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 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.     

GC content and genome length in Chargaff compliant genomes

Musto et al. [H. Musto, H. Naya, A. Zavala, H. Romero, F. Alvarez-Valin, G. Bernardi, Genomic GC level, optimal growth temperature, and genome size in prokaryotes, Biochem. Biophys. Res. Commun. 347 (2006) 1-3] recently reported a linear correlation between GC content and genome length. The regression model was heteroscedactic which suggested that the relationship might be more clearly defined. Alternative regression models (R²>0.95) were fitted to a set of over 900 sequences compliant with Chargaff’s second parity rule. The new models suggest that the relationship between GC content and genome length is more complex than was originally suggested. While similar models can be derived for non-Chargaff compliant genomes, their interpretation is likely to be more difficult.     

High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes

The causes of the variation between genomes in their guanine (G) and cytosine (C) content is one of the central issues in evolutionary genomics. The thermal adaptation hypothesis conjectures that, as G:C pairs in DNA are more thermally stable than adenonine:thymine pairs, high GC content may he a selective response to high temperature. A compilation of data on genomic GC content and optimal growth temperature for numerous prokaryotes failed to demonstrate the predicted correlation. By contrast, the GC content of Structural RNAs is higher at high temperatures. The issue that we address here is whether more freely evolving sites in exons (i.e. codonic third positions) evolve in the same manner as genomic DNA as a whole, Showing no correlated response, or like structural RNAs showing a strong correlation. The latter pattern would provide strong support for the thermal adaptation hypothesis, as the variation in GC content between orthologous genes is typically most profoundly seen at codon third sites (GC3). Simple analysis of completely sequenced prokaryotic genomes shows that GC3, but not genomic GC, is higher on average in thermophilic species. This demonstrates, if nothing else, that the results from the two measures cannot be presumed to be the same. A proper analysis, however, requires phylogenetic control. Here, therefore, we report the results of a comparative analysis of GC composition and optimal growth temperature for over 100 prokaryotes. Comparative analysis fails to show, in either Archea or Eubacteria, any hint of connection between optimal growth temperature and GC content in the genome as a whole, in protein-coding regions or, more crucially at GC. Conversely, comparable analysis confirms that GC content of structural RNA is strongly correlated with optimal temperature. Against the expectations of the thermal adaptation hypothesis, within prokaryotes GC content in protein-coding genies, even at relatively freely evolving sites, cannot be considered an adaptation to the thermal environment.     

Correspondence analysis of amino acid usage within the family Bacillaceae

When the amino acid usage of all completely sequenced prokaryotes is studied by multivariate analysis (MVA), it is known that the genomic molar content of guanine plus cytosine (GC) and optimal growth temperature (Topt) have a dominant effect. Furthermore, these two factors are associated to the first two axes of different MVA, and thus, nearly independent among them. However, it was recently shown that for several Families of prokaryotes there are significant and positive correlations between GC and Topt. This trend is particularly clear within Bacillaceae, where there are species displaying a broad range of variations for these two factors. In this paper we report that (a) Topt and genomic GC are the main factors shaping amino acid usage but are not independent between them, (b) the usage of cysteine is the second source of variability, and finally (c) the global hydrophobicity of the encoded proteins of each species is the third main factor.     

Correlations between genomic GC levels and optimal growth temperatures: some comments

Regarding the existence of any specific correlation between optimal growth temperature and genomic GC levels, Musto et al. [FEBS Lett. 573 (2004) 73] have recently performed analysis on 20 prokaryotic families and showed that in most of the families there exists a positive correlation between these two parameters. On the basis of these results they claimed that optimal growth temperature is one of the factors that influence genomic GC composition in prokaryotes. In a subsequent article, Marashi and Ghalanbor [Biochem. Biophys. Res. Commun. 325 (2004) 381] have demonstrated that the correlation values change substantially when very few points in some of the families were excluded from the data set of Musto et al. [FEBS Lett. 573 (2004) 73]. But Marashi and Ghalanbor have not provided any reason behind this. The points excluded by Marashi and Ghalanbor are actually the outliers in the data set, which strongly affect the correlation coefficients. But the presence of outliers in large data set hardly had any effect on the correlation values. Marashi and Ghalanbor have excluded points from only those families that have small sample sizes and observed a substantial change in correlation coefficient values. Therefore, we argue that any conclusion drawn for a small sample size having outliers is always questionable. Although Musto's approach is a novel one, but to make any generalization one needs to be careful about the flawlessness in the data set.     

基于流式细胞术的海菜花属植物基因组大小测定

利用改良的裂解液P1,以中国古代莲(Nelumbo nucifera Gaertn.Fruct.et Semin)为外标,采用流式细胞术(FCM)对海菜花属(Ottelia Pers.)6个代表性物种及3个存疑类群的基因组大小(C值)进行测定,并对海菜花属系统发育关系进行评估。结果显示:所测定的材料中,水菜花(Ottelia cordata(Wall.)Dandy)C值最小(6.759 pg),灌阳水车前(O.guanyangensis Z.Z.Li,S.Wu&Q.F.Wang)C值最大(12.929 pg);对基因组大小与该属系统发育树进行比较分析,结果发现该属植物基因组大小与系统发育关系具有一致性;对海菜花属3个存疑类群进行分子系统学研究,结果发现存疑类群与嵩明海菜花(Ottelia acuminata var.songmingensis Z.T.Jiang,H.Li&Z.L.Dao)及灌阳水车前的关系最近,而与水菜花的关系较远,这与基因组大小变异相一致。根据基因组大小进一步推测3个存疑类群很可能为二倍体。本研究结果可为海菜花属植物的系统学研究提供新资料,同时为该属植物基因组学研究提供基础数据。     

Compositional Properties and Thermal Adaptation of SRP-RNA in Bacteria and Archaea

Previous studies have reported a positive correlation between the GC content of the double-stranded regions of structural RNAs and the optimal growth temperature (OGT) in prokaryotes. These observations led to the hypothesis that natural selection favors an increase in GC content to ensure the correct folding and the structural stability of the molecule at high temperature. To date these studies have focused mainly on ribosomal and transfer RNAs. Therefore, we addressed the question of the relationship between GC content and OGT in a different and universally conserved structural RNA, the RNA component of the signal recognition particle (SRP). To this end we generated the secondary structures of SRP-RNAs for mesophilic, thermophilic, and hyperthermophilic bacterial and archaeal species. The analysis of the GC content in the stems and loops of the SRP-RNA of these organisms failed to detect a relationship between the GC contents in the stems of this structural RNA and the growth temperature of bacteria. By contrast, we found that in archaea the GC content in the stem regions of SRP-RNA is highest in hyperthermophiles, intermediate in thermophiles, and lower in mesophiles. In these organisms, we demonstrated a clear positive correlation between the GC content of the stem regions of their SRP-RNAs and their OGT. This correlation was confirmed by a phylogenetic nonindependence analysis. Thus we conclude that in archaea the increase in GC content in the stem regions of SRP-RNA is an adaptation response to environmental temperature.     

On the origin of genomic adaptation at high temperature for prokaryotic organisms

For a long time, the central issue of evolutionary genomics was to find out the adaptive strategy of nucleic acid molecules of various microorganisms having different optimal growth temperatures (Topt). Long-standing controversies exist regarding the correlations between genomic G+C content and Topt, and this debate has not been yet settled. We address this problem by considering the fact that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting: (i) nucleic acid thermostability and (ii) stability of codon-anticodon interactions. In the present study, we analyzed 16 prokaryotic genomes having intermediate G+C content and widely varying optimal growth temperatures. Results show that elevated growth temperature imposes selective constraints not only on nucleic acid level but also affects the stability of codon-anticodon interaction. We observed a decrease in the frequency of SSC and SSG codons with the increase in Topt to avoid the formation of side-by-side GC base pairs in the codon-anticodon interaction, thereby making it impossible for a genome to increase GC composition uniformly through the whole coding sequence. Thus, we suggest that any attempt to obtain a generalized relation between genomic GC composition and optimal growth temperature would hardly evolve any satisfactory result.     

Identification of Horizontally-transferred Genomic Islands and Genome Segmentation Points by Using the GC Profile Method

The nucleotide composition of genomes undergoes dramatic variations among all three kingdoms of life. GC content, an important characteristic for a genome, is related to many important functions, and therefore GC content and its distribution are routinely reported for sequenced genomes. Traditionally, GC content distribution is assessed by computing GC contents in windows that slide along the genome. Disadvantages of this routinely used window-based method include low resolution and low sensitivity. Additionally, different window sizes result in different GC content distribution patterns within the same genome. We proposed a windowless method, the GC profile, for displaying GC content variations across the genome. Compared to the window-based method, the GC profile has the following advantages: 1) higher sensitivity, because of variation-amplifying procedures; 2) higher resolution, because boundaries between domains can be determined at one single base pair; 3) uniqueness, because the GC profile is unique for a given genome and 4) the capacity to show both global and regional GC content distributions. These characteristics are useful in identifying horizontally-transferred genomic islands and homogenous GC-content domains. Here, we review the applications of the GC profile in identifying genomic islands and genome segmentation points, and in serving as a platform to integrate with other algorithms for genome analysis. A web server generating GC profiles and implementing relevant genome segmentation algorithms is available at: www.zcurve.net.     

Oxygen and Guanine–Cytosine Profiles in Marine Environments

One of the historic debates in molecular evolution concerns the strong variation in the genomic guanine–cytosine (GC) content of prokaryotes, which ranges from approximately 20–75%: Is this factor selectively neutral, or is it the result of natural selection? In a previous article published by our group, we showed that inside well-defined taxonomic groups of prokaryotes, strictly aerobic organisms tend to display higher genomic GC levels than strictly anaerobic species. In the present study, we examined the GC content of fragments of DNA obtained from microbial communities along a well-defined environmental gradient: a 4,000-m vertical profile in the North Pacific subtropical gyre. The patterns of GC distribution might be associated with oxygen concentrations in the seawater column. These results give further support to the link between a physiologic trait (aerobic respiration) and genomic GC content.     

r/K selection of GC content in prokaryotes

The guanine/cytosine (GC) content of prokaryotic genomes is species-specific, taking values from 16% to 77%. This diversity of selection for GC content remains contentious. We analyse the correlations between GC content and a range of phenotypic and genotypic data in thousands of prokaryotes. GC content integrates well with these traits into r/K selection theory when phenotypic plasticity is considered. High GC-content prokaryotes are r-strategists with cheaper descendants thanks to a lower average amino acid metabolic cost, colonize unstable environments thanks to flagella and a bacillus form and are generalists in terms of resource opportunism and their defence mechanisms. Low GC content prokaryotes are K-strategists specialized for stable environments that maintain homeostasis via a high-cost outer cell membrane and endospore formation as a response to nutrient deprivation, and attain a higher nutrient-to-biomass yield. The lower proteome cost of high GC content prokaryotes is driven by the association between GC-rich codons and cheaper amino acids in the genetic code, while the correlation between GC content and genome size may be partly due to functional diversity driven by r/K selection. In all, molecular diversity in the GC content of prokaryotes may be a consequence of ecological r/K selection.     

Molecular confirmation of the genomic constitution of <Emphasis Type="Italic">Douglasdeweya</Emphasis> (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification

A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae.     

Examination of Genome Homogeneity in Prokaryotes Using Genomic Signatures

Background

DNA word frequencies, normalized for genomic AT content, are remarkably stable within prokaryotic genomes and are therefore said to reflect a “genomic signature.” The genomic signatures can be used to phylogenetically classify organisms from arbitrary sampled DNA. Genomic signatures can also be used to search for horizontally transferred DNA or DNA regions subjected to special selection forces. Thus, the stability of the genomic signature can be used as a measure of genomic homogeneity. The factors associated with the stability of the genomic signatures are not known, and this motivated us to investigate further. We analyzed the intra-genomic variance of genomic signatures based on AT content normalization (0^th order Markov model) as well as genomic signatures normalized by smaller DNA words (1^st and 2^nd order Markov models) for 636 sequenced prokaryotic genomes. Regression models were fitted, with intra-genomic signature variance as the response variable, to a set of factors representing genomic properties such as genomic AT content, genome size, habitat, phylum, oxygen requirement, optimal growth temperature and oligonucleotide usage variance (OUV, a measure of oligonucleotide usage bias), measured as the variance between genomic tetranucleotide frequencies and Markov chain approximated tetranucleotide frequencies, as predictors.

Principal Findings

Regression analysis revealed that OUV was the most important factor (p<0.001) determining intra-genomic homogeneity as measured using genomic signatures. This means that the less random the oligonucleotide usage is in the sense of higher OUV, the more homogeneous the genome is in terms of the genomic signature. The other factors influencing variance in the genomic signature (p<0.001) were genomic AT content, phylum and oxygen requirement.

Conclusions

Genomic homogeneity in prokaryotes is intimately linked to genomic GC content, oligonucleotide usage bias (OUV) and aerobiosis, while oligonucleotide usage bias (OUV) is associated with genomic GC content, aerobiosis and habitat.     

Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryote Genomes

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. Received: 2 February 1998 / Accepted: 15 June 1998     

Deoxyribonucleic acid base composition and genome size of Prochloron

The DNA base composition of the photosynthetic prokaryote Prochloron was determined (on samples collected from the natural environment) to be 40.8 mol% GC. The sharp differential melting curve indicated the absence of significant quantities of contaminating DNA from other organisms. The genome size, estimated from the renaturation kinetics of thermally denatured DNA, was 3.59×10⁹ daltons mol. wt, similar to that of many other prokaryotes. The fact that Prochloron has not yet been cultured in the laboratory cannot, therefore, be attributed to a reduced genetic information content.     

Analysis of the Relationship between Genomic GC Content and Patterns of Base Usage,Codon Usage and Amino Acid Usage in Prokaryotes: Similar GC Content Adopts Similar Compositional Frequencies Regardless of the Phylogenetic Lineages

The GC contents of 2670 prokaryotic genomes that belong to diverse phylogenetic lineages were analyzed in this paper. These genomes had GC contents that ranged from 13.5% to 74.9%. We analyzed the distance of base frequencies at the three codon positions, codon frequencies, and amino acid compositions across genomes with respect to the differences in the GC content of these prokaryotic species. We found that although the phylogenetic lineages were remote among some species, a similar genomic GC content forced them to adopt similar base usage patterns at the three codon positions, codon usage patterns, and amino acid usage patterns. Our work demonstrates that in prokaryotic genomes: a) base usage, codon usage, and amino acid usage change with GC content with a linear correlation; b) the distance of each usage has a linear correlation with the GC content difference; and c) GC content is more essential than phylogenetic lineage in determining base usage, codon usage, and amino acid usage. This work is exceptional in that we adopted intuitively graphic methods for all analyses, and we used these analyses to examine as many as 2670 prokaryotes. We hope that this work is helpful for understanding common features in the organization of microbial genomes.     

Modelling the effect of temperature and water activity on growth of Aspergillus niger strains and applications for food spoilage moulds

AIMS: To develop a model for the combined effect of water activity (a(w)) and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature. METHODS AND RESULTS: An extended combined model describing the growth of two strains of A. niger, as a function of temperature (25-30 degrees C) and a(w) (0.90-0.99) was developed. The growth rate (micro) was expressed as the increase in colony radial growth per unit of time. This extends the previous square root model showing the relationship between temperature and bacterial growth rate developed by Ratkowsky et al. (1983) and the parabolic relationship between the logarithm of the growth rate and a(w) developed by Gibson et al. (1994). A good correlation between the experimental data and the model predictions was obtained, with regression coefficients (r(2)) > 0.99. In addition, the use of this model allowed predictions of the cardinal a(w) levels: a(w(min)), and a(w(opt)). The estimation of the minimum a(w) levels (a(w(min))) was in accordance with data in the literature for similar and a range of other Aspergillus and related species, regardless of the solutes used for a(w) modification. The estimation of the optimal a(w) (a(w(opt))) and the optimal growth rate (micro(opt)) were in good agreement with the experimental results and data from the literature. CONCLUSIONS: This approach enables accurate prediction of the combined effects of environmental factors on growth of spoilage fungi for rapid prediction of cardinal limits using surface response curves. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a rapid method for predicting optimal and marginal conditions for growth of a wide range of spoilage micro-organisms in relation to interacting environmental conditions and will have applications for improving shelf-life of intermediate moisture foods.