We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 ؋ 10 ؊4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400 -500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 ␤-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum ␤-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases. DNA polymerases catalyze the template-directed incorporation of deoxynucleotides into a growing primer terminus. Errors in nucleotide incorporation by DNA polymerases are significant sources of mutation, and contribute to genetic diversity. The fidelity of replication by DNA polymerases is ensured by base pair complementarity, by dNTP-induced conformational changes, and, in some cases, by proofreading catalyzed by a 3Ј 3 5Ј exonuclease domain (1).DNA polymerase I (Pol I) is one of five different DNA polymerases that have been described so far in Escherichia coli. In vivo, Pol I plays a role in lagging-strand replication of chromosomal DNA, DNA repair, and ColE1 plasmid replication (2). Pol I has been intensively studied as a model polymerase, and extrapolation to a diversity of other DNA polymerases has been possible given the conservation in structure and catalytic mechanism across eukaryotic and prokaryotic DNA polymerases (3).Here, we engineered a highly error-prone E. coli DNA Pol I, and used this mutant polymerase as the basis of an in vivo mutagenesis system for modification of targeted sequences. Generation of enzyme variants is important in both basic studies of enzyme structure and function, and in creation of improved derivatives for medicine and technology. Several approaches for enzyme modification have been described to date. One of these approaches is random nucleotide mutagenesis, which involves the generation of large libraries harboring random mutations in vitro (4) and the subsequent identification of mutants of inter...

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.

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Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.

Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic translation (e.g. clindamycin, macrolides and tetracyclines). A priori, two prokaryotic-like organelles, the 'apicoplast' (a non-photosynthetic plastid) and the mitochondrion, are likely targets for these drugs. Without using overt mutagenesis, we selected two independent clones (ClnR-4 and ClnR-21) with strong and stable clindamycin resistance. Several lines with substantial but lower levels of resistance were also isolated with (XR-46) or without (ClnR-23) overt mutagenesis. The ClnR-4 and ClnR-21 mutants uniquely possess a G-->U point mutation at position 1857 of the apicoplast large-subunit rRNA, whereas no mutation was identified in this region for ClnR-23 or XR-46. Position 1857 corresponds to position 2061 in Escherichia coli where it is predicted to bind clindamycin. The mutation is present in all the apicoplast rDNA copies (an estimated 12 per organelle), indicative of a strong selective advantage in the presence of clindamycin. In the absence of drug, however, such a mutation is unlikely to be neutral, as the G is a critical contributor to the transpeptidation reaction and absolutely conserved in all kingdoms. This may explain why ClnR-4 shows a slight growth defect in vitro. These mutants provide direct genetic evidence that apicoplast translation is the target for clindamycin in Toxoplasma. Further, their sensitivity profiles to other antibiotics specific for the large ribosomal subunit (macrolides and chloramphenicol) and, intriguingly, the small subunit (doxycycline) argue that these drugs also target the apicoplast ribosome.

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of Rloop formation, and of the timing of recombinant gene expression.

Plasmids are self-replicating pieces of DNA that can help dissemination of non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79-80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.