Molecular evolution of sensory domains in cyanobacterial chemoreceptors

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encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa. J. Bacteriol. 176, 1128–1140 McMorran, B.J. et al. (2001) Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa. Microbiology 147, 1517 – 1524 Ochsner, U.A. et al. (2002) Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 99, 8312 – 8317 McMorran, B.J. et al. (1996) Characterisation of the pvdE gene which is required for pyoverdine synthesis in Pseudomonas aeruginosa. Gene 176, 55 – 59 de Chial, M. et al. (2003) Identification of type II and type III

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pyoverdine receptors from Pseudomonas aeruginosa. Microbiology 149, 821 – 831 27 Baysse, C. et al. (2002) Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC. FEBS Lett. 523, 23 – 28 28 Lamont, I.L. Martin, L.W. et al. (2003) Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. Microbiology 149, 833 – 842 0966-842X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0966-842X(03)00076-3

Molecular evolution of sensory domains in cyanobacterial chemoreceptors Kristin Wuichet and Igor B. Zhulin School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA

Components of the chemotaxis system encoded in multiple heomologous operons were identified in five cyanobacterial genomes. Analysis of phylogenetic profiles, genomic context, domain architectures and sequence identity reveal that sensory modules of chemoreceptors that detect environmental cues are the subject of frequent domain birth and death events and have accelerated rates of sequence evolution. This fact could explain a remarkable diversity of the sensing repertoire of chemotaxis receptors in microorganisms. The signal transduction cascade for chemotaxis in Escherichia coli is one of the best-understood regulatory pathways in nature [1,2]. It includes specialized chemoreceptors (methyl-accepting chemotaxis proteins, MCPs) that serve as sensors of various physicochemical parameters, and a two-component regulatory system, CheA – CheY, that interacts with MCPs through the CheW docking protein. Homologous chemotaxis systems are found in many distantly related species of Bacteria and Archaea. Remarkable diversity within these systems is observed in the sensory repertoire of MCPs [3], which is determined in part by various domains in MCP N-terminal sensory modules [4– 8]. Interestingly, the C-terminal cytoplasmic signaling module of MCPs is the most conserved element within the entire chemotaxis pathway [3,9]. Thus, sensory and signaling modules in MCPs appear to have different evolutionary fates. However, the questions remain: how different are these fates, and what drives the differential domain evolution within a chemoreceptor molecule? In this article, we address these questions using a unique case study: a highly conserved set of homologous MCPcontaining chemotaxis operons that we have identified in newly sequenced genomes from a closely related group of microorganisms, cyanobacteria. Cyanobacteria are a deepbranching phylum of the bacterial evolutionary tree [10] Corresponding author: Igor B. Zhulin ([email protected]). http://timi.trends.com

and several complete or nearly complete cyanobacterial genomes are now available for comparative analysis. Chemotaxis HOMOLOGS (see Glossary), including MCPs, were recently experimentally studied in a cyanobacterium Synechocystis sp. PCC6803 [11]. Using BLAST [12] searches of public protein databases, we have identified homologs of MCPs and other chemotaxis proteins in four cyanobacterial species other than Synechocystis sp. PCC6803, namely Nostoc (Anabaena) sp. PCC7120, Nostoc punctiforme, Thermosynechococcus elongatus and Trichodesmium erythraeum IMS101. In all five cyanobacterial species, MCPs are encoded in highly conserved multiple chemotaxis operons: not only are protein sequences per se conserved, but also domain architectures and the gene order in operons. This provided us with an opportunity to reveal for the first time the trends in molecular evolution of MCPs using several independent approaches, including analysis of the genomic context. Orthologous relationships between cyanobacterial chemotaxis operons All cyanobacterial chemotaxis operons contain a gene encoding the central regulator of chemotaxis, the CheA protein. We first constructed a neighbor-joining tree of all CheA homologs available in current databases and found that cyanobacterial CheA homologs form a monophyletic cluster with exception of two instances that are indicative of a lateral gene transfer (Fig. 1). Trees of a similar topology were obtained for other conserved proteins from the chemotaxis operons. Orthologous relationships between the cyanobacterial chemotaxis operons were further Glossary Homologs : genes/proteins that have a common evolutionary history. Orthologs : genes/proteins in different organisms that are direct evolutionary counterparts of each other and were inherited by speciation. Paralogs : genes/proteins in the same organism that evolved by duplication.

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Fig. 1. Phylogenetic trees of CheA proteins. Trees were constructed using conserved portions of CheA protein corresponding to a known three-dimensional structure [23]. (a) A neighbor-joining tree constructed from a CLUSTAL [24] alignment of 123 CheA homologs available from a non-redundant protein database (NCBI). Branches of cyanobacterial sequences are shown in bold. GenBank protein identification (GI) numbers for all CheA homologs can be found as supplementary information online at http://archive.bmn.com/supp/tim/zhulin.pdf. L, sequences from laterally transferred operons. (b) A maximum-likelihood tree built from an alignment of cyanobacterial CheA sequences using the ProML program from the Phylip package (http://evolution.genetics.washington.edu/phylip.html) with its default parameters. Four clusters colored in red (1), blue (2), yellow (3) and green (4) contain CheA sequences encoded in corresponding operons (1, 2, 3, 4) shown in Fig. 2. GI numbers for shown CheA homologs are as follows: Nostoc sp. (Nos) 1, 17228421; 2, 17228563; 3, 17229653; Nostoc punctiforme (Npun) 1, 23129476; 2, 23128530; 20 _1, 23129050; 20 _2, 23129053; 3, 23124633; L, 23130659; Trichodesmium erythraeum (Tery) 1, 23040288; 2, 23039943; L, 23042558; Thermosynechococcus elongatus (Telo) 1, 22297892; 3, 22298111; 4, 22298565; Synechocystis sp. (Syn) 1, 16331224; 2, 16331986; 4, 16329790.

inferred from maximum-likelihood trees built from aligned conserved domains for each protein (Fig. 1). A presence of a distinct N-terminal module in corresponding MCPs served as additional evidence for operon ORTHOLOGY (Fig. 2). Two independent methods, namely phylogenetic analysis of CheA protein (Fig. 1) and detailed domain architectures of MCPs (Fig. 2) provided identical results for identification of orthologous operons. On the basis of these results and taking into account known trends in evolution of operons [13], a simplified evolutionary scenario (a detailed one is beyond the scope of this study) would include a common ancestor of the five species that had four PARALOGOUS chemotaxis operons (Fig. 2). Operon 1 appears to be the most conserved one (Fig. 1) and is present in all five species. Operon 2 is present in all species except for T. elongatus, and is duplicated in N. punctiforme, resulting in Operon 20 . Operon 3 is missing in T. erythraeum and Synechocystis sp., whereas Operon 4 is present only in T. elongatus and Synechocystis sp. Finally, T. erythraeum and N. punctiforme seem to acquire additional chemotaxis operons by a lateral transfer. The phylogenetic data that led http://timi.trends.com

to this conclusion (Fig. 1) is further supported by the observation that the gene order and the content of the laterally transferred operon in N. punctiforme are different from those in any other cyanobacterial operon (Fig. 2). Domain birth, death and innovation in MCP sensory modules Analysis, using the PSI-BLAST [12] and SMART [14] tools, of the domain architecture revealed that MCPs from paralogous operons have very different N-terminal sensory modules, whereas sensory modules in MCPs from orthologous operons are quite similar (Fig. 2). The N-terminal module of the orthologous MCPs from Operon 1 (shown in red) is unique (i.e. no homologous domains were detected in any publicly available database). Fold recognition using the 3D-PSSM program [15] and secondary structure prediction using the JPRED2 consensus program [16] suggest that this module comprises a globular and mostly a-helical domain rather than an unstructured region. The N-terminal module of the orthologous MCPs from Operon 2

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Fig. 2. Organization of cyanobacterial chemotaxis operons and domain architectures of corresponding MCPs. (a) Classification of operons (1,2,3,4) is based on the results of phylogenetic analyses of individual proteins encoded in operons, including CheA (Fig. 1) and the C-terminal module of MCP, and on the presence of a specific N-terminal module in MCPs encoded within operons. (b) Details of domain organization of MCPs revealed by PSI-BLAST [12] and SMART [14] searches. The numbering and color code are the same as in (a). GI numbers are shown underneath each MCP.

consists of several GAF domains, known phototransducing elements [17]. Indeed, the Operon 2 MCP from Synechocystis sp. was predicted [5] and experimentally shown [10,18] to act as a receptor for phototaxis. The N-terminal modules of the MCPs from Operons 3 and 4 contain a known ligandbinding sensory domain Cache [6] and a tetratrico peptide repeat (a known site for protein – protein interactions [19]), respectively. Dramatic consequences of domain shuffling, birth and death in the sensory modules of MCPs from Operons 2, 3 and 4 are apparent, whereas no such changes can be seen in MCPs from the most conserved Operon 1 (Fig. 2). http://timi.trends.com

Evolutionary rates in individual proteins from chemotaxis operons No obvious changes were observed in the domain organization of the sensory modules of Operon 1 MCPs. Therefore, we have measured the percentage of sequence identity in these modules and compared it to that in other conserved modules of the chemotaxis proteins. Pairwise global alignments (without end gap penalty) were performed between all of the orthologous proteins or modules using the LALIGN program (http://www.ch.embnet.org/software/LALIGN_form.html) and the resulting sequence identities were averaged.

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The obtained results demonstrate that all conserved domains of the chemotaxis components of the Operon 1 have averaged sequence identity above 60%. A striking difference was observed only for the N-terminal module of the MCP, less than 25% averaged identity. Even in the closely related species Nostoc sp. and N. punctiforme, in which all conserved domains of the Operon 1 have a sequence identity above 90%, this number for the N-terminal module of MCPs drops below 50%. A similar trend was observed for other orthologous operons (with or without inclusion of the Nostoc/ N. punctiforme pairwise comparisons). For example, in Operon 3, all conserved domains have sequence identity in the range of 40–60%, whereas the N-terminal module of the MCP has 27% identity, although it contains a conserved Cache domain. For any type of multiple or pairwise comparisons, the sequence identity in the N-terminal modules of MCPs was always significantly (statistically) lower than in the rest of the chemotaxis operon. We can therefore conclude that the N-terminal modules, which are responsible for sensory properties of MCPs, have accelerated evolutionary rates. Biological implications Proteins or their domains that are involved in protein – protein interactions show a shared rate of evolution [20]. On the other hand, proteins that do not interact with each other have different evolutionary rates even if they are encoded in the same operon and participate in the same regulatory pathway [21]. The sensory modules of MCPs do not participate in crucial protein – protein interactions within the chemotaxis pathway, as do their signaling modules and all other proteins encoded in chemotaxis operons [1,2]. The absence of physical constraints imposed by such interactions will probably account for the drastic increase in the evolutionary rate and domain shuffling in these regions that are described in this study. The evolution of MCPs follows a domain birth, death and innovation model (BDIM) applied to a multidomain network [22], in which the conserved C-terminal module serves as a hub connecting the receptor to other proteins in the signal transduction pathway. Rapid BDIM-type evolution of the sensing modules of MCPs creates a greater diversity of sensing capabilities and aids the organism’s ability to respond to a wider variety of extracellular and intracellular signals. Conclusions Comparative genomic analysis revealed that sensory domains of cyanobacterial chemoreceptors evolve much faster than their signaling domains and the rest of the chemotaxis signal transduction pathway. Fast sequence evolution in sensory domains could lead to new sensing capabilities of chemoreceptors. It is likely that this trend will be observed for other sensory receptors in prokaryotes.

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Acknowledgements This study was supported by National Science Foundation grant EIA-0219079 (to I.B.Z.). http://timi.trends.com

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