Natural populations persist in complex environments, where biotic stressors, such as pathogen and insect communities, fluctuate temporally and spatially. These shifting biotic pressures generate heterogeneous selective forces that can maintain standing natural variation within a species. To directly test if genes containing causal variation for the Arabidopsis thaliana defensive compounds, glucosinolates (GSL) control field fitness and are therefore subject to natural selection, we conducted a multi-year field trial using lines that vary in only specific causal genes. Interestingly, we found that variation in these naturally polymorphic GSL genes affected fitness in each of our environments but the pattern fluctuated such that highly fit genotypes in one trial displayed lower fitness in another and that no GSL genotype or genotypes consistently out-performed the others. This was true both across locations and within the same location across years. These results indicate that environmental heterogeneity may contribute to the maintenance of GSL variation observed within Arabidopsis thaliana.DOI: http://dx.doi.org/10.7554/eLife.05604.001

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase (CheA), and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the underlying chemosensory arrays and how these assemblies engender sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the chemotaxis kinase CheA, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts.

During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric, histidine-kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and x-ray scattering studies of thermostable CheA from Thermotoga maritima find that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP nor ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains, or removal of one within a dimer, increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates inter-subunit (trans) versus intra-subunit (cis) autophosphorylation; with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans-directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and suggests that the ATP-lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo–electron microscopy. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu, are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu-to-NO distances and examine the effects of solvent conditions on Cu binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY-PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu-to-NO and Cu-to-Cu PDS measurements of CheY-PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.

Despite the growing number of studies showing that genotype × environment and epistatic interactions control fitness, the influences of epistasis × environment interactions on adaptive trait evolution remain largely uncharacterized. Across three field trials, we quantified aliphatic glucosinolate (GSL) defense chemistry, leaf damage, and relative fitness using mutant lines of Arabidopsis thaliana varying at pairs of causal aliphatic GSL defense genes to test the impact of epistatic and epistasis × environment interactions on adaptive trait variation. We found that aliphatic GSL accumulation was primarily influenced by additive and epistatic genetic variation, leaf damage was primarily influenced by environmental variation and relative fitness was primarily influenced by epistasis and epistasis × environment interactions. Epistasis × environment interactions accounted for up to 48% of the relative fitness variation in the field. At a single field site, the impact of epistasis on relative fitness varied significantly over 2 yr, showing that epistasis × environment interactions within a location can be temporally dynamic. These results suggest that the environmental dependency of epistasis can profoundly influence the response to selection, shaping the adaptive trajectories of natural populations in complex ways, and deserves further consideration in future evolutionary studies.

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to β-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable μ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]2-O22−, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

Streptomycetes are sessile bacteria that produce metabolites that impact the behavior of microbial communities. Emerging studies have demonstrated that Streptomyces spores are distributed through various mechanisms, but it remains unclear how spores are transported to their preferred microenvironments, such as plant roots. Here, we show that Streptomyces spores are capable of utilizing the motility machinery of other soil bacteria. Motility assays and microscopy studies reveal that Streptomyces spores are transported to plant tissues by interacting directly with the flagella of both gram-positive and gram-negative bacteria. Genetics experiments demonstrate that this form of motility is facilitated by structural proteins on the spore coat. These results demonstrate that nonmotile bacteria are capable of utilizing the motility machinery of other microbes to complete necessary stages of their lifecycle.