Synonymous and Nonsynonymous Substitutions in Dictyostelium discoideum Ammonium Transporter amtA Are Necessary for Functional Complementation in Saccharomyces cerevisiae.

Ammonium transporters are present in all three domains of life. They have undergone extensive horizontal gene transfer (HGT), gene duplication, and functional diversification and therefore offer an excellent paradigm to study protein evolution. We attempted to complement a mep1Δmep2Δmep3Δ strain of Saccharomyces cerevisiae (triple-deletion strain), which otherwise cannot grow on ammonium as a sole nitrogen source at concentrations of <3 mM, with amtA of Dictyostelium discoideum, an orthologue of S. cerevisiae MEP2. We observed that amtA did not complement the triple-deletion strain of S. cerevisiae for growth on low-ammonium medium. We isolated two mutant derivatives of amtA (amtA M1 and amtA M2) from a PCR-generated mutant plasmid library that complemented the triple-deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions, which are necessary for its functionality. amtA M2 bears two nonsynonymous substitutions and one synonymous substitution, all of which are necessary for functionality. Interestingly, AmtA M1 transports ammonium but does not confer methylamine toxicity, while AmtA M2 transports ammonium and confers methylamine toxicity, demonstrating functional diversification. Preliminary biochemical analyses indicated that the mutants differ in their conformations as well as their mechanisms of ammonium transport. These intriguing results clearly point out that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions in isolation. The above-described observations have significant implications for various facets of biological processes and are discussed in detail. IMPORTANCE Functional diversification following gene duplication is one of the major driving forces of protein evolution. While the role of nonsynonymous substitutions in the functional diversification of proteins is well recognized, knowledge of the role of synonymous substitutions in protein evolution is in its infancy. Using functional complementation, we isolated two functional alleles of the D. discoideum ammonium transporter gene (amtA), which otherwise does not function in S. cerevisiae as an ammonium transporters. One of them is an ammonium transporter, while the other is an ammonium transporter that also confers methylammonium (ammonium analogue) toxicity, suggesting functional diversification. Surprisingly, both alleles require a combination of synonymous and nonsynonymous substitutions for their functionality. These results bring out a hitherto-unknown pathway of protein evolution and pave the way for not only understanding protein evolution but also interpreting single nucleotide polymorphisms (SNPs).

A Reverse Genetic Approach for Studying sRNAs in Chlamydia trachomatis.

sRNAs are noncoding transcripts that play critical roles in posttranscriptional regulation in prokaryotes. In the intracellular bacterium Chlamydia, sRNAs have been identified, but functional studies have been limited to an E. coli heterologous system. We have developed an inducible sRNA overexpression system in Chlamydia trachomatis and used it to screen putative sRNAs for effects on the Chlamydia developmental cycle, which involves conversion between replicating (RB) and infectious (EB) chlamydial forms. Overexpression of 4 of 13 C. trachomatis sRNAs decreased production of infectious EBs. We performed detailed characterization of CtrR3 and CtrR7, the two sRNAs that caused the largest progeny defects in our screen. By quantifying chlamydial number and infectious progeny, and by visualizing chlamydial forms using electron microscopy, we showed that overexpression of CtrR3 prevented RB-to-EB conversion, whereas CtrR7 overexpression blocked bacterial replication. We also describe a workflow that allowed us to identify the mRNA targets of CtrR3 in Chlamydia. We first used MS2 aptamer affinity purification coupled with RNA sequencing as an unbiased approach to isolate interacting mRNAs. We then prioritized candidates based on sequence complementarity to the CtrR3 target recognition sequence, which we had identified with bioinformatic and mutational analyses. Finally, we tested putative targets with translational fusion assays in E. coli and C. trachomatis. Using this integrated approach, we provide experimental evidence that YtgB and CTL0389 are mRNA targets of CtrR3 in Chlamydia. These findings demonstrate how our C. trachomatis sRNA overexpression system can be used to investigate the functions and mRNA targets of chlamydial sRNAs. IMPORTANCE Small RNAs (sRNAs) are a class of regulatory RNAs that play important roles in bacterial physiology and pathogenesis. In the intracellular bacterium Chlamydia, however, sRNAs are poorly understood, and functional studies have been limited to a heterologous system. In this study, we developed a genetic system for studying sRNAs in Chlamydia trachomatis and used it to identify four chlamydial sRNAs whose overexpression decreased the production of infectious bacteria. We also successfully utilized this genetic system to determine the target recognition sequence and mRNA targets of an uncharacterized, chlamydial sRNA named CtrR3. Overall, this work offers a generalizable approach for investigating the role of chlamydial sRNAs in their native organism.

Glycogen synthase kinase-ß3 in ischemic neuronal death.

Glycogen synthase kinase 3ß (GSK-3ß) is implicated in diverse cellular processes such as cell signaling and survival. Accumulating lines of evidence indicate that increased GSK-3ß activity contributes to neuronal death and pathogenesis of ischemic stroke. Considering predominant roles of GSK-3ß in neuronal apoptosis, modulation of this protein kinase is a reliable strategy for ischemic neuroprotection. In this review, we survey and synthesize the current knowledge about the role of GSK-3ß in neuroprotection following the ischemic stroke.