Precise characterization of the mutation histories of evolutionary lineages is crucial for understanding the evolutionary process, yet mutation identification has been constrained by traditional techniques. We sought to identify all accumulated mutations in an experimentally evolved lineage of the cooperative bacterium Myxococcus xanthus, which constructs fruiting bodies by a process of social multicellular development in response to starvation. This lineage had undergone two major transitions in social phenotype: from an ancestral cooperator to a socially defective cheater, and from the cheater to a competitively dominant cooperator that re-evolved social and developmental proficiency. The 9.14-Mb genome of the evolved, dominant cooperator (strain ''PX'') was sequenced to Ϸ19-fold coverage by using recent "sequencing-bysynthesis" technology and partially sequenced (Ϸ45%) by using capillary technology. The resulting data revealed 15 single-nucleotide mutations relative to the laboratory ancestor of PX after the two phases of experimental evolution but no evidence of duplications, transpositions, or multiple-base deletions. No mutations were identified by capillary sequencing beyond those found by pyrosequencing, resulting in a high probability that all mutations were discovered. Seven errors in the reference strain previously sequenced by the Sanger approach were revealed, as were five mutational differences between two distinct laboratory stocks of the reference strain. A single mutation responsible for the restoration of development in strain PX was identified, whereas 14 mutations occurred during the prior phase of experimental evolution. These results provide insight into the genetic basis of two large adaptive transitions in a social bacterium. cooperation ͉ Myxococcus xanthus

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive surface proteins that mediate adhesion to host cells in a broad range of Gram-negative pathogens. Although their sizes may differ by more than one order of magnitude, they all follow the same basic head-stalk-anchor architecture, where the head mediates adhesion and autoagglutination, the stalk projects the head from the bacterial surface, and the anchor provides the export function and attaches the adhesin to the bacterial outer membrane after export is complete. In complex adhesins, head and stalk domains may alternate several times before the anchor is reached. Despite extensive sequence divergence, the structures of TAA domains are highly constrained, due to the tight interleaving of their constituent polypeptide chains. We have therefore taken a "domain dictionary" approach to characterize representatives for each domain type by X-ray crystallography and use these structures to reconstruct complete TAA fibers. With SadA from Salmonella enterica, EhaG from enteropathogenic Escherichia coli (EHEC), and UpaG from uropathogenic E. coli (UPEC), we present three representative structures of a complex adhesin that occur in a conserved genomic context in Enterobacteria and is essential in the infection process of uropathogenic E. coli. Our work proves the applicability of the dictionary approach to understanding the structure of a class of proteins that are otherwise poorly tractable by high-resolution methods and provides a basis for the rapid and detailed annotation of newly identified TAAs.coiled coil | β-layer

The protein cereblon serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degradation is exploited in different clinical settings and has sparked the development of a growing number of thalidomide derivatives. Here, we probe the chemical space of cereblon binding beyond such derivatives and work out a simple set of chemical requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures for a diverse set of compounds, including commonly used pharmaceuticals, but also find that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. Our results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors and provide a framework for the circumvention of unintended cereblon binding by negative design for future pharmaceuticals.

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Cereblon serves as an ubiquitin ligase substrate receptor that can be tuned toward different target proteins by various cereblon-binding agents. This offers one of the most promising avenues for targeted protein degradation in cancer therapy, but cereblon binding can also mediate teratogenic effects. We present an effective assay that is suited for high-throughput screening of compound libraries for off-target cereblon interactions but also can guide lead optimization and rational design of novel cereblon effector molecules.