Mutational Tolerance Research Articles

One Against All: A Broadly Influenza Neutralizing Man-made Monoclonal Antibody Passes Phase I

One Against All: A Broadly Influenza Neutralizing Man-made Monoclonal Antibody Passes Phase I

Multiple endocrine neoplasia type 2 kindred with novel tandem RET mutations: Case report with an applied in silico mutational tolerance analysis

The American Thyroid Association (ATA) has established guidelines for prophylactic thyroidectomy in multiple endocrine neoplasia type 2A (MEN2A) based on rearranged during transfection (RET) mutations. In silico analysis, which uses computer modeling to predict alterations in protein structure, is a new method for studying these mutations. We describe a kindred with MEN2A, all sharing a well-documented RET mutation, p.C634Y, as well as a mutation of undetermined significance, p.I852M, which we analyzed via in silico analysis. The p.C634Y mutation resulted in severe predicted RET alterations, whereas the p.I852M resulted in only modest changes. Both mutations together resulted in only a small additional disruptive effect in protein structure beyond that which occurred with p.C634Y alone. Although in silico analysis may be helpful in quantitating changes in protein structure that occur in patients who have novel RET mutations (single or multiple), additional factors must account for the highly variable aggressiveness of the disease (C-cell hyperplasia/medullary thyroid carcinoma [MTC]) noted in our kindred. © 2016 Wiley Periodicals, Inc. Head Neck 38: E1881-E1885, 2016.

Structural and Mechanistic Insights Derived from Saturation Mutagenesis of CcdB

CcdB is a 101-residue homodimeric protein, which is part of the toxin-antitoxin (CcdB-CcdA) module important in F-plasmid maintenance in E. coli. We generated a site saturation mutagenesis library of CcdB. This mutant library was subjected to phenotypic screening in a strain with and without the F-plasmid and sequenced using Illumina sequencing. From a comparison of mutational tolerance at each residue in these two strains, we were able to identify the active site residues of CcdB involved in Gyrase binding, solely from mutant phenotypes. Average mutational tolerance at each individual residue further correlated well with residue depth.It is important to understand the mechanism by which binding of antitoxin to toxin in complex with its cellular results in toxin release and target rejuvenation. While an allosteric mechanism has been proposed previously, a transient ternary complex of Gyrase:CcdB:CcdA, which is an essential component for allostery, could not be experimentally detected (De Jonge et al, 2009). We identified CcdB residues important in CcdA binding from the deep sequencing data. This information was used to design SPR experiments to probe for ternary complex formation. We were able to detect a transient ternary complex between the C-terminal residues (61-72) of CcdA and the CcdB-Gyrase complex. The present study led to further insights into the binding mechanism of CcdA to CcdB and the rejuvenation mechanism whereby CcdA extracts CcdB from its complex with Gyrase.Thus saturation mutagenesis combined with deep sequencing is a powerful tool to elucidate the structural and functional determinants of a protein.

Mutagenic Effects of Ultraviolet Radiation on Growth and Agronomic Characters in Maize Cultivars

Ultraviolet light has strong genotoxic effect to induce mutations for developing high genetic variability in yields, early maturity and other characters in crops. The study investigated the mutagenic effects of ultraviolet (UV) radiation on growth, yield, agronomic and mutation tolerance of six maize cultivars. Maize seeds were exposed to UV radiation, and planted in 7 kg soils in the polythene bags, while unexposed served as control. The effect of UV radiation on the first order interaction between weeks after planting (WAP) and treatments was only significant (p<0.05) on plant height. The interaction between treatments and cultivars also produced significant effect on all growth characters except leaf width. There was reduction in growth characters at 8 and 10 WAP for number of leaves, and at 10 WAP for plant height, leaf length and leaf width. All the growth and agronomic characters at 100 minutes of UV radiation were significantly higher (p<0.05) than other exposure periods. The exposure period of stover weight at 100 minutes were significantly higher (p<0.05) than other periods, while grain weight and total number of grains at 20 minutes exposure significantly higher, but not different from control. The height of ART/OB/98/SW1 was significantly higher than other cultivars. ART/OB/98/SW6, 10–1–Y and ART/OB/98/SW1 were the most tolerant cultivars to the mutagenic effect of the UV radiation. OBA 98 had the most significant genotypic effect for yield characters, while the genotypic influence on plant stand, plant aspect, mutation tolerance and plant harvest was highly significant. The grain weight per stand for OBA 98 and ART/OB/98/SW1 were significantly (p<0.05) higher than other cultivars, while the total number of grains for ART/0B/98/SW1 and ART/OB/98/SW6 were significantly higher and different from other cultivars. ART/OB/98/SW6 had the least values of days to plant emergence, mutation tolerance and other agronomic characters compared to other cultivars. The plant height was positive and strongly correlated (p<0.01) with leaf length, leaf width and number of leaves with r = 0.95, 0.96, 0.89 respectively. Only the periods of exposure of the UV radiation was positive and strongly correlated with leaf width (r = 0.79). The association between the stover weight and periods of exposure was positive and insignificant, while the correlation between total numbers of grains and grain weight per stand was positive and strong (r = 0.99). Therefore, quality protein maize cultivars should be improved by introgression of favourable genes of drought tolerance, grain yield and related characters through induced mutation of UV radiation.

CRISPR interference and priming varies with individual spacer sequences.

CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed ‘naïve’ adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.

Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum.

BackgroundMethanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes.ResultsThe methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by experimental evolution in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1.ConclusionThe methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0558-6) contains supplementary material, which is available to authorized users.

Genetic instability in budding and fission yeast-sources and mechanisms.

Cells are constantly confronted with endogenous and exogenous factors that affect their genomes. Eons of evolution have allowed the cellular mechanisms responsible for preserving the genome to adjust for achieving contradictory objectives: to maintain the genome unchanged and to acquire mutations that allow adaptation to environmental changes. One evolutionary mechanism that has been refined for survival is genetic variation. In this review, we describe the mechanisms responsible for two biological processes: genome maintenance and mutation tolerance involved in generations of genetic variations in mitotic cells of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. These processes encompass mechanisms that ensure the fidelity of replication, DNA lesion sensing and DNA damage response pathways, as well as mechanisms that ensure precision in chromosome segregation during cell division. We discuss various factors that may influence genome stability, such as cellular ploidy, the phase of the cell cycle, transcriptional activity of a particular region of DNA, the proficiency of DNA quality control systems, the metabolic stage of the cell and its respiratory potential, and finally potential exposure to endogenous or environmental stress.

In Silico Analysis of NF2 Gene Missense Mutations in Neurofibromatosis Type 2: From Genotype to Phenotype.

Computer-based (in silico) protein modeling to examine genotype-phenotype relationships for a given mutation has been applied to many genes but never to NF2. Missense mutations in the merlin protein occur in approximately 9% of patients with neurofibromatosis type 2 (NF2). Within this subset of patients, no genotype-phenotype correlations have been established. The aim of this study was to determine if genotype correlates with phenotype in the cohort of NF2 patients with missense mutations as a first step to defining a method to predict clinical phenotype from genotype for these patients. We analyzed 45 patients with NF2 as a result of missense mutations drawn from the United Kingdom NF2 registry. Our analysis included 17 different NF2 mutations from NF2 patients and six single-nucleotide polymorphisms (SNP)--presumed benign because they are observed in the dbSNP National Center for Biotechnology Information database and 1000 Genomes. We analyzed the mutations using three mutation tolerance prediction approaches: Align GVGD, SIFT, and PolyPhen-2. The mutation sites were also modeled on the three-dimensional crystal structure of merlin to investigate the spatial relationship of NF2-causing mutations. Two mutation tolerance predictors (SIFT and PolyPhen-2) were able to distinguish NF2-causing mutations from non-NF2-causing SNPs (p < 0.05). Mapping mutations on the molecular structure of merlin suggest that mutations resulting in greater structural conflicts within the protein are more likely to correlate with severe phenotypes. This work is a step toward a better understanding of genotype-phenotype relationships in NF2 caused by missense mutations using a computer-based methodology.

Dissecting enzyme function with microfluidic-based deep mutational scanning

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

260. AAV Ancestral Reconstruction Library Enables Selection of Broadly Infectious Viral Variants

Adeno-associated virus (AAV) vectors have demonstrated clinical efficacy in treating several diseases. Improved vectors may extend these landmark successes to other indications, and protein engineering approaches are enhancing the gene delivery properties of AAV vectors to address such unmet medical needs. To access new viral sequences with potentially enhanced infectious properties, and to gain insights into AAV's evolutionary history, we computationally designed and experimentally characterized an ancestral reconstruction of the AAV capsid. To this end, we first generated a phylogenetic tree of AAV sequences through Bayesian Markov chain Monte Carlo simulations, then selected a node for reconstruction based on its relative confidence value and proximity to AAV serotypes of clinical interest. We next used HandAlign, a Markov chain Monte Carlo alignment sampler, to predict the ancestral sequence of the most likely alignment at the selected node. This generated a reconstruction where most amino acids were predicted with high confidence, while 32 of the positions were predicted with low confidence. In order to address uncertainty in these positions, we synthesized the inferred ancestral capsid as a large combinatorial library incorporating degenerate residues at the 32 variable amino acid sites. We then characterized the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to six rounds of selection on a panel of representative cell lines. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general the selected ancestral libraries were broadly infectious across the cell line panel, indicating that they favor promiscuity over specificity. Unlike many current serotypes, these ancestral AAVs do not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Additionally, ancestral libraries were found to be as susceptible to neutralizing antibodies as extant serotypes, suggesting that such ancestral variant candidates exhibit immunogenic properties similar to modern descendants. Interestingly, ancestral AAVs retained infectivity at temperatures that completely ablated infectivity of modern serotypes; this higher thermostability may have supported increased evolvability through mutational tolerance. Finally, selected variants mediated 19-31 fold higher gene expression in muscle compared to AAV1, a serotype clinically utilized for muscle delivery, highlighting their potential promise for gene therapy.

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF's tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.

In silico analysis of RET variants in medullary thyroid cancer: from the computer to the bedside.

The American Thyroid Association (ATA) medullary thyroid cancer (MTC) guidelines group RET variants, in the setting of familial medullary thyroid cancer and multiple endocrine neoplasia type 2, into 4 classes of severity based on epidemiological data. The aim of this study was to determine if genotype correlates with phenotype in RET missense mutations. In silico mutational tolerance prediction. Academic research hospital. We analyzed all RET variants currently listed in the ATA guidelines for the management of MTC using 2 computer-based (in silico) mutation tolerance prediction approaches: PolyPhen-2 HumVar and PolyPhen-2 HumDiv. Our analysis also included 27 different RET single-nucleotide polymorphisms resulting in missense variants. There was a statistically significant difference in the overall HumDiv score between ATA groups A and B (P = .025) and a statistically significant different HumVar score between benign polymorphisms and ATA group A (P = .023). Overall, RET variants associated with a less aggressive clinical phenotype generally had a lower Hum Div/Var score. Polyphen-2 Hum Div/Var may provide additional clinical data to help distinguish benign from MEN2/familial medullary thyroid carcinoma-causing RET variants as well as less aggressive phenotypes (ATA A) from more aggressive ones (ATA B-C). In silico genetic analyses, with proper validation, may predict the phenotypic severity of RET variants, providing clinicians with a tool to aid clinical decision making in cases in which the RET variant is currently unknown or little epidemiological data are available.

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation.

High-throughput profiling of point mutations across the HIV-1 genome.

BackgroundThe HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance.ResultsHere we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions – representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket.ConclusionThe approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0124-6) contains supplementary material, which is available to authorized users.

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

The continuing challenge of HIV-1 treatment resistance in patients creates a need for the development of new antiretroviral inhibitors. The HIV nucleocapsid (NC) protein is a potential therapeutic target. NC is necessary for viral RNA packaging and in the early stages of viral infection. The high level of NC amino acid conservation among all HIV-1 clades suggests a low tolerance for mutations. Thus, NC mutations that could arise during inhibitor treatment to provide resistance may render the virus less fit. Disruption of NC function provides a unique opportunity to strongly dampen replication at multiple points during the viral life cycle with a single inhibitor. Although NC exhibits desirable features for a potential antiviral target, the structural flexibility, size, and the presence of two zinc fingers makes small molecule targeting of NC a challenging task. In this review, we discuss the recent advances in strategies to develop inhibitors of NC function and present a perspective on potential novel approaches that may help to overcome some of the current challenges in the field.

The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin.

Influenza is notable for its evolutionary capacity to escape immunity targeting the viral hemagglutinin. We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability. We created mutant viruses that incorporate most of the ≈10(4) amino-acid mutations to hemagglutinin from A/WSN/1933 (H1N1) influenza. After passaging these viruses in tissue culture to select for functional variants, we used deep sequencing to quantify mutation frequencies before and after selection. These data enable us to infer the preference for each amino acid at each site in hemagglutinin. These inferences are consistent with existing knowledge about the protein's structure and function, and can be used to create a model that describes hemagglutinin's evolution far better than existing phylogenetic models. We show that hemagglutinin has a high inherent tolerance for mutations at antigenic sites, suggesting that this is one factor contributing to influenza's antigenic evolution.

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring ‘self’ DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition.

Obligate Insect Endosymbionts Exhibit Increased Ortholog Length Variation and Loss of Large Accessory Proteins Concurrent with Genome Shrinkage

Extreme genome reduction has been observed in obligate intracellular insect mutualists and is an assumed consequence of fixed, long-term host isolation. Rapid accumulation of mutations and pseudogenization of genes no longer vital for an intracellular lifestyle, followed by deletion of many genes, are factors that lead to genome reduction. Size reductions in individual genes due to small-scale deletions have also been implicated in contributing to overall genome shrinkage. Conserved protein functional domains are expected to exhibit low tolerance for mutations and therefore remain relatively unchanged throughout protein length reduction while nondomain regions, presumably under less selective pressures, would shorten. This hypothesis was tested using orthologous protein sets from the Flavobacteriaceae (phylum: Bacteroidetes) and Enterobacteriaceae (subphylum: Gammaproteobacteria) families, each of which includes some of the smallest known genomes. Upon examination of protein, functional domain, and nondomain region lengths, we found that proteins were not uniformly shrinking with genome reduction, but instead increased length variability and variability was observed in both the functional domain and nondomain regions. Additionally, as complete gene loss also contributes to overall genome shrinkage, we found that the largest proteins in the proteomes of nonhost-restricted bacteroidetial and gammaproteobacterial species often were inferred to be involved in secondary metabolic processes, extracellular sensing, or of unknown function. These proteins were absent in the proteomes of obligate insect endosymbionts. Therefore, loss of genes encoding large proteins not required for host-restricted lifestyles in obligate endosymbiont proteomes likely contributes to extreme genome reduction to a greater degree than gene shrinkage.

A Comprehensive, High-Resolution Map of a Gene’s Fitness Landscape

Mutations are central to evolution, providing the genetic variation upon which selection acts. A mutation’s effect on the suitability of a gene to perform a particular function (gene fitness) can be positive, negative, or neutral. Knowledge of the distribution of fitness effects (DFE) of mutations is fundamental for understanding evolutionary dynamics, molecular-level genetic variation, complex genetic disease, the accumulation of deleterious mutations, and the molecular clock. We present comprehensive DFEs for point and codon mutants of the Escherichia coli TEM-1 β-lactamase gene and missense mutations in the TEM-1 protein. These DFEs provide insight into the inherent benefits of the genetic code’s architecture, support for the hypothesis that mRNA stability dictates codon usage at the beginning of genes, an extensive framework for understanding protein mutational tolerance, and evidence that mutational effects on protein thermodynamic stability shape the DFE. Contrary to prevailing expectations, we find that deleterious effects of mutation primarily arise from a decrease in specific protein activity and not cellular protein levels.