Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.antibiotic resistance | evolutionary innovation | molecular evolution | protein promiscuity | phenotype microarray

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.

BackgroundAcute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease.MethodsThis study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand.ResultsGlobal analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens.ConclusionsThe broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

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