rv3400 codes for β‐phosphoglucomutase in Mycobacterium tuberculosis

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