Valérie Pezo

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

Binary Genetic Cassettes for Selecting XNA-Templated DNA Synthesis In Vivo†

Information transfer between natural nucleic acids (DNA and RNA) and xenobiotic nucleic acids (XNA) is rapidly gaining momentum for extending the range of chemical constitutions and the format of molecular evolution accessible to living organisms. Artificial coding by nucleic acid analogues previously focused on structural alterations of base pairs to expand the alphabet of genetic messages. Studies were mostly conducted ex vivo and few experiments have succeeded in vivo thus far. Kool and collaborators demonstrated that size-expanded nucleobases can serve as template for DNA synthesis in E. coli. Substitution of thymine for 5chlorouracil in a whole genome could be performed through automated evolution of E. coli. Conveying genetic information to DNA from an XNA with a chemically deviant backbone is amenable to tight metabolic selection, as demonstrated for hexitol nucleic acid (HNA) using the thymidylate synthase screen in E. coli. We have now shown that various combinations of only the two bases guanine and thymine can be used to encode the active site of thymidylate synthase. This finding was exploited to simplify the synthesis of XNA to be assayed as templates for DNA biosynthesis in vivo, by halving the alphabet needed for this purpose. It could thus be demonstrated that cyclohexenyl nucleic acid (CeNA) can serve in vivo as template, mobilizing a limited effort of chemical synthesis. Further simplification of the binary system to uracil and hypoxanthine enabled to reprogram E. coli with templates simultaneously bearing noncanonical bases and a noncanonical backbone, namely arabinofuranosyl nucleic acid (AraNA) and HNA. A functional thyA gene encoding thymidylate synthase is absolutely required by E. coli cells to grow in nutrient medium devoid of thymine or thymidine (TLM, thymidineless medium). We took advantage of this selection scheme for constructing a plasmid carrying a defective thyA gene in which the six codons specifying the active site around the cysteine at position 146 have been deleted, leaving a gap when digested with the restriction enzymes NheI and NsiI. Mosaic DNA oligonucleotides in which several of the six codons are carried by an XNA backbone can be tested for informational transfer simply by selecting for active thyA genes after transformation of the thyA-deficient strain G929 with heteroduplex ligation products (Figure 1). Up to six contiguous HNA nucleotides were found to serve as a short template for E. coli replication enzymes.

Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

A Metabolic Prototype for Eliminating Tryptophan From The Genetic Code

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

Probing Ambiguous Base‐Pairs by Genetic Transformation with XNA Templates

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5‐Anhydro‐2,3‐dideoxy‐2‐(5‐nitroindazol‐1‐yl)‐D‐arabino‐hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.