Intrinsically disordered regions (IDRs) are characterized by their lack of stable secondary or tertiary structure and comprise a large part of the eukaryotic proteome. Although these regions play a variety of signaling and regulatory roles, they appear to be rapidly evolving at the primary sequence level. To understand the functional implications of this rapid evolution, we focused on a highly diverged IDR in Saccharomyces cerevisiae that is involved in regulating multiple conserved MAPK pathways. We hypothesized that under stabilizing selection, the functional output of orthologous IDRs could be maintained, such that diverse genotypes could lead to similar function and fitness. Consistent with the stabilizing selection hypothesis, we find that diverged, orthologous IDRs can mostly recapitulate wildtype function and fitness in S. cerevisiae. We also find that the electrostatic charge of the IDR is correlated with signaling output and, using phylogenetic comparative methods, find evidence for selection maintaining this quantitative molecular trait despite underlying genotypic divergence.C urrent predictions suggest that close to 40% of all proteins in eukaryotic organisms are either entirely disordered or contain sizeable regions that are disordered, meaning they do not autonomously fold into defined secondary or tertiary structures (1, 2). These intrinsically disordered regions (IDRs) are thought to have important implications for protein function (3, 4) and are known to play regulatory roles, often through short linear motifs (SLiMs) that control protein-protein interactions, localization, degradation, and posttranslational modifications (5, 6). Although proteome-wide studies have provided in silico evidence for conservation of length (7) and composition (8) in some IDRs, reports of increased rates of insertions and deletions (9-13) and amino acid substitutions (14) in IDRs are indicative of their rapid evolution compared with ordered regions. In addition, although some SLiMs are indeed conserved in IDRs (15-17), others appear in clusters where precise position and number are not conserved (18)(19)(20). Although it is reasonable to assume that conservation of sequence in IDRs is indicative of functional conservation of SLiMs, it is more difficult to interpret the functional consequences of IDRs that are highly diverged at the sequence level: These may represent either nonfunctional sequences evolving in the absence of constraint or weakly constrained functional elements that are gained or lost in a compensatory manner [undergoing evolutionary turnover (as described in refs. 18, 21)], such that they are not conserved at the amino acid sequence level.Like IDRs, noncoding DNA often shows relatively rapid evolution and weak constraints at the sequence level (22). Interestingly, IDRs show other parallels with noncoding DNA (18,23,24). For example, nonconserved clusters of phosphorylation sites in IDRs are reminiscent of nonconserved transcription factor binding sites in enhancers. Although these enhancers and the bi...

Salmonella Typhimurium (S. Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S. Tm replication by conducting parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S. Tm. Screening an S. Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella.

Graphical AbstractHighlights d Host-to-host transmission selects for AIEC with greater motility and acetate metabolism d Hypermotility mediates higher invasive and inflammatory host responses d Improved acetate utilization is a common trait for many hypomotile isolates d Crohn's disease E. coli isolates also exhibit increased motility and acetate utilization SUMMARY Crohn's disease (CD) is an inflammatory bowel disease influenced by bacteria. Adherent-invasive E. coli (AIEC) is associated with CD, yet the adaptations facilitating AIEC gut colonization are unknown. AIEC isolates exhibit high genetic diversity, suggesting strains evolve independently across different gut environments. We tracked the adaptive evolution of AIEC in a murine model of chronic colonization across multiple hosts and transmission events. We detected evolved lineages that outcompeted the ancestral strain in the host through independent mechanisms. One lineage was hypermotile because of a mobile insertion sequence upstream of the master flagellar regulator, flhDC, which enhanced AIEC invasion and establishment of a mucosal niche. Another lineage outcompeted the ancestral strain through improved use of acetate, a short-chain fatty acid in the gut. The presence of hypermotile and acetate-consuming lineages discriminated E. coli isolated from CD patients from healthy controls, suggesting an evolutionary trajectory that distinguishes AIEC from commensal E. coli.

Salmonella Typhimurium has a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to the various host environments it colonizes. The genome of Salmonella Typhimurium has undergone multiple gene acquisition events and has accrued changes in non-coding DNA that have undergone selection by regulatory evolution. Together, at least 17 horizontally acquired pathogenicity islands (SPIs), prophage-associated genes, and changes in core genome regulation contribute to the virulence program of Salmonella. Here, we review the latest understanding of these elements and their contributions to pathogenesis, emphasizing the regulatory circuitry that controls niche-specific gene expression. In addition to an overview of the importance of SPI-1 and SPI-2 to host invasion and colonization, we describe the recently characterized contributions of other SPIs, including the antibacterial activity of SPI-6 and adhesion and invasion mediated by SPI-4. We further discuss how these fitness traits have been integrated into the regulatory circuitry of the bacterial cell through cis-regulatory evolution and by a careful balance of silencing and counter-silencing by regulatory proteins. Detailed understanding of regulatory evolution within Salmonella is uncovering novel aspects of infection biology that relate to host-pathogen interactions and evasion of host immunity.

No abstract