- Email: [email protected]
Thermophiles like hot T
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TRENDS in Microbiology
5 Miller, C. et al. (2004) SOS response induction by b-lactams and bacterial defense against antibiotic lethality. Science 305, 1629– 1631 6 Miller, C. et al. (2003) DpiA binding to the replication origin of Escherichia coli plasmids and chromosomes destabilizes plasmid inheritance and induces the bacterial SOS response. J. Bacteriol. 185, 6025–6031 7 Perez-Capilla, T. et al. (2005) SOS-independent induction of dinB transcription by b-lactam-mediated inhibition of cell wall synthesis in Escherichia coli. J. Bacteriol. 187, 1515–1518 8 Layton, J.C. and Foster, P.L. (2003) Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol. Microbiol. 50, 549–561 9 Layton, J.C. and Foster, P.L. (2005) Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli. J. Bacteriol. 187, 449–457 10 Maiques, E. et al. (2006) b-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus. J. Bacteriol. 188, 2726–2729 11 Aertsen, A. et al. (2004) An SOS response induced by high pressure in Escherichia coli. J. Bacteriol. 186, 6133–6141 12 Aertsen, A. and Michiels, C.W. (2005) Mrr instigates the SOS response after high pressure stress in Escherichia coli. Mol. Microbiol. 58, 1381–1391
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13 Makovets, S. et al. (1999) Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes. Proc. Natl. Acad. Sci. U. S. A. 96, 9757–9762 14 Slack, A. et al. (2006) On the mechanism of gene amplification induced under stress in Escherichia coli. PLoS Genet. 2, e48 (http://genetics. plosjournals.org) 15 Kuzminov, A. (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage l. Microbiol. Mol. Biol. Rev. 63, 751–813 16 Courcelle, J. et al. (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158, 41–64 17 Beaber, J.W. et al. (2004) SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427, 72–74 18 Cayrol, C. et al. (1995) Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase. Proc. Natl. Acad. Sci. U. S. A. 92, 11806–11809 19 Godoy, V.G. et al. (2006) Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression. EMBO J. 25, 868–879 0966-842X/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.08.006
Genome Analysis
Thermophiles like hot T Ryan Lieph, Felipe A. Veloso and David S. Holmes Center for Bioinformatics and Genome Biology, Millennium Institute of Fundamental and Applied Biology, Life Science Foundation and Andre´s Bello University, Av. Zanartu 1482, Santiago, Chile
A plethora of mechanisms confer protein stability in thermophilic microorganisms and, recently, it was suggested that these mechanisms might be divided along evolutionary lines. Here, a multi-genome comparison shows that there is a statistically significant increase in the proportion of NTN codons correlated with increasing optimal growth temperature for both Bacteria and Archaea. NTN encodes exclusively non-polar, hydrophobic amino acids and indicates a common underlying use of hydrophobicity for stabilizing proteins in Bacteria and Archaea that transcends evolutionary origins. However, some microorganisms do not follow this trend, suggesting that alternate mechanisms (e.g. intracellular electrolytes) might be used for protein stabilization. These studies highlight the usefulness of large-scale comparative genomics to uncover novel relationships that are not immediately obvious from protein structure studies alone. Protein thermostability: a mechanistic distinction between Bacteria and Archaea? Protein structure information has revealed an extensive repertoire of mechanisms that help to maintain protein integrity and function at high temperatures [1]. Thermophilic proteins can exhibit higher core hydrophobicity [2], Corresponding author: Holmes, D.S. ([email protected]) Available online 28 August 2006. www.sciencedirect.com
greater numbers of ionic interactions [3], increased packing density [4], additional networks of hydrogen bonds [5], decreased lengths of surface loops [6], stabilization by heatstable chaperones [7], an increase in disulfide bond formation [8] and a general shortening of length [9]. Changes in amino acid composition that account for some of these mechanisms can be detected in proteome-wide surveys. These include elevated levels of lysine, valine and glutamic acid, arginine-to-lysine replacement and a decrease in glutamine and histidine in thermophiles that cannot be explained by genomic G+C compositional biases or by universal trends of amino acid gain and loss in protein evolution [9–13]. Recently, it was suggested that mechanisms for conferring protein stability could be divided along evolutionary lines [14]. Thermophilic Archaea, as exemplified by Pyrococcus furiosus, are postulated to have evolved in hot places and to use a structure-based method for protein stability: proteins of high density that result from the contribution of numerous mechanisms including higher core hydrophobicity and tighter atom packing. By contrast, thermophilic Bacteria such as Thermotoga maritima are thought to have entered hot environments after evolving elsewhere. It has been suggested that they employ a sequence-based method of protein stability in which protein density is similar to that of mesophiles but with strategically placed salt-bridges to confer thermostability. Here, we suggest that at least one mechanism for protein
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Box 1. Terminology Optimum growth temperatures are defined as: Mesophiles: 20–45 8C Thermophiles: 45–80 8C Hyperthermophiles: >80 8C NTN codons (where N is any nucleotide) specify only the hydrophobic, non-polar amino acids: phenylalanine, methionine, valine, leucine, and isoleucine. In this article, codon usage is predicted from genome sequence information and, therefore, thymine (T) has been used for codon nomenclature instead of uracil (U).
stabilization – increased hydrophobicity – transcends this proposed evolutionary distinction. Increased hydrophobicity confers protein thermostability in Bacteria and Archaea NTN codons (where N represents any nucleotide) encode only the hydrophobic, non-polar amino acids (Box 1). A bias in the use of NTN codons and its relationship to increased hydrophobicity was first reported for mitochondrial DNA sequences where it was linked to the increased proportion of membrane-spanning proteins in these organelles [15]. To determine if increased hydrophobicity is a common mechanism for protein stabilization throughout the Bacteria and Archaea, a multi-genome comparison was carried out to calculate the frequency of NTN codons in the genomes of 163 organisms (see online Supplementary Material, part 1). The frequency of 175 million codons from 0.5 million genes of 14 hyperthermophiles, 142 mesophiles and seven thermophiles was determined using the Comprehensive Microbial Resource [16]. The occurrence of each nucleotide of every codon was evaluated as a function of genomic G+C composition and the optimal growth temperature of each organism (Figure 1). No correlation of genome G+C composition with optimum growth temperature was detected for either Bacteria or
Archaea in agreement with previous observations using smaller datasets [17]. However, according to one-way ANOVA (analysis of variance), a statistically significant trend (p-value <2.2 1016) in the increase of the mean relative frequency of NTN codons was observed with increasing optimal growth temperature (mesophiles 30.1%, thermophiles 30.6%, hyperthermophiles 33%) in both Bacteria and Archaea. ANOVA is known to be robust even when distributions show departures from normality, if the sample size is large and the departures are symmetrical (as in this case). Nevertheless, to reaffirm the significance of the trend, the non-parametric KruskalWallis test was also performed, which led to the same conclusion (see Supplementary Material, part 2). The apparently small 3% difference between mesophiles and hyperthermophiles implies an additional nine hydrophobic residues in an average protein of 300 amino acids in length. The trend described was not observed for either NNT or TNN codons (see Supplementary Material, part 3). What is special about NTN codons? The correlation of increased NTN codons with lifestyle temperature is not just found in specific categories of proteins but is distributed across all protein role categories (data not shown; available from the author on request). NTN encodes exclusively non-polar, hydrophobic amino acids, which points to the common use of increased hydrophobicity for stabilizing proteins in both Bacteria and Archaea. This suggests that at least one mechanism for thermal stabilization of proteins transcends the mechanistic distinction proposed for protein thermostability based on the phylogenetic history of Bacteria and Archaea [14]. The constraint on second position codon space provoked by the increased use of NTN codons is also one of the influences on thermophilic DNA sequence evolution and should be considered when interpreting phylogenetic signals.
Figure 1. Frequency (%) of NTN codons per genome of 163 completely sequenced Bacteria and Archaea plotted as a function of genomic (coding) G+C composition. Least squares curves have been fitted to the data. Additional information on other genomes represented in this figure is available in the online Supplementary Material, part 1. www.sciencedirect.com
Update
TRENDS in Microbiology
NTN encodes isoleucine, leucine, methionine, phenylalanine and valine. An analysis of the proteome composition of our dataset of 163 genome sequences shows that the main difference between mesophiles and hyperthermophiles lies in an increase in isoleucine (from 6.23% to 8.06%, mesophile to thermophile respectively), phenylalanine (from 4.02% to 4.27%) and valine (from 7.06% to 8.11%) (see Supplementary Material, part 4). The modest increments of these amino acids could account for why they were overlooked in earlier proteome-wide studies. The hydrophobic amino acid tryptophan is not encoded by the NTN group, instead, it is encoded by TGG and does not show any significant correlation with optimum growth temperature (from 1.18% to 1.09%); however, the uncharged polar amino acid tyrosine increased from 2.97% in mesophiles to 4.06% in hyperthermophiles (other trends in amino acid composition are shown in the online Supplementary Material, part 4). This does not undermine the importance of the contribution of less frequent (but perhaps strategically positioned) tryptophans to thermostability. However, it does suggest that the NTN codon group of amino acids shares some particular property not manifested by tryptophan, which has been exploited in evolution to stabilize proteins against thermal denaturation. It also provokes the question as to whether this consideration could have been an influence during the evolution of the genetic code. Not all thermophiles comply with the NTN increase Although the trend of the increasing frequency of NTN codons with lifestyle temperature is clear, there is some variation of NTN codon frequency in both Bacteria and Archaea. For example, the hyperthermophilic archaeon Methanopyrus kandleri AV 19 is an outlier with a lower NTN content than might be expected (Figure 1). This microorganism has a high intracellular concentration (1.1 M) of the trivalent anion cyclic 2,3–diphosphoglycerate, which has been suggested to stabilize proteins against thermal denaturation [18]. M. kandleri has fewer predicted laterally transferred genes than other Archaea [19], which could suggest that it is difficult for it to stabilize proteins encoded by laterally transferred genes from organisms that use increased hydrophobicity as a major mechanism for protein stabilization. Another possible outlier is the thermophilic bacterium Thermus thermophilus HB27, which exhibits more NTN codons than might be expected for its lifestyle and genomic G+C content (Figure 1). This suggests that increased hydrophobicity could be a particularly important contributor to protein thermostability in this microorganism. Unlike M. kandleri, lateral gene transfer from thermophilic Archaea is thought to be common in T. thermophilus [20]. This would be more likely to occur if both the donor and recipient shared common mechanisms for conferring protein thermostability, which, in this case, is the proposed increase in hydrophobicity through the increased use of NTN codons. What about halophiles? Halophiles use two mechanisms to protect cellular integrity against high salt: one involves the selective influx of www.sciencedirect.com
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Box 2. Outstanding questions The observed increase in NTN codons and hydrophobicity is displayed by both Archaea and Bacteria. Did it arise independently in the two kingdoms as a mechanism for stabilizing proteins or was it inherited from a common thermophilic ancestor? What light do protein structural studies shed on the role of the specific hydrophobic amino acids encoded by NTN codons in conferring protein thermostability? What is the influence on DNA sequence evolution of the need to maintain a higher relative frequency of NTN codons in thermophiles and how will this consideration influence the interpretation of phylogenetic signals? As more genome sequences become available, what other thermophilic outliers will be discovered that do not comply with the NTN rule? Will these reveal novel underlying mechanisms for conferring protein stability? Will additional niche-specific codon signals be discovered in psychrophiles and other groups of organisms such as acidophiles and alkalophiles? Will the preliminary suggestion that NTN compliance in thermophiles could influence the frequency of lateral gene transfer be supported by future studies?
potassium ions and the other involves the use of osmolytes [21]. Do they also use increased hydrophobicity to stabilize their proteins? The genomes of four mesophilic halophiles have been completely sequenced: the Archaea Halobacterium sp. NRC-1 [22], Haloarcula marimorti [23] and Natronomonas pharaonis [24] and the bacterium Bacillus halodurans [25]. However, none exhibits an increase in the use of NTN codons (Figure 1), which suggests that, unlike thermophiles, increased hydrophobicity is not a mechanism used by halophiles to stabilize proteins. Interestingly, the thermophile T. thermophilus, which has a particularly large increase in the use of NTN codons, also exhibits salt tolerance, using trehalose to stabilize its proteins [26]. This supports the argument that different mechanisms might be used to stabilize proteins against the dual challenges imposed by thermophilic and saline environments. Concluding remarks and future perspectives The multi-genome comparison reported here indicates that there is a statistically significant increase in the proportion of NTN codons correlated with increasing optimal growth temperature for both Bacteria and Archaea. Because NTN encodes exclusively non-polar, hydrophobic amino acids, it points to a common use of hydrophobicity for stabilizing proteins in both Bacteria and Archaea that transcends evolutionary origins. However, additional codon signals surely await discovery as more sequenced genomes become available for comparison, and outstanding questions remain to be answered (Box 2). Acknowledgements This work is supported by Fondecyt 1050063 and by a Microsoft 1 Sponsored Research Program.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tim. 2006.08.004.
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References 1 Kumar, S. and Nussinov, R. (2001) How do thermophilic proteins deal with heat? Cell. Mol. Life Sci. 58, 1216–1233 2 Schumann, J. et al. (1993) Stabilization of creatinase from Pseudomonas putida by random mutagenesis. Protein Sci. 2, 1612–1620 3 Vetriani, C. et al. (1998) Protein thermostability above 100 8C: a key role for ionic interactions. Proc. Natl. Acad. Sci. U. S. A. 95, 12300–12305 4 Russell, R.J. et al. (1997) The crystal structure of citrate synthase from ˚ the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 A resolution. Biochemistry 36, 9983–9994 5 Jaenicke, R. and Bohm, G. (1998) The stability of proteins in extreme environments. Curr. Opin. Struct. Biol. 8, 738–748 6 Thompson, M.J. and Eisenberg, D. (1999) Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability. J. Mol. Biol. 290, 595–604 7 Haslbeck, M. et al. (2005) Some like it hot: the structure and function of small heat-shock proteins. Nat. Struct. Mol. Biol. 12, 842–846 8 Beeby, M. et al. (2005) The genomics of disulfide bonding and protein stabilization in thermophiles. PLoS Biol. 3, e309 9 Tekaia, F. et al. (2002) Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene 297, 51–60 10 Kreil, D.P. and Ouzounis, C.A. (2001) Identification of thermophilic species by the amino acid compositions deduced from their genomes. Nucleic Acids Res. 29, 1608–1615 11 Farias, S.T. et al. (2004) Thermo-search: lifestyle and thermostability analysis. In Silico Biol. 4, 377–380 12 Berezovsky, I.N. et al. (2005) Entropic stabilization of proteins and its proteomic consequences. PloS Comput Biol. 1, e47 13 Singer, G.A. and Hickey, D.A. (2003) Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content. Gene 317, 39–47 14 Berezovsky, I.N. and Shakhnovich, E.I. (2005) Physics and evolution of thermophilic adaptation. Proc. Natl. Acad. Sci. U. S. A. 102, 12742– 12747
15 Naylor, G.J. et al. (1995) Hydrophobicity and phylogeny. Nature 373, 565–566 16 Peterson, J.D. et al. (2001) The Comprehensive Microbial Resource. Nucleic Acids Res. 29, 123–125 17 Hurst, L.D. and Merchant, A.R. (2001) High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc. Biol. Sci. 268, 493–497 18 Shima, S. et al. (1998) Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri. Arch. Microbiol. 170, 469–472 19 Slesarev, A.I. et al. (2002) The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens. Proc. Natl. Acad. Sci. U. S. A. 99, 4644–4649 20 Omelchenko, M.V. et al. (2005) Comparative genomics of Thermus thermophilus and Deinococcus radiodurans: divergent routes of adaptation to thermophily and radiation resistance. BMC Evol. Biol. 5, 57 21 Santos, H. and da Costa, M.S. (2002) Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol. 4, 501–509 22 Ng, W.V. et al. (2000) Genome sequence of Halobacterium species NRC1. Proc. Natl. Acad. Sci. U. S. A. 97, 12176–12181 23 Baliga, N.S. et al. (2004) Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res. 14, 2221–2234 24 Falb, M. et al. (2005) Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome Res. 15, 1336– 1343 25 Takami, H. et al. (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res. 28, 4317–4331 26 Nunes, O.C. et al. (1995) Compatible solutes in the thermophilic Bacteria Rhodothermus marinus and ‘‘Thermus thermophilus’’. Appl. Environ. Microbiol. 61, 2351–2357 0966-842X/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.08.004
Letters
IL-10 and susceptibility to Coccidioides immitis infection Joshua Fierer VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA; and Department of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA
The Trends in Microbiology article by Romani and Puccetti [1] on the role of interleukin (IL)-10 in fungal infections emphasized the excellent research that their group has done on the immune response to the opportunistic fungal pathogens Candida albicans and Aspergillus fumigatus. However, we recommend caution in generalizing their observations to all fungal infections. C. albicans is a commensal organism in humans and symptomatic mucosal infections of this fungus are primarily caused by disturbances of the normal bacterial flora, which can occur after treatment with antibiotics, or in individuals with T cell deficiencies, such as those with AIDS or chronic mucocutaneous candidiasis. Invasive Candida infections are almost always iatrogenic or caused by neutropenia or congenital disorders of polymorphonuclear leukocyte Corresponding author: Fierer, J. ([email protected]) Available online 8 August 2006. www.sciencedirect.com
function and are not a result of problems with T cellmediated immunity. This is also true of invasive infections caused by the nearly ubiquitous environmental fungus, A. fumigatus. It is possible that IL-10-producing T cells that block inflammation are an adaptation to prevent unnecessary inflammation being directed against essentially nonpathogenic fungi that are part of the normal flora or commonly encountered in the environment. By contrast, production of IL-10 might be detrimental if the fungus is a primary pathogen. We have studied the immune response to one such pathogenic fungus, Coccidioides immitis, a dimorphic fungus that is found in the Sonoran deserts in the western hemisphere [2]. Coccidioides species infect thousands of people each year in the southwest of the USA [3]. To study the genetics of resistance to C. immitis, several strains of inbred mice were infected and it was found that C57BL/6 and other susceptible mice make high
TRENDS in Microbiology
5 Miller, C. et al. (2004) SOS response induction by b-lactams and bacterial defense against antibiotic lethality. Science 305, 1629– 1631 6 Miller, C. et al. (2003) DpiA binding to the replication origin of Escherichia coli plasmids and chromosomes destabilizes plasmid inheritance and induces the bacterial SOS response. J. Bacteriol. 185, 6025–6031 7 Perez-Capilla, T. et al. (2005) SOS-independent induction of dinB transcription by b-lactam-mediated inhibition of cell wall synthesis in Escherichia coli. J. Bacteriol. 187, 1515–1518 8 Layton, J.C. and Foster, P.L. (2003) Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol. Microbiol. 50, 549–561 9 Layton, J.C. and Foster, P.L. (2005) Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli. J. Bacteriol. 187, 449–457 10 Maiques, E. et al. (2006) b-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus. J. Bacteriol. 188, 2726–2729 11 Aertsen, A. et al. (2004) An SOS response induced by high pressure in Escherichia coli. J. Bacteriol. 186, 6133–6141 12 Aertsen, A. and Michiels, C.W. (2005) Mrr instigates the SOS response after high pressure stress in Escherichia coli. Mol. Microbiol. 58, 1381–1391
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13 Makovets, S. et al. (1999) Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes. Proc. Natl. Acad. Sci. U. S. A. 96, 9757–9762 14 Slack, A. et al. (2006) On the mechanism of gene amplification induced under stress in Escherichia coli. PLoS Genet. 2, e48 (http://genetics. plosjournals.org) 15 Kuzminov, A. (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage l. Microbiol. Mol. Biol. Rev. 63, 751–813 16 Courcelle, J. et al. (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158, 41–64 17 Beaber, J.W. et al. (2004) SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427, 72–74 18 Cayrol, C. et al. (1995) Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase. Proc. Natl. Acad. Sci. U. S. A. 92, 11806–11809 19 Godoy, V.G. et al. (2006) Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression. EMBO J. 25, 868–879 0966-842X/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.08.006
Genome Analysis
Thermophiles like hot T Ryan Lieph, Felipe A. Veloso and David S. Holmes Center for Bioinformatics and Genome Biology, Millennium Institute of Fundamental and Applied Biology, Life Science Foundation and Andre´s Bello University, Av. Zanartu 1482, Santiago, Chile
A plethora of mechanisms confer protein stability in thermophilic microorganisms and, recently, it was suggested that these mechanisms might be divided along evolutionary lines. Here, a multi-genome comparison shows that there is a statistically significant increase in the proportion of NTN codons correlated with increasing optimal growth temperature for both Bacteria and Archaea. NTN encodes exclusively non-polar, hydrophobic amino acids and indicates a common underlying use of hydrophobicity for stabilizing proteins in Bacteria and Archaea that transcends evolutionary origins. However, some microorganisms do not follow this trend, suggesting that alternate mechanisms (e.g. intracellular electrolytes) might be used for protein stabilization. These studies highlight the usefulness of large-scale comparative genomics to uncover novel relationships that are not immediately obvious from protein structure studies alone. Protein thermostability: a mechanistic distinction between Bacteria and Archaea? Protein structure information has revealed an extensive repertoire of mechanisms that help to maintain protein integrity and function at high temperatures [1]. Thermophilic proteins can exhibit higher core hydrophobicity [2], Corresponding author: Holmes, D.S. ([email protected]) Available online 28 August 2006. www.sciencedirect.com
greater numbers of ionic interactions [3], increased packing density [4], additional networks of hydrogen bonds [5], decreased lengths of surface loops [6], stabilization by heatstable chaperones [7], an increase in disulfide bond formation [8] and a general shortening of length [9]. Changes in amino acid composition that account for some of these mechanisms can be detected in proteome-wide surveys. These include elevated levels of lysine, valine and glutamic acid, arginine-to-lysine replacement and a decrease in glutamine and histidine in thermophiles that cannot be explained by genomic G+C compositional biases or by universal trends of amino acid gain and loss in protein evolution [9–13]. Recently, it was suggested that mechanisms for conferring protein stability could be divided along evolutionary lines [14]. Thermophilic Archaea, as exemplified by Pyrococcus furiosus, are postulated to have evolved in hot places and to use a structure-based method for protein stability: proteins of high density that result from the contribution of numerous mechanisms including higher core hydrophobicity and tighter atom packing. By contrast, thermophilic Bacteria such as Thermotoga maritima are thought to have entered hot environments after evolving elsewhere. It has been suggested that they employ a sequence-based method of protein stability in which protein density is similar to that of mesophiles but with strategically placed salt-bridges to confer thermostability. Here, we suggest that at least one mechanism for protein
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Box 1. Terminology Optimum growth temperatures are defined as: Mesophiles: 20–45 8C Thermophiles: 45–80 8C Hyperthermophiles: >80 8C NTN codons (where N is any nucleotide) specify only the hydrophobic, non-polar amino acids: phenylalanine, methionine, valine, leucine, and isoleucine. In this article, codon usage is predicted from genome sequence information and, therefore, thymine (T) has been used for codon nomenclature instead of uracil (U).
stabilization – increased hydrophobicity – transcends this proposed evolutionary distinction. Increased hydrophobicity confers protein thermostability in Bacteria and Archaea NTN codons (where N represents any nucleotide) encode only the hydrophobic, non-polar amino acids (Box 1). A bias in the use of NTN codons and its relationship to increased hydrophobicity was first reported for mitochondrial DNA sequences where it was linked to the increased proportion of membrane-spanning proteins in these organelles [15]. To determine if increased hydrophobicity is a common mechanism for protein stabilization throughout the Bacteria and Archaea, a multi-genome comparison was carried out to calculate the frequency of NTN codons in the genomes of 163 organisms (see online Supplementary Material, part 1). The frequency of 175 million codons from 0.5 million genes of 14 hyperthermophiles, 142 mesophiles and seven thermophiles was determined using the Comprehensive Microbial Resource [16]. The occurrence of each nucleotide of every codon was evaluated as a function of genomic G+C composition and the optimal growth temperature of each organism (Figure 1). No correlation of genome G+C composition with optimum growth temperature was detected for either Bacteria or
Archaea in agreement with previous observations using smaller datasets [17]. However, according to one-way ANOVA (analysis of variance), a statistically significant trend (p-value <2.2 1016) in the increase of the mean relative frequency of NTN codons was observed with increasing optimal growth temperature (mesophiles 30.1%, thermophiles 30.6%, hyperthermophiles 33%) in both Bacteria and Archaea. ANOVA is known to be robust even when distributions show departures from normality, if the sample size is large and the departures are symmetrical (as in this case). Nevertheless, to reaffirm the significance of the trend, the non-parametric KruskalWallis test was also performed, which led to the same conclusion (see Supplementary Material, part 2). The apparently small 3% difference between mesophiles and hyperthermophiles implies an additional nine hydrophobic residues in an average protein of 300 amino acids in length. The trend described was not observed for either NNT or TNN codons (see Supplementary Material, part 3). What is special about NTN codons? The correlation of increased NTN codons with lifestyle temperature is not just found in specific categories of proteins but is distributed across all protein role categories (data not shown; available from the author on request). NTN encodes exclusively non-polar, hydrophobic amino acids, which points to the common use of increased hydrophobicity for stabilizing proteins in both Bacteria and Archaea. This suggests that at least one mechanism for thermal stabilization of proteins transcends the mechanistic distinction proposed for protein thermostability based on the phylogenetic history of Bacteria and Archaea [14]. The constraint on second position codon space provoked by the increased use of NTN codons is also one of the influences on thermophilic DNA sequence evolution and should be considered when interpreting phylogenetic signals.
Figure 1. Frequency (%) of NTN codons per genome of 163 completely sequenced Bacteria and Archaea plotted as a function of genomic (coding) G+C composition. Least squares curves have been fitted to the data. Additional information on other genomes represented in this figure is available in the online Supplementary Material, part 1. www.sciencedirect.com
Update
TRENDS in Microbiology
NTN encodes isoleucine, leucine, methionine, phenylalanine and valine. An analysis of the proteome composition of our dataset of 163 genome sequences shows that the main difference between mesophiles and hyperthermophiles lies in an increase in isoleucine (from 6.23% to 8.06%, mesophile to thermophile respectively), phenylalanine (from 4.02% to 4.27%) and valine (from 7.06% to 8.11%) (see Supplementary Material, part 4). The modest increments of these amino acids could account for why they were overlooked in earlier proteome-wide studies. The hydrophobic amino acid tryptophan is not encoded by the NTN group, instead, it is encoded by TGG and does not show any significant correlation with optimum growth temperature (from 1.18% to 1.09%); however, the uncharged polar amino acid tyrosine increased from 2.97% in mesophiles to 4.06% in hyperthermophiles (other trends in amino acid composition are shown in the online Supplementary Material, part 4). This does not undermine the importance of the contribution of less frequent (but perhaps strategically positioned) tryptophans to thermostability. However, it does suggest that the NTN codon group of amino acids shares some particular property not manifested by tryptophan, which has been exploited in evolution to stabilize proteins against thermal denaturation. It also provokes the question as to whether this consideration could have been an influence during the evolution of the genetic code. Not all thermophiles comply with the NTN increase Although the trend of the increasing frequency of NTN codons with lifestyle temperature is clear, there is some variation of NTN codon frequency in both Bacteria and Archaea. For example, the hyperthermophilic archaeon Methanopyrus kandleri AV 19 is an outlier with a lower NTN content than might be expected (Figure 1). This microorganism has a high intracellular concentration (1.1 M) of the trivalent anion cyclic 2,3–diphosphoglycerate, which has been suggested to stabilize proteins against thermal denaturation [18]. M. kandleri has fewer predicted laterally transferred genes than other Archaea [19], which could suggest that it is difficult for it to stabilize proteins encoded by laterally transferred genes from organisms that use increased hydrophobicity as a major mechanism for protein stabilization. Another possible outlier is the thermophilic bacterium Thermus thermophilus HB27, which exhibits more NTN codons than might be expected for its lifestyle and genomic G+C content (Figure 1). This suggests that increased hydrophobicity could be a particularly important contributor to protein thermostability in this microorganism. Unlike M. kandleri, lateral gene transfer from thermophilic Archaea is thought to be common in T. thermophilus [20]. This would be more likely to occur if both the donor and recipient shared common mechanisms for conferring protein thermostability, which, in this case, is the proposed increase in hydrophobicity through the increased use of NTN codons. What about halophiles? Halophiles use two mechanisms to protect cellular integrity against high salt: one involves the selective influx of www.sciencedirect.com
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Box 2. Outstanding questions The observed increase in NTN codons and hydrophobicity is displayed by both Archaea and Bacteria. Did it arise independently in the two kingdoms as a mechanism for stabilizing proteins or was it inherited from a common thermophilic ancestor? What light do protein structural studies shed on the role of the specific hydrophobic amino acids encoded by NTN codons in conferring protein thermostability? What is the influence on DNA sequence evolution of the need to maintain a higher relative frequency of NTN codons in thermophiles and how will this consideration influence the interpretation of phylogenetic signals? As more genome sequences become available, what other thermophilic outliers will be discovered that do not comply with the NTN rule? Will these reveal novel underlying mechanisms for conferring protein stability? Will additional niche-specific codon signals be discovered in psychrophiles and other groups of organisms such as acidophiles and alkalophiles? Will the preliminary suggestion that NTN compliance in thermophiles could influence the frequency of lateral gene transfer be supported by future studies?
potassium ions and the other involves the use of osmolytes [21]. Do they also use increased hydrophobicity to stabilize their proteins? The genomes of four mesophilic halophiles have been completely sequenced: the Archaea Halobacterium sp. NRC-1 [22], Haloarcula marimorti [23] and Natronomonas pharaonis [24] and the bacterium Bacillus halodurans [25]. However, none exhibits an increase in the use of NTN codons (Figure 1), which suggests that, unlike thermophiles, increased hydrophobicity is not a mechanism used by halophiles to stabilize proteins. Interestingly, the thermophile T. thermophilus, which has a particularly large increase in the use of NTN codons, also exhibits salt tolerance, using trehalose to stabilize its proteins [26]. This supports the argument that different mechanisms might be used to stabilize proteins against the dual challenges imposed by thermophilic and saline environments. Concluding remarks and future perspectives The multi-genome comparison reported here indicates that there is a statistically significant increase in the proportion of NTN codons correlated with increasing optimal growth temperature for both Bacteria and Archaea. Because NTN encodes exclusively non-polar, hydrophobic amino acids, it points to a common use of hydrophobicity for stabilizing proteins in both Bacteria and Archaea that transcends evolutionary origins. However, additional codon signals surely await discovery as more sequenced genomes become available for comparison, and outstanding questions remain to be answered (Box 2). Acknowledgements This work is supported by Fondecyt 1050063 and by a Microsoft 1 Sponsored Research Program.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tim. 2006.08.004.
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Letters
IL-10 and susceptibility to Coccidioides immitis infection Joshua Fierer VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA; and Department of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA
The Trends in Microbiology article by Romani and Puccetti [1] on the role of interleukin (IL)-10 in fungal infections emphasized the excellent research that their group has done on the immune response to the opportunistic fungal pathogens Candida albicans and Aspergillus fumigatus. However, we recommend caution in generalizing their observations to all fungal infections. C. albicans is a commensal organism in humans and symptomatic mucosal infections of this fungus are primarily caused by disturbances of the normal bacterial flora, which can occur after treatment with antibiotics, or in individuals with T cell deficiencies, such as those with AIDS or chronic mucocutaneous candidiasis. Invasive Candida infections are almost always iatrogenic or caused by neutropenia or congenital disorders of polymorphonuclear leukocyte Corresponding author: Fierer, J. ([email protected]) Available online 8 August 2006. www.sciencedirect.com
function and are not a result of problems with T cellmediated immunity. This is also true of invasive infections caused by the nearly ubiquitous environmental fungus, A. fumigatus. It is possible that IL-10-producing T cells that block inflammation are an adaptation to prevent unnecessary inflammation being directed against essentially nonpathogenic fungi that are part of the normal flora or commonly encountered in the environment. By contrast, production of IL-10 might be detrimental if the fungus is a primary pathogen. We have studied the immune response to one such pathogenic fungus, Coccidioides immitis, a dimorphic fungus that is found in the Sonoran deserts in the western hemisphere [2]. Coccidioides species infect thousands of people each year in the southwest of the USA [3]. To study the genetics of resistance to C. immitis, several strains of inbred mice were infected and it was found that C57BL/6 and other susceptible mice make high