Defining the core essential genome of<i>Pseudomonas aeruginosa</i>

Poulsen, Bradley E.; Yang, Rui; Clatworthy, Anne E.; White, T. L.; Osmulski, Sarah J.; Li, Li; Peñaranda, Cristina; Shoresh, Noam; Hung, Deborah T.

doi:10.1101/396689

Cited by 8 publications

(7 citation statements)

References 61 publications

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“…All sequencing read count data and analysis files are available (https://data.broadinstitute.org/fitness/) and on the NCBI Sequence Read Archive (65). The analysis pipeline, including sequencing summarization and the FiTnEss software, is freely available (https://github.com/broadinstitute/FiTnEss) (66).…”

Section: Methodsmentioning

confidence: 99%

Defining the core essential genome of Pseudomonas aeruginosa

Poulsen

Yang

Clatworthy

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

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Section: Methodsmentioning

confidence: 99%

Defining the core essential genome of Pseudomonas aeruginosa

Poulsen

Yang

Clatworthy

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

158

175

View full text Add to dashboard Cite

show abstract

“…This kind of in-depth investigation can ultimately help to uncover how bacterial species, particularly diverse species that include both pathogens and non-pathogens, can become harmful or antibiotic resistant, for example via horizontal transfer of pathogen-associated genetic material. TIS in nine strains of P. aeruginosa determined the core essential genome in five media, and highlighted that essentiality of some genes depends on genomic context 90 . The influence of genetic background on phenotype is further illustrated by examples that highlight how genes involved in responding to antibiotic stress can be strain specific.…”

Section: Nature Reviews | Geneticsmentioning

confidence: 99%

A decade of advances in transposon-insertion sequencing

et al. 2020

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It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and highthroughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.

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“…to establish growth‐decoupling switches for bioproduction; Durante‐Rodríguez et al , ) or for the assessment of fundamental questions in metabolism (e.g. related to gene essentiality; Poulsen et al , ). As an example, we applied the versatile CRISPRi system described in this study to control the expression of the conditionally essential gene pyrF .…”

Section: Discussionmentioning

confidence: 99%

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Batianis

Kozaeva

Damalas

et al. 2020

Microbial Biotechnology

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Summary Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

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Defining the core essential genome ofPseudomonas aeruginosa

Cited by 8 publications

References 61 publications

Defining the core essential genome of Pseudomonas aeruginosa

Defining the core essential genome of Pseudomonas aeruginosa

A decade of advances in transposon-insertion sequencing

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

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