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.     

Correlations between genomic GC levels and optimal growth temperatures are not 'robust'

Musto et al. [FEBS Lett. 573 (2004) 73] studied the correlations between GC levels and optimal growth temperatures in 20 prokaryotic families. They reported that positive correlations are generally observed, and many of these are significant. Here, we have shown that these correlations are not "robust," i.e., correlation coefficients and/or significance of correlations can be considerably influenced by exclusion of very few (even as small as one) species from each dataset. The sensitivity of correlations is assumed as a result of high levels of bias in the family datasets. We concluded that solely based on these data, one cannot establish that GC contents of prokaryotic genomes increase as a result of growth temperature increments.     

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.     

On the methodological weakness of 'the effective number of codons': a reply to Marashi and Najafabadi

In a recent publication [Biochem. Biophys. Res. Commun. 317 (2004) 957] it was proposed that the 'effective number of codons' (Nc) in a gene should be calculated by summing the individual amino acid Nc's using rounding whenever the codon homozygosities are lower than the reciprocal value of the number of members of the synonymous families. This led Marashi and Najafabadi to examine the consequences of individual re-adjustment when comparing observed Nc with the expected Nc under assumptions of no selection, and C=G and A=T [Biochem. Biophys. Res. Commun. 324 (2004) 1]. Clearly, the present methodology has some weaknesses; in this work, I discuss these in relation to the observations by Marashi and Najafabadi, and finally an alternative method for the calculation of Nc is introduced with the purpose of eliminating the need for re-adjustments.     

Amyloid fibril formation propensity is inherent into the hexapeptide tandemly repeating sequence of the central domain of silkmoth chorion proteins of the A-family

Peptide-analogues of the A and B families of silkmoth chorion proteins form amyloid fibrils under a variety of conditions [Iconomidou, V.A., Vriend, G. Hamodrakas, S.J. 2000. Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145; Iconomidou,V.A., Chryssikos, G.D.,Gionis, V., Vriend, G., Hoenger, A., Hamodrakas, S.J., 2001. Amyloid-like fibrils from an 18-residue peptide-analogue of a part of the central domain of the B-family of silkmoth chorion protein. FEBS Lett. 499, 268-273; Hamodrakas, S.J. Hoenger, A., Iconomidou, V. A., 2004 . Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid crystalline intermediate phase. J. Struct. Biol. 145, 226-235.], which led us to propose that silkmoth chorion is a natural protective amyloid. In this study, we designed and synthesized two mutant peptide-analogues of the central conservative domain of the A family: (a) one, cA_m1, with a length half of that of the central domain of the A family, which folds and self-assembles, in various conditions, into amyloid fibrils very similar in properties and structure to the fibrils formed by the cA peptide, which corresponds to the entire length of the A family central domain [Iconomidou, V.A., Vriend, G. Hamodrakas, S.J. 2000. Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145.], in full support of our previous proposal, (b) the second, cA_m2, differing from cA_m1 at three positions, where three glutamates have replaced two valines and one alanine residues, does not form amyloid fibrils in any conditions. It appears that (a) the amyloidogenic properties of silkmoth chorion peptides are encoded into the tandemly repeating hexapeptides comprising the central domain of silkmoth chorion proteins, and, that (b) suitable mutations, properly and carefully designed, greatly affect the strong amyloidogenic properties inherent in certain aminoacid sequences and may inhibit amyloid formation.     

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.     

Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145].     

R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays

Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay.     

RankProd: a bioconductor package for detecting differentially expressed genes in meta-analysis

While meta-analysis provides a powerful tool for analyzing microarray experiments by combining data from multiple studies, it presents unique computational challenges. The Bioconductor package RankProd provides a new and intuitive tool for this purpose in detecting differentially expressed genes under two experimental conditions. The package modifies and extends the rank product method proposed by Breitling et al., [(2004) FEBS Lett., 573, 83-92] to integrate multiple microarray studies from different laboratories and/or platforms. It offers several advantages over t-test based methods and accepts pre-processed expression datasets produced from a wide variety of platforms. The significance of the detection is assessed by a non-parametric permutation test, and the associated P-value and false discovery rate (FDR) are included in the output alongside the genes that are detected by user-defined criteria. A visualization plot is provided to view actual expression levels for each gene with estimated significance measurements. AVAILABILITY: RankProd is available at Bioconductor http://www.bioconductor.org. A web-based interface will soon be available at http://cactus.salk.edu/RankProd     

ADPglucose pyrophosphorylase's N-terminus: structural role in allosteric regulation

We studied the functional role of the Escherichia coli ADPglucose pyrophosphorylase's N-terminus in allosteric regulation, and the particular effects caused by its length. Small truncated mutants were designed, and those lacking up to 15-residues were active and highly purified for further kinetic analyses. Ndelta3 and Ndelta7 did not change the kinetic parameters with respect to the wild-type. Ndelta11 and Ndelta15 enzymes were insensitive to allosteric regulation and highly active in the absence of the activator. Co-expression of two polypeptides corresponding to the N- and C-termini generated an enzyme with activation properties lower than those of the wild-type [C.M. Bejar, M.A. Ballicora, D.F. Gómez Casati, A.A. Iglesias, J. Preiss, The ADPglucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains, FEBS Lett. 573 (2004) 99-104]. Here, we characterized a Ndelta15 co-expression mutant, in which the allosteric regulation was restored to wild-type levels. Unusual allosteric effects caused by either an N-terminal truncation or co-expression of individual domains may respond to structural changes favoring an up-regulated or a down-regulated conformation rather than specific activator or inhibitor sites' disruption.     

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria. Reply to a commentary

Costa, L.E., Reynafarje, B. and Lehninger, A.L. [(1984) J. Biol. Chem. 259, 4802-4811] have reported 'second-generation' measurements of the H+/O ratio approaching 8.0 for vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria. In a Commentary in this Journal [Krab, K., Soos, J. and Wikstr?m, M. (1984) FEBS Lett. 178, 187-192] it was concluded that the measurements of Costa et al. significantly overestimated the true H+/O stoichiometry. It is shown here that the mathematical simulation on which Krab et al. based this claim is faulty and that data reported by Costa et al. had already excluded the criticism advanced by Krab et al. Also reported are new data, obtained under conditions in which the arguments of Krab et al. are irrelevant, which confirm that the H+/O ratio for succinate oxidation extrapolated to level flow is close to 8.     

NMR distance measurements in DNA duplexes: sugars and bases have the same correlation times

To evaluate whether the sugar moieties of short DNA duplexes exhibit local motion of sufficient amplitude to affect interproton distance measurements, we have carried out a series of time-dependent NOESY experiments at increasingly shorter mixing times on dodecamer DNA duplexes. By use of the cytosine H5-H6 vector as a known distance in the bases and the geminal 2'H-2'H vector as a known distance in the sugars, the corresponding apparent cross-relaxation rates were sampled at various mixing times. While the ratio of the inverse sixth power of these two fixed distances is in the range 6-7, when the system is sampled at 100 ms the apparent initial rate of growth of the 2'H-2'H NOESY crosspeak is only 1.9-2.0 times faster than that of the H5-H6 crosspeak--in agreement with the results of Clore and Gronenborn [Clore, G. M., & Gronenborn, A. M. (1984) FEBS Lett. 172, 219; (1984) FEBS Lett. 175, 117] and of Gronenborn and Clore [Gronenborn, A. M., & Clore, G. M. (1985) Prog. NMR Spectrosc. 17, 1]. This observation was interpreted to indicate the existence of internal mobility with a 3-fold shorter correlation time for the sugar moieties in DNA and led to the use of this shorter correlation time to estimate sugar-sugar proton distances and many sugar-base proton distances in subsequent DNA structure determination. We have examined 2'H-2"H cross-relaxation and H5-H6 cross-relaxation at 100, 90, 60, 30, and 15 ms in dodecamer DNA duplexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibration of methionine into the Met-tRNA pool of rabbit reticulocyte lysates: Additional evidence that deacylation of ribosomal subunit-bound Met-tRNAf does occur in heme-deficiency

We have measured the rate of equilibration of [³⁵S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301–305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNA_f that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation.     

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.     

Involvement of calcium, calmodulin and phospholipase A in the alteration of membrane dynamics and superoxide production of human neutrophils stimulated by phorbol myristate acetate

We have reported an increased fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in the phorbol myristate acetate-stimulated plasma membrane of human neutrophils [FEBS Lett. (1982) 144, 199–203]. We now present evidence that both the increased fluorescence polarization and the production of super-oxide radicals by human neutrophils require calcium, calmodulin and phospholipase activity.     

Conserved quantitative stability/flexibility relationships (QSFR) in an orthologous RNase H pair

Many reports qualitatively describe conserved stability and flexibility profiles across protein families, but biophysical modeling schemes have not been available to robustly quantify both. Here we investigate an orthologous RNase H pair by using a minimal distance constraint model (DCM). The DCM is an all atom microscopic model [Jacobs and Dallakyan, Biophys J 2005;88(2):903-915] that accurately reproduces heat capacity measurements [Livesay et al., FEBS Lett 2004;576(3):468-476], and is unique in its ability to harmoniously calculate thermodynamic stability and flexibility in practical computing times. Consequently, quantified stability/flexibility relationships (QSFR) can be determined using the DCM. For the first time, a comparative QSFR analysis is performed, serving as a paradigm study to illustrate the utility of a QSFR analysis for elucidating evolutionarily conserved stability and flexibility profiles. Despite global conservation of QSFR profiles, distinct enthalpy-entropy compensation mechanisms are identified between the RNase H pair. In both cases, local flexibility metrics parallel H/D exchange experiments by correctly identifying the folding core and several flexible regions. Remarkably, at appropriately shifted temperatures (e.g., melting temperature), these differences lead to a global conservation in Landau free energy landscapes, which directly relate thermodynamic stability to global flexibility. Using ensemble-based sampling within free energy basins, rigidly, and flexibly correlated regions are quantified through cooperativity correlation plots. Five conserved flexible regions are identified within the structures of the orthologous pair. Evolutionary conservation of these flexibly correlated regions is strongly suggestive of their catalytic importance. Conclusions made herein are demonstrated to be robust with respect to the DCM parameterization.     

Mitochondrial iron metabolism in plants: frataxin comes into play

Friedreich ataxia (FRDA), an autosomal recessive neurological dysfunction that severely impairs motor coordination and reduction of life expectancy in humans, is caused by a deficiency in frataxin, a nuclear-encoded mitochondrial protein. Recently, a frataxin ortholog has been identified in Arabidopsis thaliana, named AtFH, with a transit peptide for localization in mitochondria and 65% sequence identity with human frataxin (Busi et al. FEBS Lett 576:141–144, 2004). Complementation of S. cerevisiae mutant strain Δyfh1 deficient in frataxin with AtFH, proved that the plant isoform is a functional protein, able to restore normal respiration and growth rates in the mutant yeast (Busi et al. FEBS Lett 576:141–144, 2004). AtFH is localized in mitochondria as its animal counterparts (Busi et al. Plant J 48:873–882, 2006); it is expressed mainly in flowers and developing embryos and it is an essential protein, since the knocking out of AtFH gene causes arrest of embryo development at the globular stage (Vazzola et al. FEBS Lett 581:667–672, 2007). A T-DNA insertional A.thaliana mutant showing a greater than 50% reduction of AtFH protein content, named atfh-1, has impaired activity of two mitochondrial enzymes possessing [Fe-S] clusters: aconitase and succinate dehydrogenase (Busi et al. Plant J 48:873–882, 2006). The results obtained in the last ten years on animal systems can contribute, without any doubt, to the elucidation of the role of frataxin in plant mitochondria; however, mitochondria of photosynthetically active cells, differently from animal ones, are not the major source of Reactive Oxygen Species (ROS) which could suggest possible differences in function between plant and animal frataxin.     

Primary structure of the alpha-subunit of vacuolar-type Na(+)-ATPase in Enterococcus hirae. Amplification of a 1000-bp fragment by polymerase chain reaction.

A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by the polymerase chain reaction method, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na(+)-ATPase alpha-subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of Na(+)-ATPase alpha-subunit is highly homologous to the corresponding sequences of large (alpha) subunits of vacuolar (archaebacterial) type H(+)-ATPases, supporting our proposal [Kakinuma, Y. and Igarashi, K. (1990) FEBS Lett. 271, 97-101] that the Na(+)-ATPase of this organism belongs to the vacuolar-type ATPase.