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Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

ABSTRACT: Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

SUBMITTER: Baym M

PROVIDER: S-EPMC5109470 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

Baym Michael M Shaket Lev L Anzai Isao A IA Adesina Oluwakemi O Barstow Buz B

Nature communications 20161110

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30 ...[more]

PMID: 27830751

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Project description:UnlabelledDespite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth.ImportanceThe ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.

Project description:Background: Microbial fuel cells (MFCs) are a novel bioelectrochemical devices that can use exoelectrogens as biocatalyst to convert various organic wastes into electricity. Among them, acetate, a major component of industrial biological wastewater and by-product of lignocellulose degradation, could release eight electrons per mole when completely degraded into CO2 and H2O, which has been identified as a promising carbon source and electron donor. However, Shewanella oneidensis MR-1, a famous facultative anaerobic exoelectrogens, only preferentially uses lactate as carbon source and electron donor and could hardly metabolize acetate in MFCs, which greatly limited Coulombic efficiency of MFCs and the capacity of bio-catalysis. Results: Here, to enable acetate as the sole carbon source and electron donor for electricity production in S. oneidensis, we successfully constructed three engineered S. oneidensis (named AceU1, AceU2, and AceU3) by assembling the succinyl-CoA:acetate CoA-transferase (SCACT) metabolism pathways, including acetate coenzyme A transferase encoded by ato1 and ato2 gene from G. sulfurreducens and citrate synthase encoded by the gltA gene from S. oneidensis, which could successfully utilize acetate as carbon source under anaerobic and aerobic conditions. Then, biochemical characterizations showed the engineered strain AceU3 generated a maximum power density of 8.3 ± 1.2 mW/m2 with acetate as the sole electron donor in MFCs. In addition, when further using lactate as the electron donor, the maximum power density obtained by AceU3 was 51.1 ± 3.1 mW/m2, which approximately 2.4-fold higher than that of wild type (WT). Besides, the Coulombic efficiency of AceU3 strain could reach 12.4% increased by 2.0-fold compared that of WT, which demonstrated that the engineered strain AceU3 can further utilize acetate as an electron donor to continuously generate electricity. Conclusion: In the present study, we first rationally designed S. oneidensis for enhancing the electron generation by using acetate as sole carbon source and electron donor. Based on synthetic biology strategies, modular assembly of acetate metabolic pathways could be further extended to other exoelectrogens to improve the Coulombic efficiency and broaden the spectrum of available carbon sources in MFCs for bioelectricity production.

Dataset Information

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

Publications

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

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