Christopher G. Knight

Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity

Understanding the connections among genotype, phenotype, and fitness through evolutionary time is a central goal of evolutionary genetics. Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations and show substantial morphological and fitness differences. Previous work identified genes contributing to the evolutionary success of WS, in particular the di-guanylate cyclase response regulator, WspR. Here we scrutinize the Wsp signal transduction pathway of which WspR is the primary output component. The pathway has the hallmarks of a chemosensory pathway and genetic analyses show that regulation and function of Wsp is analogous to the Che chemotaxis pathway from Escherichia coli. Of significance is the methyltransferase (WspC) and methylesterase (WspF) whose opposing activities form an integral feedback loop that controls the activity of the kinase (WspE). Deductions based on the regulatory model suggested that mutations within wspF were a likely cause of WS. Analyses of independent WS genotypes revealed numerous simple mutations in this single open reading frame. Remarkably, different mutations have different phenotypic and fitness effects. We suggest that the negative feedback loop inherent in Wsp regulation allows the pathway to be tuned by mutation in a rheostat-like manner.

Array-based evolution of DNA aptamers allows modelling of an explicit sequence-fitness landscape

Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44 131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.

A novel mode of ecdysozoan growth in Caenorhabditis elegans

SUMMARY Whereas growth in many ecdysozoa is associated with only molting, larval growth in nematodes, specifically Caenorhabditis elegans, is thought to be continuous and exponential. However, this has never been closely investigated. Here we report several detailed studies of growth in wild‐type and dwarf C. elegans strains. We find that apparent exponential growth between hatching and adulthood comprises a series of linear phases, one per larval stage, with the linear growth rate increasing at successive molts. Although most structures grow continuously, the buccal cavity does not; instead, it grows saltationally at molts, like arthropod structures. We speculate that these saltational changes in mouth size permit changes in growth rate and that molting exists in nematodes to facilitate rapid growth. We study the cellular basis of this growth in the hypodermis. At each larval stage, lateral seam cells produce daughters that fuse with hyp7, a syncytium covering most of the worm. We find that seam cells and fusing daughter cells obtain larger sizes in successive molts. The total seam cell volume remains constant relative to the size of the worm. However, fusing daughter cells contributes only a very small amount directly to hypodermal growth, suggesting that most hyp7 growth must be intrinsic. Thus, dwarfism mutations studied principally act via adult syncytial growth, with cell size being near normal in both dbl‐1 and dpy‐2 mutant worms. We speculate that the main function of seam cell proliferation may be to supply the hypodermis with additional genomes for the purpose of growth.

Association of parameter, software, and hardware variation with large-scale behavior across 57,000 climate models

In complex spatial models, as used to predict the climate response to greenhouse gas emissions, parameter variation within plausible bounds has major effects on model behavior of interest. Here, we present an unprecedentedly large ensemble of >57,000 climate model runs in which 10 parameters, initial conditions, hardware, and software used to run the model all have been varied. We relate information about the model runs to large-scale model behavior (equilibrium sensitivity of global mean temperature to a doubling of carbon dioxide). We demonstrate that effects of parameter, hardware, and software variation are detectable, complex, and interacting. However, we find most of the effects of parameter variation are caused by a small subset of parameters. Notably, the entrainment coefficient in clouds is associated with 30% of the variation seen in climate sensitivity, although both low and high values can give high climate sensitivity. We demonstrate that the effect of hardware and software is small relative to the effect of parameter variation and, over the wide range of systems tested, may be treated as equivalent to that caused by changes in initial conditions. We discuss the significance of these results in relation to the design and interpretation of climate modeling experiments and large-scale modeling more generally.

Evolution of germ-line signals that regulate growth and aging in nematodes

We show that a signal from the germ line represses growth in the nematode Caenorhabditis elegans. Laser-microbeam ablation of cells that give rise to the germ line causes adults to become giant. Ablation of these cells in self-sterile mutant worms also causes gigantism, suggesting that the germ line represses growth because it is the source of a growth-antagonizing signal rather than because of a sink of resources required for reproduction. The C. elegans germ line also emits a signal that represses longevity. This longevity-repressing signal requires the activity of DAF-16, a forkhead/winged-helix transcription factor, but we find that that the growth-repressing signal does not. The growth-repressing signal also does not require the activity of DBL-1, a transforming growth factor β-related protein that promotes growth in worms. By ablating the germ-line precursors of other species of free-living nematodes, we also found that both the growth-repressing and longevity-repressing signals are evolutionarily variable. Some species have both signals; others have just one or the other. We suggest that variation in germ-line signaling contributes to body size and life-history diversity in the nematodes.

Absolute Quantification of the Glycolytic Pathway in Yeast: DEPLOYMENT OF A COMPLETE QconCAT APPROACH

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.

TESTING LIFE-HISTORY PLEIOTROPY IN CAENORHABDITIS ELEGANS

Abstract Much life‐history theory assumes that alleles segregating in natural populations pleiotropically affect life‐history traits. This assumption, while plausible, has rarely been tested directly. Here we investigate the genetic relationship between two traits often suggested to be connected by pleiotropy: maternal body size and fertility. We carry out a quantitative trait locus (QTL) analysis on two isolates of the free‐living nematode Caenorhabditis elegans, and identify two body size and three fertility QTLs. We find that one of the fertility QTLs colocalizes with the two body size QTLs on Chromosome IV. Further analysis, however, shows that these QTLs are genetically separable. Thus, none of the five body size or fertility QTLs identified here shows detectable pleiotropy for the assayed traits. The evolutionary origin of these QTLs, possible candidate loci, and the significance for life‐history evolution are discussed.

Mutation rate plasticity in rifampicin resistance depends on Escherichia coli cell–cell interactions

Variation of mutation rate at a particular site in a particular genotype, in other words mutation rate plasticity (MRP), can be caused by stress or ageing. However, mutation rate control by other factors is less well characterized. Here we show that in wild-type Escherichia coli (K-12 and B strains), the mutation rate to rifampicin resistance is plastic and inversely related to population density: lowering density can increase mutation rates at least threefold. This MRP is genetically switchable, dependent on the quorum-sensing gene luxS—specifically its role in the activated methyl cycle—and is socially mediated via cell–cell interactions. Although we identify an inverse association of mutation rate with fitness under some circumstances, we find no functional link with stress-induced mutagenesis. Our experimental manipulation of mutation rates via the social environment raises the possibility that such manipulation occurs in nature and could be exploited medically.

Making the right connections: biological networks in the light of evolution

Our understanding of how evolution acts on biological networks remains patchy, as is our knowledge of how that action is best identified, modelled and understood. Starting with network structure and the evolution of protein–protein interaction networks, we briefly survey the ways in which network evolution is being addressed in the fields of systems biology, development and ecology. The approaches highlighted demonstrate a movement away from a focus on network topology towards a more integrated view, placing biological properties centre‐stage. We argue that there remains great potential in a closer synergy between evolutionary biology and biological network analysis, although that may require the development of novel approaches and even different analogies for biological networks themselves.

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions, local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.