Madeleine Bouzon

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.

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains.

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.

Laminin-mediated adhesion in metastatic rat rhabdomyosarcoma cell lines involves prominent interactions with the laminin E8 fragment

In vitro attachment assays were carried out to assess adhesion between two basement membrane proteins, type IV collagen and laminin, and rat rhabdomyosarcoma (RMS) cell lines with different metastatic potentials. Whereas cells did not adhere to type IV collagen, adhesion to laminin appeared to be very sensitive as maximal adhesion was achieved in dose-response assays with only nanograms of laminin. Adhesion was mediated by interactions between coated laminin and cell surface components, probably receptors, but not endogenous laminin. Laminin-mediated adhesion of RMS cell lines was compared with that of the MCF-7 (human mammary carcinoma) and the L6 (rat myoblast) cell lines. In dose-response assays, RMS cell lines required 10 times less laminin to reach half-maximal attachment rates than MCF-7 and L6 cell lines. Two laminin fragments, P1 and E8, which are structurally and immunologically distinct as shown byα-helix content, SDS-PAGE and monoclonal antibody mapping, supported adhesion by RMS cells and L6 myoblasts, but MCF-7 adhered only to P1. This fragment was 10 times less active than laminin in RMS cell lines. Attachment in dose-response assays and adhesion inhibition studies by antibodies revealed that E8 accounted for the activity of laminin in RMS cell adhesion. Adhesion in the RMS cell lines was dominated by interaction with E8 regardless of metastatic potential.

Laminin-induced capping and receptor expression at cell surface in a rat rhabdomyosarcoma cell line: Involvement in cell adhesion and migration on laminin substrates☆

The present study reveals the dynamic distribution of membrane laminin receptors induced by laminin binding in a rat rhabdomyosarcoma cell line RMS S4. The treatment of the cells with soluble laminin did not modify cell adhesion to laminin-coated substrates in in vitro attachment assays. Fluorescent labeling of membrane-bound laminin revealed that occupied receptors were induced to cluster and cap. New free membrane binding sites were made evident after capping of bound laminin by a double labeling technique. Cytochalasin D (CD) treatment prevented the capping process. The adhesion of CD-treated cells to laminin-coated substrates was inhibited by cell preincubation with soluble laminin. Cycloheximide treatment had no effect on the ability of RMS S4 cells to adhere to adsorbed laminin after preincubation in the presence of soluble laminin. These results taken as a whole suggest that free receptors may arise from an intracellular pool that could be maintained by membrane receptor recycling. Since capping and motility seem related events, migration of RMS S4 cells on laminin was studied in the agarose drop assay. Immobilized laminin stimulated basic cell motility by more than 200%. E8 laminin fragment retained partially the motility stimulating property of laminin while P1 pepsinic fragment had no effect. The presence of constantly available receptors at the cell surface could be determinant in the ability of cells to migrate on laminin substrates.

Synthesis of specially designed probes to broaden transketolase scope

Efficient, biocompatible, stereospecific strategies were developed to prepare eight probes to assay transketolase (TK) variants with new substrate specificities. The structure of these probes combines a sugar moiety (D‐threo or L‐erythro ketose, or D‐threo aldose) with the side chain of an amino acid (Ala, Leu, Val, Met, Thr) for in vivo detection of new TK activities using amino acid auxotrophs. To obtain D‐threo ketose probes, biocatalysts, such as transketolase and fructose‐6‐phosphate aldolase Ala129Ser, were used whereas L‐erythro ketoses and D‐threo aldose probes were synthesized by the way of organocatalysis or Sharpless dihydroxylation as sustainable alternative key steps to biocatalysis.

Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.

A Synthetic Alternative to Canonical One-Carbon Metabolism

One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO2. The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

Amino acid precursors for the detection of transketolase activity in Escherichia coli auxotrophs

Probes were developed for the in vivo detection of transketolase activity by the use of a complementation assay in Escherichia coli auxotrophs They combine the d-threo ketose moiety recognised by transketolase and the side chain of leucine or methionine. These compounds were donor substrates of yeast transketolase leading to the release of the corresponding alpha-hydroxyaldehydes which could be converted in E. coli by a cascade of reactions into leucine or methionine required for cellular growth.

In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli

Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2. Importantly, the biosynthesis of serine from formate and CO2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.