Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries

Goodman, Andrew L.; Wu, Meng; Gordon, Jeffrey I.

doi:10.1038/nprot.2011.417

Cited by 159 publications

(186 citation statements)

References 14 publications

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“…To identify colonization determinants, it was first necessary to develop tools to globally analyze mutant pool composition in V. fischeri. The Bacteroides thetaiotaomicron mutagenesis vector pSAM was modified so that its resistance cassette and transposase promoter functioned in V. fischeri (10,12). The resulting vector, V. fischeri mariner transposon delivery vector (pMarVF1), can be conjugated from Escherichia coli into V. fischeri, generating stable erythromycinresistant insertions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Global discovery of colonization determinants in the squid symbiontVibrio fischeri

Brooks

Gyllborg

Cronin

et al. 2014

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

102

120

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Animal epithelial tissue becomes reproducibly colonized by specific environmental bacteria. The bacteria (microbiota) perform critical functions for the host's tissue development, immune system development, and nutrition; yet the processes by which bacterial diversity in the environment is selected to assemble the correct communities in the host are unclear. To understand the molecular determinants of microbiota selection, we examined colonization of a simplified model in which the light organ of Euprymna scolopes squid is colonized exclusively by Vibrio fischeri bacteria. We applied highthroughput insertion sequencing to identify which bacterial genes are required during host colonization. A library of over 41,000 unique transposon insertions was analyzed before and after colonization of 1,500 squid hatchlings. Mutants that were reproducibly depleted following squid colonization represented 380 genes, including 37 that encode known colonization factors. Validation of select mutants in defined competitions against the wild-type strain identified nine mutants that exhibited a reproducible colonization defect. Some of the colonization factors identified included genes predicted to influence copper regulation and secretion. Other mutants exhibited defects in biofilm development, which is required for aggregation in host mucus and initiation of colonization. Biofilm formation in culture and in vivo was abolished in a strain lacking the cytoplasmic chaperone DnaJ, suggesting an important role for protein quality control during the elaboration of bacterial biofilm in the context of an intact host immune system. Overall these data suggest that cellular stress responses and biofilm regulation are critical processes underlying the reproducible colonization of animal hosts by specific microbial symbionts.H umans and other animals are often sterile before birth, from which point they immediately proceed to acquire environmental bacteria (1). The bacteria that reproducibly colonize animal hosts are critical for host tissue development, immune system development, and nutrient acquisition. The selection process by which the functional symbionts take residence in the animal from among the great diversity of environmental microbes is poorly understood, so model systems have been especially valuable to examine how specific patterns of colonization are shaped by the genetic makeup of the bacteria and the host environment (2).The light organ of the Hawaiian bobtail squid, Euprymna scolopes, is colonized exclusively by the Gram-negative luminous bacterium Vibrio fischeri. The host inhabits seawater containing 10 6 bacteria per milliliter, with V. fischeri comprising at most 0.02% of the environmental population (3). E. scolopes hatch without symbionts, but then rapidly acquire environmental bacteria and proceed to select for V. fischeri in a "winnowing" process that ensures colonization by only the specific symbiont (4). The squid-Vibrio system thus presents an opportunity to investigate the processes that underlie acquisition of s...

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Section: Resultsmentioning

confidence: 99%

Global discovery of colonization determinants in the squid symbiontVibrio fischeri

Brooks

Gyllborg

Cronin

et al. 2014

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

102

120

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“…This makes it possible to use high throughput sequencing to define the precise location and abundance of each transposon mutant in the library (SI Methods). Comparing the number of reads for each mutant in an "output" population after a given selection to the number of reads generated from an "input" population provides information about the effect each transposon insertion had on the fitness of the organism under the selection condition applied (20,21).…”

Section: Nonsimultaneous Detection Of Viruses and Community Rearrangementioning

confidence: 99%

Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut

Reyes

McNulty

et al. 2013

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

Self Cite

299

269

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Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/ hosts. To characterize their role as agents shaping gut microbial community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage-bacterial host dynamics in the gut.T he human gut is home to tens of trillions of microbial cells representing all three domains of life, although most are bacteria. These organisms collaborate and compete for functional niches and physical locations (habitats). Together, they form a continuously functioning microbial metabolic "organ." The microbial diversity, interpersonal variation, and dynamism of the human gut microbiota make the task of identifying the factors that define community configurations extremely challenging.In some ecosystems, phages maintain high bacterial strain level diversity through lysis of their host strains (constant diversity dynamics model; refs 1, 2). The resulting emptied niche is filled with either an evolved resistant bacterial strain or a taxonomically closely related bacterial species. These dynamics have been observed in open marine environments (1). In contrast, a recent study of 37 healthy adults indicated that a person's fecal microbiota was remarkably stable, with 60% of bacterial strains retained over the course of 5 y (3). Stability followed a power law dynamic that when extrapolated suggests that most strains in an individual's gut community are retained for decades (3). In a metagenomic analysis of virus-like particles (VLPs) purified from the fecal microbiota of healthy adult monozygotic twins and their mothers, sampled over the course of a year, viral community structure exh...

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“…To profile the input, INSeq libraries were prepared using two different methods. In the first, approximately 10 9 cells were prepared following 4 h, as previously described (12). In the second, a sample of 10 6 bacteria that was used to inoculate the day-of-hatch chicks was grown overnight and the input library was generated from that growth.…”

Section: Construction Of C Jejuni Inseq Library a C Jejunimentioning

confidence: 99%

High-Throughput Sequencing of Campylobacter jejuni Insertion Mutant Libraries Reveals mapA as a Fitness Factor for Chicken Colonization

Johnson

Livny

DiRita

2014

Journal of Bacteriology

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bCampylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture.

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Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries

Cited by 159 publications

References 14 publications

Global discovery of colonization determinants in the squid symbiontVibrio fischeri

Global discovery of colonization determinants in the squid symbiontVibrio fischeri

Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut

High-Throughput Sequencing of Campylobacter jejuni Insertion Mutant Libraries Reveals mapA as a Fitness Factor for Chicken Colonization

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