Evolutionary Robustness of an Optimal Phenotype: Re-evolution of Lysis in a Bacteriophage Deleted for Its Lysin Gene

Optimality models are frequently used to create expectations about phenotypic evolution based on the fittest possible phenotype. However, they often ignore genetic details, which could confound these expectations. We experimentally analyzed the ability of organisms to evolve towards an optimum in an experimentally tractable system, lysis time in bacteriophage T7. T7 lysozyme helps lyse the host cell by degrading its cell wall at the end of infection, allowing viral escape to infect new hosts. Artificial deletion of lysozyme greatly reduced fitness and delayed lysis, but after evolution both phenotypes approached wild-type values. Phage with a lysis-deficient lysozyme evolved similarly. Several mutations were involved in adaptation, but most of the change in lysis timing and fitness increase was mediated by changes in gene 16, an internal virion protein not formerly considered to play a role in lysis. Its muralytic domain, which normally aids genome entry through the cell wall, evolved to cause phage release. Theoretical models suggest there is an optimal lysis time, and lysis more rapid or delayed than this optimum decreases fitness. Artificially constructed lines with very rapid lysis had lower fitness than wild-type T7, in accordance with the model. However, while a slow-lysing line also had lower fitness than wild-type, this low fitness resulted at least partly from genetic details that violated model assumptions.     

Codon Bias is a Major Factor Explaining Phage Evolution in Translationally Biased Hosts

The size and diversity of bacteriophage populations require methodologies to quantitatively study the landscape of phage differences. Statistical approaches are confronted with small genome sizes forbidding significant single-phage analysis, and comparative methods analyzing full phage genomes represent an alternative but they are of difficult interpretation due to lateral gene transfer, which creates a mosaic spectrum of related phage species. Based on a large-scale codon bias analysis of 116 DNA phages hosted by 11 translationally biased bacteria belonging to different phylogenetic families, we observe that phage genomes are almost always under codon selective pressure imposed by translationally biased hosts, and we propose a classification of phages with translationally biased hosts which is based on adaptation patterns. We introduce a computational method for comparing phages sharing homologous proteins, possibly accepted by different hosts. We observe that throughout phages, independently from the host, capsid genes appear to be the most affected by host translational bias. For coliphages, genes involved in virion morphogenesis, host interaction and ssDNA binding are also affected by adaptive pressure. Adaptation affects long and small phages in a significant way. We analyze in more detail the Microviridae phage space to illustrate the potentiality of the approach. The small number of directions in adaptation observed in phages grouped around ϕX174 is discussed in the light of functional bias. The adaptation analysis of the set of Microviridae phages defined around ϕMH2K shows that phage classification based on adaptation does not reflect bacterial phylogeny. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-Term Experimental Evolution in Escherichia coli. XIII. Phylogenetic History of a Balanced Polymorphism

We investigated the phylogenetic history of a balanced polymorphism that evolved in an experimental population of Escherichia coli. Previous work showed that two ecologically and morphologically distinct types, designated L (large) and S (small), arose by generation 6000 and coexisted for more than 12,000 generations thereafter. Here, we performed RFLP analyses using Insertion Sequence elements to resolve the phylogenetic history of L and S. Specifically, we sought to determine whether the derived S morph was monophyletic, indicating a long history of coexistence with L or, alternatively, S was repeatedly regenerated from L, indicating a series of periods with only transiently stable coexistence. Phylogenetic analysis of some 200 clones collected throughout the history of this population demonstrates that S is monophyletic. We then performed competition assays using clones of both morphs from different generations to determine whether either or both lineages continued to undergo genetic adaptation. Indeed, both lineages continued to adapt, and their continued evolution contributed to fluctuations in their relative abundance over evolutionary time. Based on their phylogenetic history and independent evolutionary trajectories, S and L fulfill Cohan’s criteria for being different asexual species.[Reviewing Editor: Niles Lehman]     

Evolution of genome size in fishes: a phylogenetic test of the Hinegardner and Rosen hypothesis

Despite remarkable advances in genomic studies over the past few decades, surprisingly little is known about the processes governing genome evolution at macroevolutionary timescales. In a seminal paper, Hinegardner and Rosen (Am Nat 106:621–644, 1972) suggested that taxa characterized by larger genomes should also display disproportionately stronger fluctuations in genome size. Therefore, according to the Hinegardner and Rosen (HR) hypothesis, there should be a negative correlation between average within-family genome size and its corresponding coefficient of variation (CV), a prediction that was supported by their analysis of the genomes of 275 species of fish. In this study we reevaluate the HR hypothesis using an expanded dataset (2050 genome size records). Moreover, in addition to the use of standard linear regression techniques, we also conducted modern comparative analyses that take into account phylogenetic non-independence. Our analyses failed to confirm the negative relationship detected in the original study, suggesting that the evolution of genome size in fishes might be more complex than envisioned by the HR hypothesis. Interestingly, the frequency distribution of fish genome sizes was strongly skewed, even on a logarithmic scale, suggesting that the dynamics underlying genome size evolution are driven by multiplicative phenomena, which might include chromosomal rearrangements and the expansion of transposable elements.     

Compositional Correlations in the Chicken Genome

This paper analyses the compositional correlations that hold in the chicken genome. Significant linear correlations were found among the regions studied—coding sequences (and their first, second, and third codon positions), flanking regions (5′ and 3′), and introns—as is the case in the human genome. We found that these compositional correlations are not limited to global GC levels but even extend to individual bases. Furthermore, an analysis of 1037 coding sequences has confirmed a correlation among GC₃, GC₂, and GC₁. The implications of these results are discussed. Received: 9 December 1998 / Accepted: 18 April 1999     

Identification and Functional Analysis of Genome Mutations in a Fluoride-Resistant Streptococcus mutans Strain

It is known that fluoride-resistant microorganisms are different from fluoride-sensitive ones in growth, adherence and metabolic activity. It was hypothesized that these phenotypic differences were due to stable genotypic changes in the fluoride-resistant strains. However, until now, no studies have reported these genotypic changes. The aim of this study is to identify such changes in a fluoride-resistant Streptococcus mutans strain (C180-2FR) using whole-genome shotgun (WGS) sequencing and to examine the potential function of the identified mutations by comparing gene expression between the fluoride-sensitive (C180-2) and C180-2FR strains. We performed 50 bp paired-end Illumina shotgun sequencing for both strains. Through extensive bioinformatic analysis, we were able to identify 8 single nucleotide polymorphisms (SNPs) in the genome of C180-2FR, which were further confirmed by Sanger sequencing. Expression of the genes containing or in proximity to the SNPs in C180-2 and C180-2FR was then quantified by real-time PCR. A gene cluster containing genes coding for fluoride antiporters was up-regulated 10-fold in C180-2FR when compared to that in C180-2, independent of growth phase. Two SNPs are located in this gene cluster, one in its promoter region and the other in its protein-coding region. In addition, one gene, which codes for a putative glycerol uptake facilitator protein, was found to be down-regulated by 60% in C180-2FR at an early growth phase. The promoter region of this gene contained a SNP. No difference in expression was found for the other SNP-containing genes. In summary, using WGS sequencing, we were able to uncover genetic changes in the genome of a fluoride-resistant strain. These findings can provide new insights into the mechanism of microbial fluoride resistance.     

The Consistent Phylogenetic Signal in Genome Trees Revealed by Reducing the Impact of Noise

Phylogenetic trees based on gene repertoires are remarkably similar to the current consensus of life history. Yet it has been argued that shared gene content is unreliable for phylogenetic reconstruction because of convergence in gene content due to horizontal gene transfer and parallel gene loss. Here we test this argument, by filtering out as noise those orthologous groups that have an inconsistent phylogenetic distribution, using two independent methods. The resulting phylogenies do indeed contain small but significant improvements. More importantly, we find that the majority of orthologous groups contain some phylogenetic signal and that the resulting phylogeny is the only detectable signal present in the gene distribution across genomes. Horizontal gene transfer or parallel gene loss does not cause systematic biases in the gene content tree.     

Long Repeats in a Huge Genome: Microsatellite Loci in the Grasshopper Chorthippus biguttulus

It is commonly believed that both the average length and the frequency of microsatellites correlate with genome size. We have estimated the frequency and the average length for 69 perfect dinucleotide microsatellites in an insect with an exceptionally large genome: Chorthippus biguttulus (Orthoptera, Acrididae). Dinucleotide microsatellites are not more frequent in C. biguttulus, but repeat arrays are 1.4 to 2 times longer than in other insect species. The average repeat number in C. biguttulus lies in the range of higher vertebrates. Natural populations are highly variable. At least 30 alleles per locus were found and the expected heterozygosity is above 0.95 at all three loci studied. In contrast, the observed heterozygosity is much lower (≤0.51), which could be caused by long null alleles. [Reviewing Editor: Dr. Dmitri Petrov] Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AY532396–AY532400.     

山羊草属异源多倍体植物基因组进化的ＲＡＰＤ分析

和２４个随机引物对山羊草属（Ａegilops L.)异源多倍体物种对其祖先二倍体物进行ＲＡＰＤ分析,对扩增出的３１３条带进行聚类分析发现,含Ｄ基因组的多倍体与二倍体祖先Ａe.squarrosa(DD)在聚类图上聚为一支;除Ａe.juvenalis(DDMMUU)聚到上一支外,含Ｕ基因组的多倍 与二倍体祖先Ae.umbellulata(ＵＵ）在聚类图上聚为另一支;多倍体与其他二倍体均不聚在一起,表明多倍体分别与Ａe.squarrosa(DD)、Ae.umbellulata(UU)具有较近的亲缘关系,这说明多倍体开之后,Ｄ和Ｕ基因组变化较小,而其他基因组则发生了较大的变化。     

Little Evidence for Synergism Among Deleterious Mutations in a Nonsegmented RNA Virus

Several models have been proposed to account for the segmentation of RNA viruses. One of the best known models suggests that segmentation, and mixing of segments during coinfections, is a way to eliminate deleterious mutations from the genome. However, for validity, this model requires that deleterious mutations interact in a synergistic way. That is, two mutations together should have a more deleterious effect than the result of adding their individual effects. Here I present evidence that deleterious mutations in foot-and-mouth disease virus produce a decline in fitness but that the relationship between the number of mutations fixed and the magnitude of fitness decline is compatible mainly with a nonsynergistic model. However, the statistical uncertainties associated with the data still give some room for the existence of very weak synergistic epistasis. Received: 2 November 1998 / Accepted: 19 April 1999     

Correlation of mycelial growth rate with other phenotypic characters in evolved genotypes of Aspergillus nidulans

Fungal populations can adapt to their environment by the generation and fixation of spontaneous beneficial mutations. In this study we examined whether adaptation, measured as an increased mycelial growth rate, has correlated responses in the filamentous fungus Aspergillus nidulans with several other metric characters that could be important fitness components (colony forming units, germination speed, and biomass formation). Studying 60 populations that had evolved over 800 generations by experimental evolution, we find that only mycelial growth rate increased during adaptation to growing on solid medium. We further found that among evolved strains colony forming units is negatively correlated with mycelial growth rate and that colony forming units and biomass formation show a positive correlation. Our results give insight into changes in fungal phenotype as a result of adaptation and suggest that mycelial growth rate is the only available target of selection.     

Modelling the evolution of genomes with integrated external and internal functions

The genomes that organisms transmit between generations contain information about different kinds of functions. The genome with the "best" mix and number of genes for these functions is the one that natural selection favours. Here I introduce a new way to model simple organisms with genes for external and internal functions, and use it to study the evolution of genome size. The external functions are exemplified by resource use and the internal functions by mutation control (repair). It is shown that even with a suitable proportion of genes for mutation control, the genomes in the organisms do not forever incorporate genes that increase resource use. Instead they evolve towards an optimal genome of limited size. The optimal proportion of genes for mutation control is shown to have an upper limit given by the ease with which transmission accuracy is improved by adding extra genes for this purpose to the genome. The model illustrates how natural selection on genomes integrates systems for the transmission of genetic information with systems relating to the external adaptation of the organism. It also opens up for other, more detailed theoretical investigations of genome functions.     

The Mystery of Two Straight Lines in Bacterial Genome Statistics

In special coordinates (codon position-specific nucleotide frequencies), bacterial genomes form two straight lines in 9-dimensional space: one line for eubacterial genomes, another for archaeal genomes. All the 348 distinct bacterial genomes available in Genbank in April 2007, belong to these lines with high accuracy. The main challenge now is to explain the observed high accuracy. The new phenomenon of complementary symmetry for codon position-specific nucleotide frequencies is observed. The results of analysis of several codon usage models are presented. We demonstrate that the mean-field approximation, which is also known as context-free, or complete independence model, or Segre variety, can serve as a reasonable approximation to the real codon usage. The first two principal components of codon usage correlate strongly with genomic G+C content and the optimal growth temperature, respectively. The variation of codon usage along the third component is related to the curvature of the mean-field approximation. First three eigenvalues in codon usage PCA explain 59.1%, 7.8% and 4.7% of variation. The eubacterial and archaeal genomes codon usage is clearly distributed along two third order curves with genomic G+C content as a parameter.     

Substantial Regional Variation in Substitution Rates in the Human Genome: Importance of GC Content, Gene Density, and Telomere-Specific Effects

This study presents the first global, 1-Mbp-level analysis of patterns of nucleotide substitutions along the human lineage. The study is based on the analysis of a large amount of repetitive elements deposited into the human genome since the mammalian radiation, yielding a number of results that would have been difficult to obtain using the more conventional comparative method of analysis. This analysis revealed substantial and consistent variability of rates of substitution, with the variability ranging up to twofold among different regions. The rates of substitutions of C or G nucleotides with A or T nucleotides vary much more sharply than the reverse rates, suggesting that much of that variation is due to differences in mutation rates rather than in the probabilities of fixation of C/G vs. A/T nucleotides across the genome. For all types of substitution we observe substantially more hotspots than coldspots, with hotspots showing substantial clustering over tens of Mbp’s. Our analysis revealed that GC-content of surrounding sequences is the best predictor of the rates of substitution. The pattern of substitution appears very different near telomeres compared to the rest of the genome and cannot be explained by the genome-wide correlations of the substitution rates with GC content or exon density. The telomere pattern of substitution is consistent with natural selection or biased gene conversion acting to increase the GC-content of the sequences that are within 10–15 Mbp away from the telomere.Reviewing Editor: Dr. Jerzy Jurka

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This revised version was published online in July 2005 with corrected page numbers.     

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.