Rupert Mutzel

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)

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP‐dependent protein kinase fused to the promoter and N‐terminal‐proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP‐dependent protein kinase, to facilitate early development.

Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia : sequence, expression in heterologous hosts, and purification of the enzyme

The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined. The cpo codes for a protein of 278 amino acids (aa). The matuŕe enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304. Expression studies showed that the cpo from P. pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans. Based on the overproduction of the CPO in E. coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P. pyrrocinia. Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S. aureofaciens ATCC10762 revealed an identity of 38%.

Ca2+/Calmodulin-independent Activation of Calcineurin from Dictyostelium by Unsaturated Long Chain Fatty Acids

This study describes a novel mode of activation for the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin fromDictyostelium discoideum we found a reversible, Ca2+/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca2+/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 μm with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca2+/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca2+/calmodulin action by binding to the calmodulin-binding site.

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.

Overexpression, purification and characterization of Dictyostelium calcineurin A.

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Vectors for expression of truncated coding sequences in Escherichia coli.

We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.

Aberrant stalk development and breakdown of tip dominance in Dictyostelium cell lines with RNAi-silenced expression of calcineurin B

BackgroundCalcineurin, the Ca2+/calmodulin-dependent protein phosphatase, plays important roles in various cellular processes in lower and higher eukaryotes. Here we analyze the role of calcineurin in the development of Dictyostelium discoideum by RNAi-mediated manipulation of its expression.ResultsThe cnbA gene of Dictyostelium discoideum which encodes the regulatory B subunit (CNB) of calcineurin was silenced by RNAi. We found a variety of silencing levels of CNB in different recombinant cell lines. Reduction of CNB expression in a given cell line was correlated with developmental aberrations. Cell lines with strongly reduced protein levels developed slower than wild type cells and formed short stalks and spore heads with additional tips. Formation of short stalks results from incomplete vacuolization of prestalk cells during terminal differentiation. Expression of the stalk-specific gene ecmB was reduced in mutant cells. Aberrant stalk development is a cell autonomous defect, whereas the breakdown of tip dominance can be prevented by the presence of as low as 10% wild type cells in chimeras.ConclusionSilencing of calcineurin B in Dictyostelium by expression of RNAi reveals an unexpected link between increased intracellular calcium levels, possibly triggered by the morphogen DIF, activation of calcineurin, and the terminal stage of morphogenesis.

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.