Intimate evolution of proteins. Proteome atomic content correlates with genome base composition

Discerning the significant relations that exist within and among genome sequences is a major step toward the modeling of biopolymer evolution. Here we report the systematic analysis of the atomic composition of proteins encoded by organisms representative of each kingdoms. Protein atomic contents are shown to vary largely among species, the larger variations being observed for the main architectural component of proteins, the carbon atom. These variations apply to the bulk proteins as well as to subsets of ortholog proteins. A pronounced correlation between proteome carbon content and genome base composition is further evidenced, with high G+C genome content being related to low protein carbon content. The generation of random proteomes and the examination of the canonical genetic code provide arguments for the hypothesis that natural selection might have driven genome base composition.     

How Prochlorococcus bacteria use nitrogen sparingly in their proteins

Organisms use proteins to perform an enormous range of functions that are essential for life. Proteins are usually composed of 20 different kinds of amino acids that each contain between one and four nitrogen atoms. In aggregate, the nitrogen atoms that are bound in proteins typically account for a substantial fraction of the nitrogen in a cell. Many organisms obtain the nitrogen that they use to make proteins from the environment, where its availability can vary greatly. These observations prompt the question: can environmental nitrogen scarcity lead to adaptive evolution in the nitrogen content of proteins? In this issue, Gilbert & Fagan (2011) address this question in the marine cyanobacteria Prochlorococcus, examining a variety of ways in which cells might be thrifty with nitrogen when making proteins. They show that different Prochlorococcus strains vary substantially in the average nitrogen content of their encoded proteins and relate this variation to nitrogen availability in different marine habitats and to genomic base composition (GC content). They also consider biases in the nitrogen content of different kinds of proteins. In most Prochlorococcus strains, a group of proteins that are commonly induced during nitrogen stress are poor in nitrogen relative to other proteins, probably reflecting selection for reduced nitrogen content. In contrast, ribosomal proteins are nitrogen rich relative to other Prochlorococcus proteins, and tend to be down‐regulated during nitrogen limitation. This suggests the possibility that decaying ribosomal proteins act as a source of nitrogen‐rich amino acids during periods of nitrogen stress. This work contributes to our understanding of how nutrient limitation might lead to adaptive variation in the composition of proteins and signals that marine microbes hold great promise for testing hypotheses about protein elemental costs in the future.     

Contrasting mechanisms of proteomic nitrogen thrift in Prochlorococcus

Organisms limited by carbon, nitrogen or sulphur can reduce protein production costs by transitions to less costly amino acids, or by reducing protein expression. These alternative mechanisms of nutrient thrift might respond differently to selection, but this possibility remains untested. We hypothesized that relatively invariant sequence composition responds to long-term variation in nutrient concentrations, whereas dynamic expression profiles vary with nutrient predictability. Prolonged nutrient scarcity favours proteome-wide nutrient reduction. Under stable, nonfluctuating nutrient availability, reduction of nutrient content typically occurs in proteins upregulated when nutrient availability is low, e.g. assimilation and catabolism. We suggest that fluctuating nutrient availability favours mechanisms involving short-term downregulation of nutrient-rich proteins. We analysed protein nitrogen content in six high-light, low-nutrient adapted (HL) vs. six low-light, high-nutrient adapted (LL) Prochlorococcus (marine cyanobacteria) strains, alongside expression data under experimental nitrogen and phosphorus limitation in two strains, MED4 (HL) vs. MIT9313 (LL). HL strains contained less nitrogen, but DNA GC content confounded this relationship. While anabolic and catabolic proteins had normal nitrogen content, most strains showed reduced nitrogen in typical nitrogen stress response proteins. In the experimental data set, though, proteins upregulated under nitrogen limitation were nitrogen-poor only in MIT9313, not MED4. MIT9313 responded similarly to nitrogen and phosphorus limitation, with slow, sustained downregulation of nitrogen-rich ribosomal proteins. In contrast, under nitrogen but not phosphorus limitation, MED4 rapidly downregulated ribosomal proteins. MED4's specific, rapid nitrogen response suggests adaptation to fluctuating conditions, supporting previous work. Thus, we identify contrasting proteomic nitrogen thrift mechanisms within Prochlorococcus consistent with different nutrient regimes.     

Signatures of nitrogen limitation in the elemental composition of the proteins involved in the metabolic apparatus

Nitrogen (N) is a fundamental component of nucleotides and amino acids and is often a limiting nutrient in natural ecosystems. Thus, study of the N content of biomolecules may establish important connections between ecology and genomics. However, while significant differences in the elemental composition of whole organisms are well documented, how the flux of nutrients in the cell has shaped the evolution of different cellular processes remains poorly understood. By examining the elemental composition of major functional classes of proteins in four multicellular eukaryotic model organisms, we find that the catabolic machinery shows substantially lower N content than the anabolic machinery and the rest of the proteome. This pattern suggests that ecological selection for N conservation specifically targets cellular components that are highly expressed in response to nutrient limitation. We propose that the RNA component of the anabolic machineries is the mechanistic force driving the elemental imbalance we found, and that RNA functions as an intracellular nutrient reservoir that is degraded and recycled during starvation periods. A comparison of the elemental composition of the anabolic and catabolic machineries in species that have experienced different levels of N limitation in their evolutionary history (animals versus plants) suggests that selection for N conservation has preferentially targeted the catabolic machineries of plants, resulting in a lower N content of the proteins involved in their catabolic processes. These findings link the composition of major cellular components to the environmental factors that trigger the activation of those components, suggesting that resource availability has constrained the atomic composition and the molecular architecture of the biotic processes that enable cells to respond to reduced nutrient availability.     

Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.     

Evidence for the adaptive evolution of the carbon fixation gene rbcL during diversification in temperature tolerance of a clade of hot spring cyanobacteria

Determining the molecular basis of enzyme adaptation is central to understanding the evolution of environmental tolerance but is complicated by the fact that not all amino acid differences between ecologically divergent taxa are adaptive. Analysing patterns of nucleotide sequence evolution can potentially guide the investigation of protein adaptation by identifying candidate codon sites on which diversifying selection has been operating. Here, I test whether there is evidence for molecular adaptation of the carbon fixation gene rbcL for a clade of hot spring cyanobacteria in the genus Synechococcus that has diverged in thermotolerance. Amino acid replacements during Synechococcus radiation have resulted in an increase in the number of hydrophobic residues in the RbcLs of more thermotolerant strains. A similar increase in hydrophobicity has been observed for many thermostable proteins. Maximum likelihood models which allow for heterogeneity among codon sites in the ratio of nonsynonymous to synonymous nucleotide substitutions estimated a class of amino acid sites as a target of positive selection. Depending on the model, a single amino acid site that interacts with a flexible element involved in the opening and closing of the active site was estimated with either low or moderate support to be a member of this class. Site-directed mutagenesis approaches are being explored in order to directly test its adaptive significance.     

Degradation kinetics of 4-amino naphthalene-1-sulfonic acid by a biofilm-forming bacterial consortium under carbon and nitrogen limitations

By decolorization of azo dyes, caused by reductive cleavage of the azo linkage, toxic or recalcitrant amines are generated. The present study deals with the effect of the inflowing medium composition (C:N ratio) on the kinetic behavior of a bacterial biofilm-forming consortium, able to use as carbon, nitrogen and sulfur source, the molecule of 4-aminonaphthalene-1-sulfonic acid (4ANS), which is one of the most recalcitrant byproducts generated by decolorization of azo dyes. All the experiments were carried out at room temperature in a lab-scale packed-bed biofilm reactor. Because environmental conditions affect the bioreactor performance, two mineral salts media containing 4ANS, with distinct C:N ratios; 0.68 (carbon as the limiting nutrient) and 8.57 (nitrogen as the limiting nutrient) were used to evaluate their effect on 4ANS biodegradation. By HPLC and COD measurements, the 4ANS removal rates and removal efficiencies were determined. The cultivable bacterial strains that compose the consortium were identified by their 16S rDNA gene sequence. With the enrichment technique used, a microbial consortium able to use efficiently 4ANS as the sole carbon source and energy, nitrogen and sulfur, was selected. The bacterial strains that constitute the consortium were isolated and identified. They belong to the following genera: Bacillus, Arthrobacter, Microbacterium, Nocardioides, and Oleomonas. The results obtained with this consortium showed, under nitrogen limitation, a remarkable increase in the 4ANS removal efficiency η(ANS), and in the 4ANS volumetric removal rates R (V,4ANS), as compared to those obtained under carbon limitation. Differences observed in bioreactor performance after changing the nutrient limitation could be caused by changes in biofilm properties and structure.     

Applications of carbon nanotubes for cancer research

Carbon nanotubes have many unique properties such as high surface area, hollow cavities, and excellent mechanical and electrical properties. Interfacing carbon nanotubes with biological systems could lead to significant applications in various disease diagnoses. Significant progress in interfacing carbon nanotubes with biological materials has been made in key areas such as aqueous solubility, chemical and biological functionalization for biocompatibility and specificity, and electronic sensing of proteins. In addition, the bioconjugated nanotubes combined with the sensitive nanotube-based electronic devices would enable sensitive biosensors toward medical diagnostics. Furthermore, recent findings of improved cell membrane permeability for carbon nanotubes would also expand medical applications to therapeutics using carbon nanotubes as carriers in gene delivery systems. This article reviews the current trends in biological functionalization of carbon nanotubes and their potential applications for breast cancer diagnostics. The article also reports the applications of confocal microscopy for use in understanding the interactions of biological materials such as antibodies on carbon nanotubes that are specific to surface receptors in breast cancer cells. Furthermore, a nanotube-field-effect transistor is demonstrated for electronic sensing of antibodies that are specific to surface receptors in cancer cells.     

Protein Structures and Neutral Theory of Evolution

Abstract

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent “memorized” random sequences while biological evolution reduces to the “editing” these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirements do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and unpolar amino acid residues can form α-helices and β-strands with lengths and arrangement along the chain similar to those in real globular proteins. These α- and β-regions in random sequences can form three-dimensional folding patterns also similar to those in real proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active sites of proteins and for their stability under physiological conditions.     

碳源与氮源限制下细菌代谢调节研究进展

营养限制是微生物最常面临的环境胁迫之一。除了在营养物质匮乏的海洋、冰川、沙漠、深层地表等自然环境中,越来越多的人工环境也出现了营养限制的特征,例如各类微污染水体、提标改造的废水生物处理系统等。基质浓度极大地影响着包括细菌在内的许多微生物的生长、代谢及群落结构,最终导致其功能的改变。为了在营养限制条件下维持生存,微生物首先需感知营养供给的减少,其后通过基因、蛋白质、信号分子、代谢产物等对各代谢过程进行全局调控,最后改变基质亲和力、生长速率、运动能力、形态等以适应营养不足。胞内各种信号物质及其触发的响应是微生物应对营养胁迫的关键。本文分别梳理了以细菌为代表的微生物应对碳源、氮源限制时的关键信号物质、受体蛋白/调控过程及响应结果,并分析了碳氮限制响应过程中的相互作用,以期为极端环境微生物的认识、营养限制条件下微生物的应用,尤其是低浓度污染物生物处理、生物监测等领域提供理论基础。     

Threshold elemental ratios of carbon and phosphorus in aquatic consumers

Inadequate supply of one or more mineral elements can slow the growth of animal consumers and alter their physiology, life history and behaviour. A key concept for understanding nutrient deficiency in animals is the threshold elemental ratio (TER), at which growth limitation switches from one element to another. We used a stoichiometric model that coupled animal bioenergetics and body elemental composition to estimate TER of carbon and phosphorus (TER_C:P) for 41 aquatic consumer taxa. We found a wide range in TER_C:P (77–3086, ratio by atoms), which was generated by interspecific differences in body C : P ratios and gross growth efficiencies of C. TER_C:P also varied among aquatic invertebrates having different feeding strategies, such that detritivores had significantly higher threshold ratios than grazers and predators. The higher TER_C:P in detritivores resulted not only from lower gross growth efficiencies of carbon but also reflected lower body P content in these consumers. Supporting previous stoichiometric theory, we found TER_C:P to be negatively correlated with the maximum growth rate of invertebrate consumers. By coupling bioenergetics and stoichiometry, this analysis revealed strong linkages among the physiology, ecology and evolution of nutritional demands for animal growth.     

Protein structure and neutral theory of evolution

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent "memorized" random sequences while biological evolution reduces to the "editing" these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirement do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and amino acid residues can form alpha-helices and beta-strand with lengths and arrangement along the chain similar to those in real globular proteins. These alpha- and beta-regions in random sequences can form three-dimensional folding patterns also similar to those in proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active site of protein and for their stability under physiological conditions.     

Does experimental evolution produce better biological control agents? A critical review of the evidence

Biological control of crop pests is considered a good alternative or complement to the use of pesticides. However, legislation restricts the importation of natural enemies of pests. A potential way to circumvent this limitation is by using experimental evolution and/or artificial selection to improve native biological control agents. Here, we review studies that have used these methodologies and evaluate their success. Experimental evolution or artificial selection has been used on a wide range of traits, with most focusing on improving the performance of natural enemies in ecologically relevant environments, such as in the presence of pesticides or at different temperatures. Although most studies were poorly replicated, the selected traits generally improved following the selection process. However, correlated responses (often in the form of trade‐offs) with other traits of interest were common. We suggest that the selection procedure can be improved by increasing replication and performing experimental evolution under more semi‐natural environments, to ensure that the most useful traits are being selected.     

The impact of the nucleosome code on protein-coding sequence evolution in yeast

Coding sequence evolution was once thought to be the result of selection on optimal protein function alone. Selection can, however, also act at the RNA level, for example, to facilitate rapid translation or ensure correct splicing. Here, we ask whether the way DNA works also imposes constraints on coding sequence evolution. We identify nucleosome positioning as a likely candidate to set up such a DNA-level selective regime and use high-resolution microarray data in yeast to compare the evolution of coding sequence bound to or free from nucleosomes. Controlling for gene expression and intra-gene location, we find a nucleosome-free "linker" sequence to evolve on average 5-6% slower at synonymous sites. A reduced rate of evolution in linker is especially evident at the 5' end of genes, where the effect extends to non-synonymous substitution rates. This is consistent with regular nucleosome architecture in this region being important in the context of gene expression control. As predicted, codons likely to generate a sequence unfavourable to nucleosome formation are enriched in linker sequence. Amino acid content is likewise skewed as a function of nucleosome occupancy. We conclude that selection operating on DNA to maintain correct positioning of nucleosomes impacts codon choice, amino acid choice, and synonymous and non-synonymous rates of evolution in coding sequence. The results support the exclusion model for nucleosome positioning and provide an alternative interpretation for runs of rare codons. As the intimate association of histones and DNA is a universal characteristic of genic sequence in eukaryotes, selection on coding sequence composition imposed by nucleosome positioning should be phylogenetically widespread.     

GC-biased gene conversion and selection affect GC content in the Oryza genus (rice)

Base composition varies among and within eukaryote genomes. Although mutational bias and selection have initially been invoked, more recently GC-biased gene conversion (gBGC) has been proposed to play a central role in shaping nucleotide landscapes, especially in yeast, mammals, and birds. gBGC is a kind of meiotic drive in favor of G and C alleles, associated with recombination. Previous studies have also suggested that gBGC could be at work in grass genomes. However, these studies were carried on third codon positions that can undergo selection on codon usage. As most preferred codons end in G or C in grasses, gBGC and selection can be confounded. Here we investigated further the forces that might drive GC content evolution in the rice genus using both coding and noncoding sequences. We found that recombination rates correlate positively with equilibrium GC content and that selfing species (Oryza sativa and O. glaberrima) have significantly lower equilibrium GC content compared with more outcrossing species. As recombination is less efficient in selfing species, these results suggest that recombination drives GC content. We also detected a positive relationship between expression levels and GC content in third codon positions, suggesting that selection favors codons ending with G or C bases. However, the correlation between GC content and recombination cannot be explained by selection on codon usage alone as it was also observed in noncoding positions. Finally, analyses of polymorphism data ruled out the hypothesis that genomic variation in GC content is due to mutational processes. Our results suggest that both gBGC and selection on codon usage affect GC content in the Oryza genus and likely in other grass species.     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000