Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

Millstone: software for multiplex microbial genome analysis and engineering.

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli.

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.

Design, synthesis, and testing toward a 57-codon genome.

Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.

Rational optimization of tolC as a powerful dual selectable marker for genome engineering.

Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.

Dichotomy in the evolution of pathogenicity island and bacteriophage encoded integrases from pathogenic Escherichia coli strains.

The evolutionary histories of mobile and integrative genetic elements (MIGEs), key factors in the emergence of pathogenic bacteria, remain obscure due to their widespread distribution and diversity in gene content. Pathogenicity islands (PAIs) are large chromosomal regions that encode bacterial virulence factors present in pathogenic isolates and absent from non-pathogenic isolates. We examined the utility of PAI-encoded integrases as a marker to determine PAI phylogeny and evolutionary relationships to other MIGEs particularly bacteriophages. We examined 68 tyrosine recombinase (TR) integrases from 27 PAIs and 39 phages from five fully sequenced pathogenic Escherichia coli strains in the database. In general, all PAI regions identified were integrated adjacent to tRNA loci and had a percent GC content that differed from the host chromosome, which was not the case for most phages examined. Our phylogenetic analysis demonstrated that PAI-encoded integrases clustered within a distinct lineage, separate from phage-encoded integrases, which suggested a discrete evolutionary history. The tree branch lengths among PAI integrases were shorter than those among phage integrases suggesting that PAIs may be a more recent addition to E. coli. Further phylogenetic comparisons of these 68 integrases with 53 TR integrases from other characterized MIGEs also demonstrated that PAI-encode integrases form a distinct lineage, suggesting that these PAIs in E. coli are not remnants of other MIGEs. We identified recombination directionality factors/excisionases in the proximity of many of the E. coli PAI integrases, factors that were previously not shown to be present in E. coli PAIs. Overall this work is the first to demonstrate a dichotomy in the evolution of integrases encoded on PAIs and phages from pathogenic E. coli suggesting that PAIs are an evolutionary distinct genetic element.

Excision dynamics of Vibrio pathogenicity island-2 from Vibrio cholerae: role of a recombination directionality factor VefA.

BACKGROUND: Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate. RESULTS: We determined the conditions and the factors that affect excision of VPI-2 in V. cholerae N16961. We demonstrate that excision from chromosome 1 is induced at low temperature and after sublethal UV-light irradiation treatment. In addition, after UV-light irradiation compared to untreated cells, cells showed increased expression of three genes, intV2 (VC1758), and two putative recombination directionality factors (RDFs), vefA (VC1785) and vefB (VC1809) encoded within VPI-2. We demonstrate that along with IntV2, the RDF VefA is essential for excision. We constructed a knockout mutant of vefA in V. cholerae N16961, and found that no excision of VPI-2 occurred, indicating that a functional vefA gene is required for excision. Deletion of the second RDF encoded by vefB did not result in a loss of excision. Among Vibrio species in the genome database, we identified 27 putative RDFs within regions that also encoded IntV2 homologues. Within each species the RDFs and their cognate IntV2 proteins were associated with different island regions suggesting that this pairing is widespread. CONCLUSIONS: We demonstrate that excision of VPI-2 is induced under some environmental stress conditions and we show for the first time that an RDF encoded within a pathogenicity island in V. cholerae is required for excision of the region.