Philippe Marlière

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.

Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation.

Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N‐terminal methionine residue of ribosome‐synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high‐copy‐number plasmid and selection for utilization of the substrate analogue formyl‐leucyl‐methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl‐tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294‐4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10‐formyl‐tetrahydrofolate, the formyl donor in methionyl‐tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.

Chemical Evolution of a Bacterium’s Genome

We set out to develop a generic technology for evolving the chemical constitution of microbial populations by using the simplest possible algorithm. Extant living cells polymerize a restricted set of nucleic acid precursors, namely, four nucleoside triphosphates (UTP, CTP, ATP, GTP) and four deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP). Synthetic analogues, such as 5-halogenopyrimidines, 7-deazapurines, and 8-azapurines, are known to partially replace canonical bases in cellular RNA and DNA, yet were never demonstrated to sustain unlimited self-reproduction of an organism through complete genome or transcriptome substitution. A hamster cell line serially adapted to grow in the presence of bromodeoxyuridine, while dTMP synthesis was inhibited with aminopterin, has been reported to harbor DNA highly enriched in bromouracil over thymine. However, the significance of these findings could not be ascertained owing to the absence of a direct physical measurement of the base composition of the DNA and the absence of an assay of thymidylate biosynthesis, as well as the likely presence of metabolic components, such as nucleotides in the complex growth medium of the cells. Only certain DNA viruses are known to have undergone full transliteration of a canonical base through the biosynthesis of a noncanonical nucleoside triphosphate, for example, hydroxymethylcytosine in the T4 bacteriophage, presumably to counteract the restriction enzymes of their bacterial hosts. When Weiss and coworkers attempted to substitute thymine in the DNA of Escherichia coli with uracil, over 90% replacement was reached, but further growth was prevented. Genome-scale transliteration has apparently not evolved in any known living cell, possibly owing to a chemical barrier that natural biodiversity cannot overcome. Our experimental plan consisted of the combination of tight metabolic selection with the long-term automated cultivation of fast-growing asexual bacterial populations to change a canonical DNA base for a chemical ersatz. The cultivation setup was elaborated from the GM3 fluidic format (Figure 1), which features the cyclic transfer of the culture between twin growth chambers that alternately undergo sterilization. This cycle ensures that no internal surface of the device is spared from transient periodic cleansing with a sterilizing agent (5m sodium hydroxide), and therefore that no cultivated variant can escape dilution and selection for faster growth through the formation of biofilms. The active elimination of biofilms (wall growth) has proved critical for reprogramming and improving the metabolism of microbial populations. The GM3 cultivation device was connected to two nutrient reservoirs of different composition: a relaxing medium R that contains the canonical nutrient and a stressing medium S that contains the ersatz nutrient. Liquid pulses of defined volume are sent at regular intervals of time from these reservoirs to the culture, which is kept at a constant volume. Depending upon the state of the adapting cells, as measured by turbidity recording of the population density, the culture periodically receives a pulse of fixed volume of either medium R (if the population density falls below a fixed threshold) or medium S (if the density is higher than or equal to the threshold). Successive pulses thus renew the culture at a fixed dilution rate with a nutrient-medium flow whose composition varies with respect to the growth response of the population in such a way that the lowest tolerable concentration of canonical nutrient is automatically maintained over passing generations. We designate this mode of operation as the conditional pulse-feed regime. It qualifies as a simplified and generalized version of a method pioneered by Oliver. Mutations that confer a lower requirement for the canonical nutrient or a higher survival rate under starvation are expected to accumulate in the genome of the adapting population. No attempt was made to implement a finer regulation of differential nutrient supply than the coarse-grained control by medium-switch pulse feed described above. We thus relied on the robustness of biochemical machineries and their evolution to dampen oscillations. [*] Dr. P. Marli re Heurisko USA Inc., Delaware (USA)

Complete Genome Sequence of Streptomyces cattleya NRRL 8057, a Producer of Antibiotics and Fluorometabolites

Streptomyces cattleya, a producer of the antibiotics thienamycin and cephamycin C, is one of the rare bacteria known to synthesize fluorinated metabolites. The genome consists of two linear replicons. The genes involved in fluorine metabolism and in the biosynthesis of the antibiotic thienamycin were mapped on both replicons.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli

BackgroundTo maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate.ResultsTo overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate.ConclusionsThis approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Enzymatic synthesis of 1-(2-deoxy-β-D-ribofuranosyl) imidazole-4-carboxamide, a simplified DNA building block

Abstract Coupling of silylated 4-(5)-imidazolecarboxamide (5) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranose (6) in the presence of SnCl4 in CH3CN or condensation of the sodium salt of 5 with the halogenose 6, afforded a mixture of 1-β-glycosylated 4-(5)-imidazolecarboxamide (11) and the corresponding α anomer (12). Using a crude extract of nucleoside N-deoxyribosyltransferases from Lactobacillus leichmannii only one product was formed, the N-1-β-stereoisomer (2), in a good yield.

Enzymatically Catalyzed DNA Synthesis Using l-Asp-dGMP, l-Asp-dCMP, and l-Asp-dTMP

The replacement of the pyrophosphate moiety of deoxynucleoside triphosphates by L‐aspartic acid allows incorporation of natural deoxynucleosides into DNA using HIV reverse transcriptase (RT) as enzyme, while retaining the canonical base‐pair selectivity. N‐Methylation of the L‐aspartic acid leaving group results in a reduced fidelity of incorporation.

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.

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.