Genetic code ambiguity confers a selective advantage on Acinetobacter baylyi.

A primitive genetic code, composed of a smaller set of amino acids, may have expanded via recursive periods of genetic code ambiguity that were followed by specificity. Here we model a step in this process by showing how genetic code ambiguity could result in an enhanced growth rate in Acinetobacter baylyi.

An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response.

Mistranslation in bacterial and mammalian cells leads to production of statistical proteins that are, in turn, associated with specific cell or animal pathologies, including death of bacterial cells, apoptosis of mammalian cells in culture, and neurodegeneration in the mouse. A major source of mistranslation comes from heritable defects in the editing activities of aminoacyl-tRNA synthetases. These activities clear errors of aminoacylation by deacylation of mischarged tRNAs. We hypothesized that, in addition to previously reported phenotypes in bacterial and mammalian systems, errors of aminoacylation could be mutagenic and lead to disease. As a first step in testing this hypothesis, the effect of an editing defect in a single tRNA synthetase on the accumulation of mutations in aging bacteria was investigated. A striking, statistically significant, enhancement of the mutation rate in aging bacteria was found. This enhancement comes from an increase in error-prone DNA repair through induction of the bacterial SOS response. Thus, mistranslation, as caused by an editing-defective tRNA synthetase, can lead to heritable genetic changes that could, in principle, be linked to disease.

Global incorporation of unnatural amino acids in Escherichia coli.

The incorporation of amino acid analogs is becoming increasingly useful. Site-specific incorporation of unnatural amino acids allows the application of chemical biology to protein-specific investigations and applications. However, the global incorporation of unnatural amino acids allows for tests of proteomic and genetic code hypotheses. For example, the adaptation of organisms to unnatural amino acids may lead to new genetic codes. To understand and quantify changes from such perturbations, an understanding is required of the microbiological and proteomic responses to the incorporation of unnatural amino acids. Here we describe protocols to characterize the effects of such proteome-wide perturbations.

Rapid evolution of diminished transformability in Acinetobacter baylyi.

The reason for genetic exchange remains a crucial question in evolutionary biology. Acinetobacter baylyi strain ADP1 is a highly competent and recombinogenic bacterium. We compared the parallel evolution of wild-type and engineered noncompetent lineages of A. baylyi in the laboratory. If transformability were to result in an evolutionary benefit, it was expected that competent lineages would adapt more rapidly than noncompetent lineages. Instead, regardless of competency, lineages adapted to the same extent under several laboratory conditions. Furthermore, competent lineages repeatedly evolved a much lower level of transformability. The loss of competency may be due to a selective advantage or the irreversible transfer of loss-of-function alleles of genes required for transformation within the competent population.

Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase.

The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain. A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids. Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase. Ambiguity retarded cell growth at most temperatures in rich and minimal media. These growth rate differences were seen regardless of the carbon source. Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function. Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors. Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.

Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering.

Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.

Evolving new genetic codes.

Although the genetic code is almost universal, natural variations exist that have caused evolutionary biologists to speculate about codon evolution. There are two predominant hypotheses that specify either a gradual (ambiguous intermediate) or stochastic (codon capture) change in the code. These hypotheses are similar to two biotechnology techniques that have been used to engineer the genetic code: a 'top down' approach, in which the whole organism is evolved for the ability to incorporate unnatural amino acids, and a 'bottom up' approach, in which aminoacyl-tRNA synthetases and their cognate tRNAs are engineered. The biotechnology experiments provide insights into natural codon evolution, and a combination of these approaches should enable the evolution of organisms that can incorporate unnatural amino acids throughout their proteomes.

Evolution of phage with chemically ambiguous proteomes.

BACKGROUND: The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality. RESULTS: The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue. CONCLUSIONS: A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.

Anticipatory evolution and DNA shuffling.

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.