Update References 1 Baulcombe, D. (2004) RNA silencing in plants. Nature 431, 356–363 2 Brodersen, P. and Voinnet, O. (2006) The diversity of RNA silencing pathways in plants. Trends Genet. 22, 268–280 3 Dunoyer, P. et al. (2007) Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat. Genet. 39, 848–856 4 Vaistij, F.E. et al. (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14, 857–867 5 Himber, C. et al. (2003) Transitivity-dependent and -independent cellto-cell movement of RNA silencing. EMBO J. 22, 4523–4533 6 Palauqui, J-C. et al. (1997) Systemic acquired silencing: transgenespecific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16, 4738–4745 7 Voinnet, O. et al. (1998) Systemic spread of sequence-specific transgene RNA degradation is initiated by localised introduction of ectopic promoterless DNA. Cell 95, 177–187 8 Ueki, S. and Citovsky, V. (2001) Inhibition of systemic onset of postranscriptional gene silencing by non-toxic concentrations of cadmium. Plant J. 28, 283–291 9 Brosnan, C.A. et al. (2007) Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 104, 14741–14746
Trends in Genetics Vol.24 No.4 10 Bouche, N. et al. (2006) An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 25, 3347–3356 11 Deleris, A. et al. (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313, 68–71 12 Moissiard, G. et al. (2007) Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicerlike 2, and is compromised by viral-encoded suppressor proteins. RNA 13, 1268–1278 13 Smith, L.M. et al. (2007) An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521 14 Matzke, M.A. and Birchler, J.A. (2005) RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 6, 24–35 15 Huettel, B. et al. (2007) RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants. Biochim. Biophys. Acta 1769, 358–374 16 Kanno, T. et al. (2005) Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat. Genet. 37, 761–765 17 Havelda, Z. et al. (2003) In situ characterization of Cymbidium Ringspot Tombusvirus infection-induced posttranscriptional gene silencing in Nicotiana benthamiana. J. Virol. 77, 6082–6086 0168-9525/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2008.01.005 Available online 5 March 2008
Genome Analysis
Adapting to environmental changes using specialized paralogs ˝ 2, Lejla Pasˇic´2 Gabino Sanchez-Perez1*, Alex Mira2*, Ga´bor Nyiro 2 and Francisco Rodriguez-Valera 1 2
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada Department of Microbiology, Universidad Miguel Hernandez, 03550 San Juan de Alicante, Alicante, Spain
When a bacterial species survives under changing environmental circumstances (e.g. salinity or temperature), its proteins might not function in all physicochemical conditions. We propose that prokaryotes cope with this problem by having two or more copies of the genes affected by environmental fluctuations, each one performing the same function under different conditions (i.e. ecoparalog). We identify potential examples in the bacterium Salinibacter ruber and in other species that experience wide environmental variations. In prokaryotes, evolution of paralogous sequences by duplication and subsequent divergence is a major contributor to gene genesis [1], and gene families can account for up to 50% of bacterial genomes [2]. Certain genes (e.g. elongation factors) have more than one copy in the genome, probably because there is a selective pressure to increase protein dose. In these cases, divergence is prevented by recombination between the genes, and similarities among the copies are high (100%). However, standard paralogous genes diverge and develop different specialized functions, and amino acid similarities among
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Corresponding author: Mira, A. (
[email protected]). These authors contributed equally to this paper.
them are typically 30–45% [3]. Typical examples include membrane transporters, where different paralogs have specialized in the transport of different peptides, sugars or minerals. Here, we present a third evolutionary process leading to new genes following sequence redundancy: related genes that perform the same cellular function under different ecological conditions (i.e. ecoparalogs). This concept has arisen from the in-depth analysis of the genome of the first hyperhalophilic bacterium sequenced: Salinibacter ruber [4]. Hyperhalophiles adapt to high environmental salinity by increasing the intracellular concentration of K+ rather than by the use of organic compatible solutes [5,6] and thus belong to the ’salt-in’ strategy halophiles. Standard, nonhalophilic proteins are not functional at high salinities. However, typical halophilic proteins accumulate acidic residues on their surface and show their optimal activity and stability at high salinity. The predicted isoelectric point (pI) provides an indication of the acidic nature of proteins [7]; accordingly, S. ruber has the most acidic proteome (low average pI) among bacteria [4]. However, we detected sets of paralogous genes in its genome that seem to perform the same function but differed in predicted pI values. Our study of these genes indicates that they differ in halophilicity and could therefore act as backups to maintain essential cellular functions over
Update a wider range of salinity, increasing the environmental distribution of the organism. Identification of ecoparalogs in S. ruber For two genes to be considered ecoparalogs, they should differ little in sequence so that function of their encoded proteins is identical; any variation, however, should confer a different ecological character (e.g. halophilicity). If differences in halophilicity between S. ruber paralogs were caused by chance, divergent paralogs should have the largest differences. However, low-divergence paralogs show larger differences in acidity (Supplementary Figure S1),
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suggesting that amino acid substitutions specifically provided different halophilicity to each member of a pair. Thus, we identified sets of potential ecoparalogs by selecting proteins with 45–95% similarity, identical annotation and different pI values. However, the residues exposed on the surface of the protein are the ones involved in preserving the proper hydration sphere required for protein solubility and, ultimately, its biological function [8]. Therefore, we measured the acidity of amino acids encoded by the putative ecoparalogs considering their location on the protein, when a homologous secondary structure was available. The examples shown in Figure 1 clearly indicate that the two
Figure 1. The electrostatic potential surface representation of three pairs of ecoparalogs. Differences in halophilism are apparent between these gene pairs: (a) Ferrochelatase (genes with locus tag numbers SRU_1741 and SRU_2695); (b) glutamate dehydrogenase (SRU_0505 and SRU_2255); (c) replication initiator protein dnaA (SRU_0001 and SRU_2199). The yellow arrows point the DNA binding region located in domain IV. Electrostatic potentials were calculated by using MOLMOL. The surface potentials are mapped from 0.5 kcal mol1 (red) to +0.5 kcal mol1 (blue). The numbers in parentheses indicate the density of acidic residues per 103 A˚2.
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Update ecoparalogs differ in halophilicity as shown by the charge distribution on the surface (Figure 1a,b) or on the active site (Figure 1c) and could differ dramatically in their ability to function at different ionic strengths. In one specific case, the two copies of glutamate dehydrogenase are known to have different salt activity ranges despite having very similar pI. One copy showed maximum enzymatic activity in the absence of salts, whereas the other required high salinity to be active [9], showing that a small difference in pI could result in a widely different salt response. Thus, we measured the difference in surface acidic amino acid density between the two glutamate-dehydrogenase (DH) ecoparalogs and considered this an experimentally tested lower threshold for a significant difference in halophilicity. Thus, we used as a complementary criteria to differ in pI that the acidic amino acid density on the surface of the protein products should differ by >0.2 negative residues per ˚ 2, eliminating 15 gene pairs from the list. Using these 1000 A criteria, a total of 27 pairs and 4 triplets of potential ecoparalogs were selected (Supplementary Table S1). We confirmed that the potential ecoparalogs were functional equivalents as follows. (i) By performing sequence similarity searches against the database, ecoparalogs were selected if they produced hits only to genes with a single annotation. This obviously does not guarantee that
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ecoparalogs perform the same function because of errors in genome annotation procedures, but it gives a first approximation that can be confirmed experimentally. (ii) We also confirmed that none of the copies were pseudogenes [nonsynonymous to synonymous substitution ratios (dN/dS values) significantly different from 1]. Fourteen potential ecoparalogs had dN/dS ratios >1.25, which is indicative of positive selection. In addition, we confirmed the functionality of ecoparalogs by examining the conservation of ribosome-binding sites, which are erased in pseudogenes [10] and by quantitative PCR of selected cases, showing that both copies of dnaA and phytoene DH are expressed (data not shown). Function and origin of ecoparalogs The function of identified ecoparalogs was diverse but by no means random (Supplementary Figure 2). The largest pI differential values were found among proteins with functions predicted to be on the outer membrane or the periplasmic space, where the influence of changing salinity can be dramatic for protein function and stability. For instance, some ecoparalogs were involved in cell envelope biosynthesis or extracellular binding. Interestingly, a large set of ecoparalogs were DNA-binding proteins. DNA is a highly negatively charged polymer, and several
Figure 2. Phylogeny and genomic context of Salinibacter ruber ecoparalogs. The maximum likelihood phylogeny of uridine diphosphate–glucuronate decarboxylase genes and cytochrome c oxidase subunit I genes are shown in (a) and (b), respectively. The ecoparalog in tree (a) seems to have arisen by duplication and subsequent divergence. The ecoparalog phylogeny in tree (b) is consistent with horizontal gene transfer from haloarchaea. Support for nodes correspond to 1000 bootstrap replicates and is expressed as percentage. The scale bar indicates number of positions per site. Genomic context of S. ruber ecoparalogs (gray boxes) is shown in the graphs below the corresponding trees (c) and (d). Shaded contiguous ecoparalogs have a consistent phylogeny with each other. Gray squares suggest putatively co-transcribed gene sets (same transcription direction and
