Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phyla Jay McCarren and Edward F. DeLong* Department of Civil and Environmental Engineering and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Summary Since the recent discovery of retinylidene proteins in marine bacteria (proteorhodopsins), the estimated abundance and diversity of this gene family has expanded rapidly. To explore proteorhodopsin photo- system evolutionary and distributional trends, we identified and compared 16 different proteorhodopsin- containing genome fragments recovered from natu- rally occurring bacterioplankton populations. In addition to finding several deep-branching proteor- hodopsin sequences, proteorhodopsins were found in novel taxonomic contexts, including a betaproteobac- terium and a planctomycete. Approximately one-third of the proteorhodopsin-containing genome fragments analysed, as well as a number of recently reported marine bacterial whole genome sequences, contained a linked set of genes required for biosynthesis of the rhodopsin chromophore, retinal. Phylogenetic analy- ses of the retinal biosynthetic genes suggested their co-evolution and probable coordinated lateral gene transfer into disparate lineages, including Euryarcha- eota, Planctomycetales, and three different proteobac- terial lineages. The lateral transfer and retention of genes required to assemble a functional proteor- hodopsin photosystem appears to be a coordinated and relatively frequent evolutionary event. Strong selection pressure apparently acts to preserve these light-dependent photosystems in diverse marine microbial lineages. Introduction Retinylidene proteins (commonly called rhodopsins) com- prise a large and functionally diverse family of proteins. Different varieties of these photoactive integral membrane proteins are capable of performing two known functions; ion-translocating rhodopsins can act as light-driven ion pumps, and sensory rhodopsins, in the course of light- activated conformational changes, interact with transduc- ers to direct phototaxis and motility (Spudich et al., 2000). Rhodopsins can be broadly categorized as either type I rhodopsins, which are all of microbial origin and found in all three domains of life, or type II rhodopsins, which are all animal photoreceptors (Spudich et al., 2000). Type I rhodopsins include several classes originally identified in extremely halophilic archaea: the two ion-pumping rhodopsins, halorhodopsin (HR) and bacteriorhodopsin (BR), which pump chloride ions and protons, respectively, and two sensory rhodopsins (SRI and SRII). Proteor- hodopsins (PR) are another diverse clade of type I rhodopsins first identified in an uncultivated gammapro- teobacterium (Beja et al., 2000a), but now known to be prevalent in other bacteria (de la Torre et al., 2003; Sabehi et al., 2005). Recent additions to the large type I rhodop- sin family include a monophyletic clade of fungal rhodop- sins (Ruiz-Gonzalez and Marin, 2004), another well- supported clade of sequences of unknown function from diverse microbes, and several sequences representing independent branches in the type I rhodopsin phyloge- netic trees (Fig. 1). Originally, microbial rhodopsins were typically associ- ated with Archaea, until a PR was identified in an uncul- tured gammaproteobacterium of the SAR86 clade (Beja et al., 2000a). Genome fragments from other members of the SAR86 group from diverse geographic locales, and other taxa including Alphaproteobacteria (de la Torre et al., 2003), were also shown to encode the photoprotein (Sabehi et al., 2003). Now, PR-like sequences number in the hundreds (Venter et al., 2004), and PRs are estimated to be present in 13% of all marine bacteria in the photic zone (Sabehi et al., 2005). Surprisingly, recent metage- nomic analyses revealed the presence of marine bacterial-like PRs within the genomes of marine plank- tonic Euryarchaea (Frigaard et al., 2006). Conversely, the complete genome sequence of the extremely halophilic bacterium Salinibacter ruber contained several archaeal- like rhodopsins (HR, SRI and SRII), similar to those from its archaeal neighbours that dwell in the same hypersaline Received 21 September, 2006; accepted 2 November, 2006. *For correspondence. E-mail delong@mit.edu; Tel. (+1) 617 253 0252; Fax (+1) 617 253 2679. Environmental Microbiology (2007) 9(4), 846–858 doi:10.1111/j.1462-2920.2006.01203.x © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd