Fig. 1: Knockout Sudoku was used to curate a saturating coverage transposon insertion mutant collection for Gluconobacter oxydans B58.

A The G. oxydans B58 genome contains 3283 genes. Two thousand five hundred and seventy genes were fully annotated with a BLAST hit, InterPro ID, and gene ontology (GO) group. An additional 163 genes had an annotation and GO group but lacked an InterPro ID; 399 retrieved only a BLAST hit, and 150 were unable to be assigned any annotation. B A Monte Carlo (MC) estimate (green curve) of the number of genes represented by at least one mutant as a function of the number of mutants collected demonstrated that picking 25,000 mutants would yield at least one disruption for 95% of genes, while picking 50,000 mutants would yield at least one disruption for 99% of genes. In total, we picked 49,256 single-gene disruption mutants and located at least one disruption for 2733 genes. A Monte Carlo simulation (blue curve) of picking with random drawing from the sequenced progenitor collection (PC) without replacements demonstrates that the genome coverage was truly saturated. The center of each curve is the mean value of the unique gene disruption count from 1000 simulations, while the upper and lower parts of each curve represent two standard deviations around this mean. C A one-sided Fisher’s exact test for gene ontology enrichment among the non-disrupted (putatively essential) genes revealed significant enrichment (p < 0.05, yellow line) of genes involved in translation and other ribosome-related functions. D The curated condensed collection (CC) contains 17,706 isolated colonies across 185 plates. High-throughput sequencing of the CC confirmed the location for 4419 unique disruption strains, representing disruptions in 2556 genes. Hundred and seventy-seven genes located in the PC were not located in the CC. No disruption mutant was detected in 550 genes.