Temperature effects on cellular host-microbe interactions explain continent-wide endosymbiont prevalence.

Endosymbioses influence host physiology, reproduction, and fitness, but these relationships require efficient microbe transmission between host generations to persist. Maternally transmitted Wolbachia are the most common known endosymbionts,1 but their frequencies vary widely within and among host populations for unknown reasons.2,3 Here, we integrate genomic, cellular, and phenotypic analyses with mathematical models to provide an unexpectedly simple explanation for global wMel Wolbachia prevalence in Drosophila melanogaster. Cooling temperatures decrease wMel cellular abundance at a key stage of host oogenesis, producing temperature-dependent variation in maternal transmission that plausibly explains latitudinal clines of wMel frequencies on multiple continents. wMel sampled from a temperate climate targets the germline more efficiently in the cold than a recently differentiated tropical variant (∼2,200 years ago), indicative of rapid wMel adaptation to climate. Genomic analyses identify a very narrow list of wMel alleles-most notably, a derived stop codon in the major Wolbachia surface protein WspB-that underlie thermal sensitivity of cellular Wolbachia abundance and covary with temperature globally. Decoupling temperate wMel and host genomes further reduces transmission in the cold, a pattern that is characteristic of host-microbe co-adaptation to a temperate climate. Complex interactions among Wolbachia, hosts, and the environment (GxGxE) mediate wMel cellular abundance and maternal transmission, implicating temperature as a key determinant of Wolbachia spread and equilibrium frequencies, in conjunction with Wolbachia effects on host fitness and reproduction.4,5 Our results motivate the strategic use of locally selected wMel variants for Wolbachia-based biocontrol efforts, which protect millions of individuals from arboviruses that cause human disease.6.

The Small ß-Barrel Domain: A Survey-Based Structural Analysis.

The small ß-barrel (SBB) is an ancient protein structural domain characterized by extremes: it features a broad range of structural varieties, a deeply intricate evolutionary history, and it is associated with a bewildering array of cellular pathways. Here, we present a thorough, survey-based analysis of the structural properties of SBBs. We first consider the defining properties of the SBB, including various systems of nomenclature used to describe it, and we introduce the unifying concept of an "urfold." To begin elucidating how vast functional diversity can be achieved by a relatively simple domain, we explore the anatomy of the SBB and its representative structural variants. Many SBB proteins assemble into cyclic oligomers as the biologically functional units; these oligomers often bind RNA, and typically exhibit great quaternary structural plasticity (homomeric and heteromeric rings, variable subunit stoichiometries, etc.). We conclude with three themes that emerge from the rich structure ↔ function versatility of the SBB.

Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly.

Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA∙∙∙RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways-including stress response, quorum sensing, and biofilm formation-has motivated genetic and "RNAomic" studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq∙∙∙RNA interactions (in vitro). Indeed, crystallographic and biophysical studies first established Hfq as a member of the phylogenetically conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq·RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P1 and P6, both to 1.5 Šresolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.