Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

Shiver, Anthony L.; Culver, Rebecca; Deutschbauer, Adam M.; Huang, Kerwyn Casey

doi:10.1038/s41596-021-00531-3

Cited by 27 publications

(33 citation statements)

References 22 publications

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“…First, our study was conducted in a defined medium to avoid introduction of molecules from another species into the 2-species system, such as culture media compositions that contain whole cell lysates, and it is important to consider that culture and growth conditions may affect either secreted molecule production and/or sensory responses. Next, the species being studied must have the necessary genetic tools available, such as reporter expression systems and random mutagenesis protocols, however these are becoming more widely accessible [94][95][96]. The identification of sensed products using our approach was more straightforward when there was a primary sensory molecule driving the response, such as in the case of StP, citrate, or acetoin, although fractionation of WT and mutant supernatants can reveal the molecules underlying multifactordriven responses as well.…”

Section: Plos Biologymentioning

confidence: 99%

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

Zarrella

Khare

2022

PLoS Biol

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Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter–reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these 2 species.

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Section: Plos Biologymentioning

confidence: 99%

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

Zarrella

Khare

2022

PLoS Biol

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“…Transposon sequencing of the transformants revealed >2,000 unique transposition events spread across the genome (Fig 3C and S5 Table). Given these encouraging results, we expect that a scaled-up transformation protocol will produce a transposon pool of sufficient diversity for future chemical-genomic investigation using barcode sequencing [8,33,34]. Furthermore, we expect M-TUBE should have wide applicability for generation of libraries of thousands of transposon mutants, even in bacterial species with complex growth requirements.…”

Section: Generation Of a Transposon Mutant Library In An Anaerobic Gu...mentioning

confidence: 99%

“…Electroporation is typically performed using cuvettes in an operator-dependent manner that is limited to small batches of volume 1 mL or less. Even with high efficiency, creation of a comprehensive mutant library with hundreds of thousands of mutants [8][9][10] for functional-genomics studies can require electroporation of large volumes (tens of milliliters) of saturated bacterial culture, which corresponds to hundreds of cuvette-based electroporation reactions. Performing serial electroporation with manual pipetting is a labor-intensive, time-consuming, and costly process.…”

Section: Introductionmentioning

confidence: 99%

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform

et al. 2022

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Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

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“…Moreover, isolation of single strains is necessary to uncover mutants with phenotypes that are masked in a pooled population, for example involving secretion of a molecule that is shared among the entire population regardless of genotype [ 27 – 29 ]. To facilitate isolation from a pooled library, we recently developed a protocol based on cell sorting that is effective for even strict anaerobes [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

et al. 2022

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Background Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. Results Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. Conclusion We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

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Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

Cited by 27 publications

References 22 publications

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

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