Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms

Opijnen, Tim van; Lazinski, David W.; Camilli, Andrew

doi:10.1002/0471142727.mb0716s106

Cited by 62 publications

(60 citation statements)

References 44 publications

(58 reference statements)

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“…S1AB ) . The presence of Mme I sites allows for digestion of genomic DNA containing Krmit transposon insertion sites (TIS), producing uniform 20-nt regions of adjacent chromosomal DNA (insertion tags) for next-generation sequencing and Tn-seq screens 31 . Initial tests for in vivo transposition using pKRMIT in a GAS serotype M1T1 strain revealed that Krmit transposed comparably to Oskar 28 29 , exhibiting an average transposition frequency of 4 × 10 −3 with insertions occurring exclusively within the dinucleotide TA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The complexity of the Krmit mutant libraries in M1T1 5448 was examined by Tn-seq 26 31 with modifications for its use in GAS with Krmit . Briefly, genomic DNA was isolated from the different mutant pools (T 0 to T 3 ), subjected to complete digestion by Mme I followed by ligation to Mme I adapters, and PCR amplification to produce DNA-seq libraries consisting of 176-bp Krmit -specific insertion tags containing adjacent genomic sequence, Illumina-specific sequences and one of 8 distinct barcodes for multiplexing.…”

Section: Resultsmentioning

confidence: 99%

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Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

Breton

Belew

Valdes

et al. 2015

Sci Rep

117

143

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Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

Breton

Belew

Valdes

et al. 2015

Sci Rep

117

143

View full text Add to dashboard Cite

show abstract

“…Given the global changes effected by RpoS in stationary phase (24), we wished to know which RpoS-regulated genes protected cells from SDS in stationary phase. In order to address this question, we utilized a transposon mutant pool sequencing (Tn-Seq) experiment (46). We created a pooled library of approximately 190,000 mutants, each containing an EZ-Tn5 insertion, grew this library overnight to stationary phase under either nitrogen-limiting or carbon-limiting conditions, and collected genomic DNA samples from these cultures before and after 24 h of 2% SDS treatment (Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

2017

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Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.KEYWORDS Escherichia coli, RpoS, efflux pumps, outer membrane, permeability, stationary phase

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“…Tn mutant libraries were characterized by Tn-seq, essentially as described previously (28, 29). Briefly, genomic DNA from Tn mutant libraries was isolated using the DNeasy blood and tissue kit (Qiagen) but, before the manufacturer's recommendations for Gram-negative bacteria were followed, cells were washed once with 1 M NaCl and once with PBS.…”

Section: Methodsmentioning

confidence: 99%

Essential Genes forIn VitroGrowth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing

Rosconi

Vries

Baig

et al. 2016

Appl Environ Microbiol

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The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle.IMPORTANCE A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.

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Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms

Cited by 62 publications

References 44 publications

Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

Essential Genes forIn VitroGrowth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing

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