Does the growth temperature of a prokaryote influence the purine content of its mRNAs?

The formation and breaking of hydrogen bonds between nucleic acid bases are dependent on temperature. The high G+C content of organisms was surmised to be an adaptation for high temperature survival because of the thermal stability of G:C pairs. However, a survey of genomic GC% and optimum growth temperature (OGT) of several prokaryotes revoked any direct relation between them. Significantly high purine (R=A or G) content in mRNAs is also seen as a selective response for survival among thermophiles. Nevertheless, the biological relevance of thermophiles loading their unstable mRNAs with excess purines (purine-loading or R-loading) is not persuasive. Here, we analysed the mRNA sequences from the genomes of 168 prokaryotes (as obtained from NCBI Genome database) with their OGTs ranging from -5 °C to 100 °C to verify the relation between R-loading and OGT. Our analysis fails to demonstrate any correlation between R-loading of the mRNA pool and OGT of a prokaryote. The percentage of purine-loaded mRNAs in prokaryotes is found to be in a rough negative correlation with the genomic GC% (r(2)=0.655, slope=-1.478, P<000.1). We conclude that genomic GC% and bias against certain combinations of nucleotides drive the mRNA-synonymous (sense) strands of DNA towards variations in R-loading.     

Amino Acids as Placeholders

BACKGROUND: The composition and sequence of amino acids in a protein may serve the underlying needs of the nucleic acids that encode the protein (the genome phenotype). In extreme form, amino acids become mere placeholders inserted between functional segments or domains, and--apart from increasing protein length--playing no role in the specific function or structure of a protein (the conventional phenotype). METHODS: We studied the genomes of two malarial parasites and 521 prokaryotes (144 complete) that differ widely in GC% and optimum growth temperature, comparing the base compositions of the protein coding regions and corresponding lengths (kilobases). RESULTS: Malarial parasites show distinctive responses to base-compositional pressures that increase as protein lengths increase. A low-GC% species (Plasmodium falciparum) is likely to have more placeholder amino acids than an intermediate-GC% species (P. vivax), so that homologous proteins are longer. In prokaryotes, GC% is generally greater and AG% is generally less in open reading frames (ORFs) encoding long proteins. The increased GC% in long ORFs increases as species' GC% increases, and decreases as species' AG% increases. In low- and intermediate-GC% prokaryotic species, increases in ORF GC% as encoded proteins increase in length are largely accounted for by the base compositions of first and second (amino acid-determining) codon positions. In high-GC% prokaryotic species, first and third (non-amino acid-determining) codon positions play this role. CONCLUSION: In low- and intermediate-GC% prokaryotes, placeholder amino acids are likely to be well defined, corresponding to codons enriched in G and/or C at first and second positions. In high-GC% prokaryotes, placeholder amino acids are likely to be less well defined. Increases in ORF GC% as encoded proteins increase in length are greater in mesophiles than in thermophiles, which are constrained from increasing protein lengths in response to base-composition pressures.     

Genomic Conflict Settled in Favour of the Species Rather Than the Gene at Extreme GC Percentage Values

Wada and colleagues have shown that, whether prokaryotic or eukaryotic, each gene has a "homostabilising propensity" to adopt a relatively uniform GC percentage (GC%). Accordingly, each gene can be viewed as a "microisochore" occupying a discrete GC% niche of relatively uniform base composition amongst its fellow genes. Although first, second and third codon positions usually differ in GC%, each position tends to maintain a uniform, gene-specific GC% value. Thus, within a genome, genic GC% values can cover a wide range. This is most evident at third codon positions, which are least constrained by amino acid encoding needs. In 1991, Wada and colleagues further noted that, within a phylogenetic group, genomic GC% values can also cover a wide range. This is again most evident at third codon positions. Thus, the dispersion of GC% values among genes within a genome matches the dispersion of GC% values among genomes within a phylogenetic group. Wada described the context-independence of plots of different codon position GC% values against total GC% as a "universal" characteristic. Several studies relate this to recombination. We have confirmed that third codon positions usually relate more to the genes that contain them than to the species. However, in genomes with extreme GC% values (low or high), third codon positions tend to maintain a constant GC%, thus relating more to the species than to the genes that contain them. Genes in an extreme-GC% genome collectively span a smaller GC% range, and mainly rely on first and second codon positions for differentiation as "microisochores". Our results are consistent with the view that differences in GC% serve to recombinationally isolate both genome sectors (facilitating gene duplication) and genomes (facilitating genome duplication, e.g. speciation). In intermediate-GC% genomes, conflict between the needs of the species and the needs of individual genes within that species is minimal. However, in extreme-GC% genomes there is a conflict, which is settled in favour of the species (i.e. group selection) rather than in favour of the gene (genic selection).     

The prokaryotic tree of life: past, present... and future?

No accepted phylogenetic scheme for prokaryotes emerged until the late 1970s. Prior to that, it was assumed that there was a phylogenetic tree uniting all prokaryotes, but no suitable data were available for its construction. For 20 years, through the 1980s and 1990s, rRNA phylogenies were the gold standard. However, beginning in the last decade, findings from genomic data have challenged this new consensus. Gene trees can conflict greatly, and strains of the same species can differ enormously in genome content. Horizontal gene transfer is now known to be a significant influence on genome evolution. The next decade is likely to resolve whether or not we retain the centuries-old metaphor of the tree for all of life.     

The elevated GC content at exonic third sites is not evidence against neutralist models of isochore evolution

The human genome is divided into isochores, large stretches (>300 kb) of genomic DNA with more or less consistent GC content. Mutational/neutralist and selectionist models have been put forward to explain their existence. A major criticism of the mutational models is that they cannot account for the higher GC content at fourfold-redundant silent sites within exons (GC4) than in flanking introns (GCi). Indeed, it has been asserted that it is hard to envisage a mutational bias explanation, as it is difficult to see how repair enzymes might act differently in exons and their flanking introns. However, this rejection, we note, ignores the effects of transposable elements (TEs), which are a major component of introns and tend to cause them to have a GC content different from (usually lower than) that dictated by point mutational processes alone. As TEs tend not to insert at the extremities of introns, this model predicts that GC content at the extremities of introns should be more like that at GC4 than are the intronic interiors. This we show to be true. The model also correctly predicts that small introns should have a composition more like that at GC4 than large introns. We conclude that the logic of the previous rejection of neutralist models is unsafe.     

Genomic GC level, optimal growth temperature, and genome size in prokaryotes

Two years ago, we showed that positive correlations between optimal growth temperature (T(opt)) and genome GC are observed in 15 out of the 20 families of prokaryotes we analyzed, thus indicating that "T(opt) is one of the factors that influence genomic GC in prokaryotes". Our results were disputed, but these criticisms were demonstrated to be mistaken and based on misconceptions. In a recent report, Wang et al. [H.C. Wang, E. Susko, A.J. Roger, On the correlation between genomic G+C content and optimal growth temperature in prokaryotes: data quality and confounding factors, Biochem. Biophys. Res. Commun. 342 (2006) 681-684] criticize our results by stating that "all previous simple correlation analyses of GC versus temperature have ignored the fact that genomic GC content is influenced by multiple factors including both intrinsic mutational bias and extrinsic environmental factors". This statement, besides being erroneous, is surprising because it applies in fact not to ours but to the authors' article. Here, we rebut the points raised by Wang et al. and review some issues that have been a matter of debate, regarding the influence of environmental factors upon GC content in prokaryotes. Furthermore, we demonstrate that the relationship that exists between genome size and GC level is valid for aerobic, facultative, and microaerophilic species, but not for anaerobic prokaryotes.     

Long-range correlations in prokaryotic chromosomes

One of the fascinating properties of the DNA sequences of prokaryotic and eukaryotic chromosomes is that they possess long-range order. Computational methods like spectral analysis, mutual information and DNA random walks have been used to probe long-range order via-long range correlations. This work attempts to show the advantage of using the Information Theoretic measure of mutual information for this purpose. A number Mu is found which indicates the existence of long-range order. Mu is the ratio between the value of mutual information function between two nucleotides of a DNA sequence separated by a large distance of 100 kilobases to the value expected from a randomized sequence of the same DNA. It is found that in spite of the constant shuffling of nucleotides due to insertion, deletion, inversion and recombination that occur during evolution, the chromosomal structure of prokaryotes is not always mosaic. While all archaeal chromosomes show mosaic structure and lack long-range order, a sizable fraction of the bacterial chromosomes do possess long-range order. A statistical multivariate analysis has been done to find which of the physical variables like genome size or GC% affects the organization of the chromosome or correlates with the long-range order. The existence of long-range order in bacterial chromosomes could be directly correlated to the degree of gene strand bias shown by it. Firmicutes which have low GC content also have pronounced strand bias and show long-range correlations. It is observed that the occurrence of long-range order in bacteria is independent of genome size, but depends on its GC content and gene strand bias.     

On the correlation between genomic G+C content and optimal growth temperature in prokaryotes: data quality and confounding factors

The correlation between genomic G+C content and optimal growth temperature in prokaryotes has gained renewed interest after Musto et al. [H. Musto, H. Naya, A. Zavala, H. Romero, F. Alvarex-Valin, G. Bernardi, Correlations between genomic GC levels and optimal growth temperatures in prokaryotes, FEBS Lett. 573 (2004) 73-77], reported that positive correlations exist in 15 families studied. We have reanalyzed their data and found that when genome size and data quality were adjusted for, there was no significant evidence of relationship between optimal temperature and GC content for two of the families that had previously shown strongly significant correlations. Using updated temperature optima for Halobacteriaceae species we found the correlation is insignificant in this family. For the family Enterobacteriaceae when genome size and optimal temperature are included in a multiple linear regression, only genome size is significant as a predictor of GC content. We showed that more profound statistical methods than simple two factor correlation analysis should be used for analyzing complex intrinsic and extrinsic factors that affect genomic GC content. We further found that a positive correlation between temperature and genomic GC is only evident in free-living species of low optimal growth temperatures.     

The GC content of a group of H2S- enterobacterial related to the genus Citrobacter

The deoxyribonculeic acid (DNA) of 106 strains of Enterobacteria was analysed for the guanine + cytosine (GC) content. These strains, whose origin and principal characters are described in the text, belong to the genera Citrobacter (C. freundii H2S-) and Levinea (L. malonatica and L. amalonatica). Four other groups or classes named C.D.E. and F. could not be classified on the base of the usual phenotypic criteria. DNA from the strains of Levinea has a GC% of 50.3 to 53.3, while DNA from the strains of C. freundii H2S- has a GC% of 48.6 to 51.7. The representative values from the new classes are C, 50.9%; D, 54%; E, 52.7%; F, 49.5%. For the latter a genomic heterogeneity was shown, expressing itself as two subpopulations whose average GC% are 51.7 and 48.6 respectively. Statistical analysis of the averages give a significant individuality to these new classes.     

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.     

A genomic survey shows that the haloarchaeal type tyrosyl tRNA synthetase is not a synapomorphy of opisthokonts

The haloarchaeal-type tyrosyl tRNA synthetase (tyrRS) have previously been proposed to be a molecular synapomorphy of the opisthokonts. To re-evaluate this we have performed a taxon-wide genomic survey of tyrRS in eukaryotes and prokaryotes. Our phylogenetic trees group eukaryotes with archaea, with all opisthokonts sharing the haloarchaeal-type tyrRS. However, this type of tyrRS is not exclusive to opisthokonts, since it also encoded by two amoebozoans. Whether this is a consequence of lateral gene transfer or lineage sorting remains unsolved, but in any case haloarchaeal-type tyrRS is not a synapomorphy of opisthokonts. This demonstrates that molecular markers should be re-evaluated once a better taxon sampling becomes available.     

High levels of genetic variation in natural populations of marine lower invertebrates

The predictions of neutralist and selectionist hypotheses have been tested many times in the past, but mostly using data only from organisms such as vertebrates, with generally low to average heterozygosities. The more recent discovery of particularly high levels of genetic variation in marine sponges and coelenterates provides an opportunity to use data from such species to contribute further to the understanding of the determinants of heterozygosity in natural populations. Therefore, 23 species of sponges and coelenterates from temperate, tropical and boreal waters were analysed by gel electrophoresis for an average of 14.3 enzyme loci per species. Mean heterozygosity values for each species were unusually high, ranging between 0.106 and 0.401. The means and variances of the heterozygosity estimates showed reasonable correlation with neutralist predictions (with both the stepwise mutation and the infinite alleles models). Population sizes were generally difficult to estimate with any confidence, but, for one sponge species for which this was possible, levels of heterozygosity again were similar to neutralist predictions, although the same was not apparently true for three species of sea anemone. No differences were found between heterozygosity levels of tropical and temperate species of sponges and coelenterates, thus apparently contradicting the selectionist ‘trophic resource stability’ and ‘temporal environmental variation’ hypotheses. Conversely, however, the consistently high levels of genetic variation found in coelenterates and sponges may be argued to be related to common biological characteristics, such as sessile life, great evolutionary ‘age’, limited ability to disperse and probable low homoeostatic capability. Our results seem, overall, to agree well with neutralist expectations for species with large, stable population sizes. Also, the mean heterozygosities, their variances and the observed and expected proportions of polymorphic loci seem to fit well with predictions based on the neutralist hypothesis. However, the selectionist ‘environmental grain’ and the ‘shifting balance’ hypotheses fit the data equally well. As with much earlier work, the problems in distinguishing between the various predictions of selectionist or neutralist ideas make it both difficult and unwise to draw definite conclusions.     

Biosynthesis of phosphatidylcholine in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the methylation pathway or the CDP-choline pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of rather diverse bacteria and based on genomic data, we estimate that more than 10% of all bacteria possess PC. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. A number of symbiotic (Rhizobium leguminosarum, Mesorhizobium loti) and pathogenic (Agrobacterium tumefaciens, Brucella melitensis, Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) bacteria seem to possess the PC synthase pathway and we suggest that the respective eukaryotic host functions as the provider of choline for this pathway. Pathogens entering their hosts through epithelia (Streptococcus pneumoniae, Haemophilus influenzae) require phosphocholine substitutions on their cell surface components that are biosynthetically also derived from choline supplied by the host. However, the incorporation of choline in these latter cases proceeds via choline phosphate and CDP-choline as intermediates. The occurrence of two intermediates in prokaryotes usually found as intermediates in the eukaryotic CDP-choline pathway for PC biosynthesis raises the question whether some bacteria might form PC via a CDP-choline pathway.     

The correlation between genomic G+C and optimal growth temperature of prokaryotes is robust: a reply to Marashi and Ghalanbor

We have recently shown that optimal growth temperature (T(opt)) is one of the factors that influence genomic GC in prokaryotes. Our results have been disputed by Marashi and Ghalanbor, who claim that the correlations we show are not "robust" because the elimination of some points (arbitrarily chosen) leads, in some families, to variations in the correlation coefficients and/or significance of correlations. Here, we test whether the correlation between T(opt) and genomic GC is robust by using two independent approaches: detection of possible outliers (using robust Mahalanobis distance) and usage of a non-parametric correlation coefficient that is not sensitive to the presence of outliers. The results presented here reinforce our previous proposal that T(opt) is correlated with genomic GC in prokaryotes.     

Higher tRNA diversity in thermophilic bacteria: A possible adaptation to growth at high temperature

We have done a comparative study of tRNA diversity and total tRNA genes among different strains of bacteria with respect to the optimum growth temperature of the cells. Our observation suggests that higher tRNA diversity usually occurs in thermophiles in comparison to non-thermophiles. Among psychrophiles total tRNA was observed to be more than two-fold higher than in the non-psychrophiles. Though tRNA diversity and total tRNA have recently been shown to be affected by an organism's genomic GC% and growth rate, this work is the first report on growth temperature affecting these features in bacteria. This work extends the list of molecular features undergoing adaptation due to growth temperature and supports the view that growth temperature acts as a selecting factor at the molecular level during evolution.     

Redundancy in ribonucleotide excision repair: Competition,compensation, and cooperation

The survival of all living organisms is determined by their ability to reproduce, which in turn depends on accurate duplication of chromosomal DNA. In order to ensure the integrity of genome duplication, DNA polymerases are equipped with stringent mechanisms by which they select and insert correctly paired nucleotides with a deoxyribose sugar ring. However, this process is never 100% accurate. To fix occasional mistakes, cells have evolved highly sophisticated and often redundant mechanisms. A good example is mismatch repair (MMR), which corrects the majority of mispaired bases and which has been extensively studied for many years. On the contrary, pathways leading to the replacement of nucleotides with an incorrect sugar that is embedded in chromosomal DNA have only recently attracted significant attention. This review describes progress made during the last few years in understanding such pathways in both prokaryotes and eukaryotes. Genetic studies in Escherichia coli and Saccharomyces cerevisiae demonstrated that MMR has the capacity to replace errant ribonucleotides, but only when the base is mispaired. In contrast, the major evolutionarily conserved ribonucleotide repair pathway initiated by the ribonuclease activity of type 2 Rnase H has broad specificity. In yeast, this pathway also requires the concerted action of Fen1 and pol δ, while in bacteria it can be successfully completed by DNA polymerase I. Besides these main players, all organisms contain alternative enzymes able to accomplish the same tasks, although with differing efficiency and fidelity. Studies in bacteria have very recently demonstrated that isolated rNMPs can be removed from genomic DNA by error-free nucleotide excision repair (NER), while studies in yeast suggest the involvement of topoisomerase 1 in alternative mutagenic ribonucleotide processing. This review summarizes the most recent progress in understanding the ribonucleotide repair mechanisms in prokaryotes and eukaryotes.     

On the origin of prokaryotes

It has been a tacit assumption of evolutionary theory that the closest surviving relatives of the first cellular organisms are to be found among prokaryotes. This paper draws attention to the fact that many stages of evolution appear to have been accompanied by physical loss of superfluous DNA. It is postulated that the genomes of prokaryotes—where almost every gene is represented by one copy only—represent the results of this process carried to its extreme. On this basis certain features of very early evolution which have been eliminated from prokaryotes may survive in eukaryotes. If correct, the hypothesis would require a careful re-evaluation of the assumptions underlying use of some sequence data to construct phylogenetic trees.     

A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.