Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320

Pearson, Melanie M.; Pahil, Sapna; Forsyth, Valerie; Shea, Allyson E.; Mobley, Harry L. T.

doi:10.1128/spectrum.03142-22

Cited by 4 publications

(8 citation statements)

References 49 publications

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“…Our goal was to test the fitness of an ordered transposon library built for P. mirabilis strain HI4320 ( 20 ) in a physiologically relevant condition. Because of the severe bottleneck in mice, we devised a screen that did not require pooling of mutants.…”

Section: Resultsmentioning

confidence: 99%

“…We screened 1728 individual P. mirabilis transposon mutants from an ordered transposon library ( 20 ) on human urine and murine kidney and liver agar. Stamping arrayed mutants onto agar only required 5 mice to screen this library.…”

Section: Discussionmentioning

confidence: 99%

“…Toward this goal, we present data showing the experimental bottleneck for the total number of P. mirabilis mutants that can be tested in this model without stochastic loss is much narrower than anticipated. We recently built an ordered library containing single transposon insertions in 1728 genes ( 20 ). Based on the tight experimental bottleneck, we concluded that screening this library in a murine model would not be feasible.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Organ agar serves as physiologically relevant alternative for in vivo colonization

Pearson

Shea

Smith

et al. 2023

Preprint

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Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Organ agar serves as physiologically relevant alternative for in vivo colonization

Pearson

Shea

Smith

et al. 2023

Preprint

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“…We revived strains from the nonredundant library ((32); henceforth referred to as donor library) in microplate format to maintain parity between it and that produced in this work. The donor library was generously provided by the laboratories of Vic DiRita (plates: 2, 4-6, 8-10, 12-17, [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33], Michigan State University, and Bonnie Bassler (plates: 1, 3, 7, 11, 18, 34), Princeton University. Briefly, microtiter plates from the donor library were slightly thawed and 10 µl from each well was inoculated into a deep-well microplate containing 550 µl of selective media (Luria-Bertani broth + Kanamycin (100 µg/ml) in each of the 96 wells.…”

Section: Strain Revivalmentioning

confidence: 99%

“…In contrast to conventionally laborious genetic engineering approaches for disrupting single genes to study their phenotypic effects, transposon mutagenesis allows the simultaneous construction and screening of tens of thousands of mutants. Researchers have taken advantage of transposon mutagenesis to construct nonredundant (ordered) mutant libraries, where each nonessential gene has been inactivated and the individual mutants are arrayed across the wells of 96-well microtiter plates (28)(29)(30)(31). An ordered mutant V. cholerae library was constructed in 2008 (32).…”

Section: Introductionmentioning

confidence: 99%

Deployment of aVibrio choleraeordered transposon mutant library in a quorum-competent genetic background

Grant,

Donkor,

Sontz

et al. 2023

Preprint

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Cholera is a severe diarrheal disease that claims approximately 30,500 lives annually, mainly in regions with limited access to clean water.Vibrio cholerae, the causative agent, has sparked seven pandemics in recent centuries, with the current one being the most prolonged.V. choleraepathogenesis hinges on its ability to switch between low and high cell density gene regulatory states, enabling transmission between host and the environment.Previous researchers created a transposon mutant library forV. choleraeto support investigations aimed toward uncovering the genetic determinants of its pathogenesis. However, subsequent sequencing uncovered a mutation in the geneluxOof the parent strain, rendering mutants unable to exhibit high cell density behaviors. In this study, we used chitin-independent natural transformation to move transposon insertions from these low cell density mutants into a wildtype genomic background. Library transfer was aided by a novel gDNA extraction we developed using thymol, which also showed marked vibrio-specific activity. The resulting Grant Library comprises 3,102 unique transposon mutants, covering 79.8% ofV. cholerae’sopen reading frames. Whole genome sequencing of randomly selected mutants demonstrates 100% precision in transposon transfer to cognate genomic positions of the recipient strain. Notably,luxOmutations transferred at a low frequency only with insertions nearluxO. Our research uncovered density-dependent epistasis in motility genes, relevant toV. choleraepathogenesis. Additionally, Grant Library mutants retain a plasmid conferring natural competence, enabling rapid, scarless genomic editing. In summary, the Grant Library reintroduces organismal relevant genetic contexts absent in the low cell density locked library equivalent.

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Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization

Pearson,

Shea,

Pahil

et al. 2023

Infect Immun

View full text Add to dashboard Cite

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to bacterial colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. Organ agar was also useful for identifying previously unknown links between biosynthetic genes and swarming motility. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

show abstract

Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320

Cited by 4 publications

References 49 publications

Organ agar serves as physiologically relevant alternative for in vivo colonization

Organ agar serves as physiologically relevant alternative for in vivo colonization

Deployment of aVibrio choleraeordered transposon mutant library in a quorum-competent genetic background

Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization

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