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.
Highlights
S. oneidensis possesses an extensive complement of c-type cytochrome electron transfer proteins[7], including the mtr extracellular electron transfer (EET) operon that encodes a set of proteins responsible for most of the electron flux from metabolism to external electron acceptors[4]
There remains an ongoing need for the development of new, highly accessible tools for the genetic manipulation and characterization of S. oneidensis[1,8,20] and the menagerie of esoteric microorganisms found in nature that offer unique capabilities to biological engineering and medicine
The accelerating move of microbial genetics toward nonmodel organisms has been hindered by the scarcity of low-cost, high-quality and broadly applicable genetic tools
Summary
S. oneidensis possesses an extensive complement of c-type cytochrome electron transfer proteins[7], including the mtr extracellular electron transfer (EET) operon that encodes a set of proteins responsible for most of the electron flux from metabolism to external electron acceptors[4]. This cytochrome network provides S. oneidensis with extraordinary respiratory flexibility[8] and gives it potential applications in environmental remediation[3,5], nuclear stewardship[9] and sustainable energy[10,11], while its role in the cryptic cycling of sulfur may have consequences for the fate of CO2 sequestered in deep aquifers[12]. Clonally isolated collections of mutants remain critically important for the characterization of phenotypes such as virulence factors[29,30], secondary and cryptic metabolite production[31] and behaviours synonymous with Shewanellae, such as biofilm formation[32,33] and EET34
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