# Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study

Christophe Six, Jean-Claude Thomas, Laurence Garczarek, Martin Ostrowski, Alexis Dufresne, Nicolas Blot, David J Scanlan, Frédéric Partensky

► To cite this version:

Christophe Six, Jean-Claude Thomas, Laurence Garczarek, Martin Ostrowski, Alexis Dufresne, et al.. Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study. Genome Biology, 2007, 8 (12), pp.R259. 10.1186/gb-2007-8-12-r259. hal-00338622

HAL Id: hal-00338622

https://hal.science/hal-00338622v1

Submitted on 15 Dec 2015

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Diversity and evolution of phycobilisomes in marine *Synechococcus* spp.: a comparative genomics study

Christophe Six*†, Jean-Claude Thomas*‡, Laurence Garczarek*, Martin Ostrowski§, Alexis Dufresne*, Nicolas Blot*, David J Scanlan§ and Frédéric Partensky*

Addresses: *UMR 7144 Université Paris VI and CNRS, Station Biologique, Groupe Plancton Océanique, F-29682 Roscoff cedex, France. †Mount Allison University, Photosynthetic Molecular Ecophysiology Group, Biology Department, E4L 1G7 Sackville, New Brunswick, Canada. ‡UMR 8186 CNRS and Ecole Normale Supérieure, Biologie Moléculaire des Organismes Photosynthétiques, F-75230 Paris, France. §Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.

Correspondence: Frédéric Partensky. Email: partensky@sb-roscoff.fr

Published: 5 December 2007

Genome Biology 2007, 8:R259 (doi:10.1186/gb-2007-8-12-r259)

The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2007/8/12/R259

Received: 23 July 2007

Revised: 22 October 2007

Accepted: 5 December 2007

© 2007 Six et al.; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## Abstract

**Background:** Marine *Synechococcus* owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrin I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine *Synechococcus* strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism.

**Results:** By carefully comparing the *Synechococcus* genomes, we have retrieved candidate genes potentially required for the synthesis of phycobiliproteins in each pigment type. This includes linker polypeptides, phycobilin lyases and a number of novel genes of uncharacterized function. Interestingly, strains belonging to a given pigment type have similar phycobilisome gene complements and organization, independent of the core genome phylogeny (as assessed using concatenated ribosomal proteins). While phylogenetic trees based on concatenated allophycocyanin protein sequences are congruent with the latter, those based on phycocyanin and phycoerythrin notably differ and match the *Synechococcus* pigment types.

**Conclusion:** We conclude that the phycobilisome core has likely evolved together with the core genome, while rods must have evolved independently, possibly by lateral transfer of phycobilisome rod genes or gene clusters between *Synechococcus* strains, either via viruses or by natural transformation, allowing rapid adaptation to a variety of light niches.

<footnote>* These authors contributed equally to this work.</footnote>
**Background**

Since their discovery almost 30 years ago [1,2], marine representatives of the *Synechococcus* genus have been found in the upper illuminated layer of most marine ecosystems, from coastal to offshore waters as well as from low to high latitudes [3-5]. Besides being ubiquitous, *Synechococcus* are often abundant, with cell densities ranging from a few hundred to over one million cells per milliliter of seawater [6-10].

*Synechococcus* cells owe their vivid colors mainly to their photosynthetic antenna, called phycobilisomes (PBSs). These water-soluble macromolecular complexes comprise rods surrounding a central core and are made of phycobiliproteins, which covalently bind chromophores (phycobilins) by thioether bonds to cysteinyl residues (for reviews, see [11-15]). All phycobiliproteins in cyanobacteria consist of two distinct subunits, α and β, organized either as trimeric (αβ)₃ or, in most cases, as hexameric discs (αβ)₆. The PBS core of marine *Synechococcus* is made of allophycocyanin (AP), which binds only the blue-colored chromophore phycocyanobilin (PCB; A_max = 620 nm). In some strains, phycocyanin (PC) may constitute the whole rod, as it does in many freshwater cyanobacteria (for example, *Synechococcus elongatus* PCC 7942, *Synechocystis* sp. PCC 6803). In that case, it binds only PCB and is of the C-PC type [15]. However, in most phycoerythrin (PE)-containing marine *Synechococcus* characterized so far, PC makes up the basal disc at the core-proximal end of the rods. It binds both PCB and the red-colored chromophore phycoerythrobilin (PEB; A_max = 550 nm) at a molar ratio of 1:2 and thus belongs to the R-PCII type [16]. In strain WH7805, however, the base of the rods is thought to consist of a so-called R-PCIII, an optically unusual PC that binds PCB and PEB at a molar ratio of 2:1 [15,17].

In most PE-containing *Synechococcus* strains isolated to date, the distal part of the PBS rods is composed of two types of PE (PEI and PEII). PEII always binds both PEB and the orange colored phycourobilin (PUB; A_max = 495 nm), whereas PEI binds either only PEB or both PEB and PUB [18,19]. However, Everroad and Wood [20] have recently suggested that some marine *Synechococcus* strains may contain rods with a single type of PE that binds only PEB chromophores. In addition, the higher order structure of PBSs is stabilized by linker polypeptides that contribute to the building of a protein environment around the phycobilins [14,21]. These linkers have very variable sizes (8-120 kDa) but most are in the 27-35 kDa range. In the rods, only those associated with PEII are chromophorylated with PUB [19,21].

Although the *Synechococcus* genus itself is polyphyletic, marine *Synechococcus* characterized thus far form a well-defined branch within the cyanobacteria radiation, together with the *Prochlorococcus* and *Cyanobium* genera [22-25]. This grouping, called 'Cluster 5' by Herdman and coworkers [26], is a combination of the former Marine Clusters A and B previously defined by Waterbury and Rippka [27]. Cluster 5

thus gathers coastal, euryhaline *Synechococcus* strains as well as strictly marine strains (that is, with elevated growth requirements for Na⁺, Mg⁺ and Ca⁺⁺). Subclusters 5.1 and 5.2 have also been tentatively defined by Herdman and coworkers [26] in order to separate the strictly marine PE-containing strains (5.1) from a group of euryhaline strains lacking PE (5.2), including WH5701 and WH8007. However, Fuller and coworkers [23] have shown that one clade within the subcluster 5.1 (clade VIII) gathers euryhaline strains lacking PE and Chen and coworkers [25] have isolated several new members of subcluster 5.2 into culture that do contain PE. Furthermore, the latter authors suggested that WH5701 and WH8007 might actually belong to two distinct clusters.

Among the strains containing two PE types, there is a clear consistency between phylogenies based on different molecular markers, including *rpoC1* [28], *ntcA* [29], the 16S rRNA gene [23] and the 16S-23S rDNA internal transcribed spacer [24]. However, none of these phylogenies is congruent with the whole cell ratio of PUB to PEB. This chromophore ratio is known to vary according to the light niche, with open ocean strains predominantly displaying a high PUB:PEB whereas mesotrophic or coastal strains generally have lower ratios or no PUB [6,7,30-32]. Some strains even display a variable PUB:PEB depending on the ambient light quality, that is, they are able to chromatically adapt [33]. These so-called type IV chromatic adapters are not confined to a particular phylogenetic clade within Cluster 5 [34]. This raises the question of the molecular basis of differences in whole cell PUB:PEB between *Synechococcus* strains. More generally, one might wonder whether PBS components have undertaken a different evolutionary trajectory compared to the core genome.

In order to address these questions, we studied 11 *Synechococcus* strains, belonging to various phylogenetic clades according to Fuller *et al.* [23] and representing the whole variety of PBS pigmentations known so far within Cluster 5. We compared the PBS gene complements of these strains, an approach that revealed a number of novel PBS genes, including putative lyases and linker polypeptides. By combining these genomic data with biochemical and optical properties of isolated phycobiliprotein complexes, we identified several marine *Synechococcus* pigment types and we propose putative, structural models for their corresponding PBSs. We also examined the phylogeny of each phycobiliprotein type, yielding new insights into the evolution of PBS complexes within the marine *Synechococcus* group.

**Results**

*Synechococcus* pigment types

Despite the apparently large diversity of pigmentation existing among marine *Synechococcus*, these can be partitioned into only three major types based on the phycobiliprotein composition of the rods: type 1 representatives have only PC, type 2 have PC and PEI and type 3 have PC, PEI and PEII.
<figure><img src="image_2.png" /><figcaption>**Figure 1**
The diversity of pigment types among marine *Synechococcus* spp. (a) Photograph of representative cultured strains of the major pigment types (1-3) and subtypes (3a-c) of marine *Synechococcus* grown under low white light and (b) corresponding absorption properties of whole cells. Pigment subtype 3d corresponds to type IV chromatic adapters, which are able to modify their PBS pigmentation from subtype 3b when grown under white or green light to subtype 3c when grown under blue light. The different colors of stars in panel A are a code for the different pigment types.</figcaption></figure>

Type 3 can be further subdivided into four subtypes (3a through 3d) based on the ratio of the two chromophores (PEB and PUB) bound to PEs, a ratio that can be low, medium, high or variable. Figure 1a illustrates these different pigment types or subtypes and their corresponding colors. The 11 fully sequenced marine *Synechococcus* strains cover the whole range of PBS pigmentation known so far in this group [6,23,33]. Pigment type 1 is represented by the blue-green, PE-lacking strains WH5701 and RS9917. These strains absorb light optimally in the wavelength range 600-660 nm, that is, red and orange light (Figure 1b). The genome of the fuchsia pink WH7805 strain (pigment type 2) contains a single set of PE genes encoding a PEI-like complex, as detailed below. The whole cell absorption maximum of this form of PE devoid of PUB ($A_{max}$ = 570 nm, corresponding to yellow-green light) is red-shifted relative to other PEs (Figure 1b).

All strains displaying pigment type 3 possess both PEB and PUB chromophores. Subtypes 3a through 3c differ from one another in their whole cell ratio of PUB to PEB (hereafter PUB:PEB), as assessed by their fluorescence excitation maxima ($F_{495 nm}$: $F_{550 nm}$) with emission at 580 nm (Table 1). Note that the use of this fluorescence excitation ratio is preferable to using the corresponding absorption ratio ($A_{495 nm}$: $A_{550 nm}$) to characterize these different subtypes *in vivo*, since the carotenoids zeaxanthin and β-carotene have a notable contribution to the wavelength range of the PUB absorption peak (Figure 1b). The PUB:PEB can be either low (approximately 0.4) in type 3a strains such as WH7803, medium (approximately 0.8) in type 3b strains such as RCC307 or high (>1.7) in type 3c strains such as in WH8102 and CC9605 (Table 1). Depending on this ratio, PBSs of these strains preferentially harvest either green light (550 nm) or blue-green light (495 nm) (Figure 1b). Finally, pigment type 3d includes strains with a variable PUB:PEB (0.7-1.7), depending on whether these cells are grown under white/green or blue light [33,34]. These type IV chromatic adapters include the strains CC9311, RS9916, BL107 and CC9902 as well as a number of other strains that have not yet been sequenced (including WH8020, M16.17, M11.1, RCC61 (a.k.a. Minos 11) and RS9911; Table 1 and data not shown). To this suite of pigment types can be added a 'moderately high' PUB:PEB subtype (PUB:PEB approximately 1.2), represented by strain WH8103 and which is indistinguishable by eye from, and included within, type 3c (Figure 1a). Though as yet unsequenced, the genome of WH8103 has been screened, in part, by suppression subtractive hybridization [35].

## Optical properties of phycobiliproteins

The color and specific absorption properties of whole *Synechococcus* cells (Figure 1) are mainly determined by the major phycobiliprotein form occurring in the PBS rods. Isolated PC and/or PE complexes have been previously characterized in a few marine *Synechococcus* strains, including WH7803, WH7805, WH8102, WH8103 and the chromatic adapters WH8020 (under white light only), M11.1 and M16.17 [13,16-19,34,36], as summarized in Table 1. In order to explore further the diversity and possible combinations of these phycobiliproteins in the different *Synechococcus* pigment types, we have used sucrose density gradients and isoelectric focusing to isolate PC, PEI and/or PEII from a number of other strains and then have determined their optical properties (Figures 2 and 3 and Table 1).

The PC present in WH5701 and RS9917, which formed a sky blue band on isoelectric focusing gels (not shown), had absorption ($A_{max}$ = 621 nm) and fluorescence ($F_{max}$ = 648 nm) properties typical of C-PC (Figure 2a), that is, known to bind only PCB chromophores [15]. We also found C-PC in the PE-containing, PUB-lacking strain WH8018, whereas WH7805 (which, like WH8018, displays pigment type 2) is known to possess R-PCIII [17]. R-PCIII has a molar PCB:PEB of 2:1, like the R-PCI of red algae, but a different spectrum, with an
<caption>Table I</caption>

Strain numbers, phylogenetic position and PBS characteristics of all marine Synechococcus spp. mentioned in this paper

<table><thead><tr><th>Strain name</th><th>RCC number</th><th>Subcluster</th><th>Clade</th><th>Pigment type</th><th>PUB:PEB</th><th>PEI form</th><th>PEII form</th><th>PC form</th><th>References</th></tr></thead><tbody><tr><td>WH5701†</td><td>1,084</td><td>5.2</td><td>NA</td><td>I</td><td>NA</td><td>NA</td><td>NA</td><td>C-PC</td><td>PC: this paper</td></tr><tr><td>RS9917†</td><td>556</td><td>5.1</td><td>VIII</td><td>I</td><td>NA</td><td>NA</td><td>NA</td><td>C-PC</td><td>PC: this paper</td></tr><tr><td>WH7805†</td><td>1085</td><td>5.1</td><td>VI</td><td>2</td><td>NA</td><td>A*</td><td>NA</td><td>R-PCIII</td><td>PC: [17] PE: [36]</td></tr><tr><td>WH8018</td><td>649</td><td>5.1</td><td>VI</td><td>2</td><td>NA</td><td>A*</td><td>NA</td><td>C-PC</td><td>PC: this paper PE: [36]</td></tr><tr><td>WH7803†</td><td>752</td><td>5.1</td><td>V</td><td>3a</td><td>WL: 0.440 ± 0.004 BL: 0.443 ± 0.006</td><td>A</td><td>A</td><td>R-PCII</td><td>PC: [16] PE: [18]</td></tr><tr><td>Almo03</td><td>43</td><td>5.1</td><td>I</td><td>3a</td><td>WL: 0.417 ± 0.017 BL: ND</td><td>A</td><td>A</td><td>ND</td><td>PE: this paper</td></tr><tr><td>RS9912</td><td>551</td><td>5.1</td><td>II</td><td>3a</td><td>WL: 0.435 ± 0.004 BL: 0.438 ± 0.003</td><td>A</td><td>A</td><td>ND</td><td>PE: this paper</td></tr><tr><td>RCC307†</td><td>307</td><td>5.1</td><td>X</td><td>3b</td><td>WL: 0.775 ± 0.103 BL: 0.761 ± 0.002</td><td>WL: A BL: ND</td><td>WL: B BL: ND</td><td>ND</td><td>PE: this paper</td></tr><tr><td>CC9311†</td><td>1,086</td><td>5.1</td><td>I</td><td>3d (CA)</td><td>WL: 0.719 ± 0.060 BL: 1.603 ± 0.023</td><td>ND</td><td>ND</td><td>ND</td><td>-</td></tr><tr><td>CC9902†</td><td>-</td><td>5.1</td><td>IV</td><td>3d (CA)</td><td>Variable between WL and BL</td><td>ND</td><td>ND</td><td>ND</td><td>B Palenik, personal communication</td></tr><tr><td>BL107†</td><td>515</td><td>5.1</td><td>IV</td><td>3d (CA)</td><td>WL: 0.735 ± 0.003 BL: 1.695 ± 0.149</td><td>ND</td><td>ND</td><td>ND</td><td>-</td></tr><tr><td>RS9916†</td><td>555</td><td>5.1</td><td>IX</td><td>3d (CA)</td><td>WL: 0.733 ± 0.003 BL: 1.659 ± 0.054</td><td>ND</td><td>WL: B BL: ND</td><td>ND</td><td>PE: this paper</td></tr><tr><td>WH8020</td><td>751</td><td>5.1</td><td>I</td><td>3d (CA)</td><td>WL: 0.737 ± 0.003 BL: 1.626 ± 0.042</td><td>WL: A BL: ND</td><td>WL: B BL: ND</td><td>R-PCII</td><td>PC: [16] PE: [18]</td></tr><tr><td>M11.1</td><td>790</td><td>5.1</td><td>-</td><td>3d (CA)</td><td>WL: 0.731 ± 0.004 BL: 1.849 ± 0.101</td><td>WL: B BL: B</td><td>WL: B BL: C</td><td>ND</td><td>PE: [34]</td></tr><tr><td>M16.17</td><td>793</td><td>5.1</td><td>-</td><td>3d (CA)</td><td>WL: 0.719 ± 0.015 BL: 1.826 ± 0.140</td><td>WL: B BL: B</td><td>WL: B BL: C</td><td>ND</td><td>PE: [34]</td></tr><tr><td>WH8103</td><td>29</td><td>5.1</td><td>III</td><td>3c</td><td>WL: 1.156 ± 0.014 BL: 1.154 ± 0.012</td><td>B</td><td>C</td><td>R-PCII</td><td>PC: [16] PE: [18]</td></tr><tr><td>WH8102†</td><td>539</td><td>5.1</td><td>III</td><td>3c</td><td>WL: 1.856 ± 0.117 BL: 1.903 ± 0.128</td><td>B</td><td>C</td><td>ND</td><td>PE: [19]</td></tr><tr><td>CC9605†</td><td>753</td><td>5.1</td><td>II</td><td>3c</td><td>WL: 2.136 ± 0.083 BL: 1.999 ± 0.187</td><td>B</td><td>C</td><td>ND</td><td>PE: this paper</td></tr><tr><td>Oli31</td><td>44</td><td>5.1</td><td>VII</td><td>3c</td><td>WL: 1.741 ± 0.012 BL: 1.774 ± 0.046</td><td>B</td><td>C</td><td>ND</td><td>PE: this paper</td></tr></tbody></table>

Subcluster and clade numbers are as defined in [23]. Strains are ordered by pigment type (1-3), as defined by their PBS rod composition, and subtype (3a-d) as defined by their whole cell PUB to PEB fluorescence excitation ratio (PUB:PEB ± standard deviation; n = 2 to 4). Phycobiliproteins have been classified into different forms, based on their respective chromophorylation (see text). References in the last column specify which PBP is described in which publication. CA, type IV chromatic adapter; A*, red-shifted PE; NA, not applicable; ND, not determined; WL, white light acclimation; BL, blue light acclimation. †Sequenced genomes.

$A_{max}$ at 555 nm and a shoulder at 590 nm [17]. Our isolation protocol did not allow us to obtain a pure PC fraction from any of the PEII-containing strains, because the PC band was always contaminated by variable amounts of PEII. It is known, however, that *Synechococcus* sp. WH7803, like WH8020 and WH8103, possesses a R-PCII type PC with a molar PEB:PCB of 2:1; it has absorption peaks at 533, 554 and 615 nm and maximal fluorescence emission at 646 nm [16].

Several types of PEI can be distinguished based on their different optical properties. The major phycobiliprotein found in WH7805 and WH8018, a PEI-like phycobiliprotein, exhibited an $A_{max}$ at 556 nm and an $F_{max}$ at 577 nm (Figure 2b). We

have called it PEI-A* to distinguish it from the PEI-A found in *Synechococcus* strains displaying the 3a and 3b pigment types. PEI-A has blue-shifted optical properties ($A_{max}$ = 550 nm; $F_{max}$ = 572 nm; Figure 2c) compared to PEI-A*, though both forms bind only PEB chromophores. PEI-B, which has a molar PUB:PEB of 2:3 [18], has been found in all strains exhibiting pigment type 3c examined thus far, as well as in some chromatic adapters, including M11.1 and M16.17 [34]. It has maximal absorption at 493 and 563 nm and fluorescence at 573 nm (Figure 2d).

Similarly, one can distinguish three optical types of PEII differing by their PUB:PEB. All have two absorption maxima (or
<figure><img src="image_3.png" /><figcaption>**Figure 2**</figcaption></figure>

Absorption (continuous line) and fluorescence (dotted line) properties of isolated PBP complexes. (a) C-PC (as in *Synechococcus* spp. RS9917, WH5701 and WH8018); (b) PEI-A* (as in *Synechococcus* spp. WH8018 and WH7805); (c) PEI-A (as in *Synechococcus* spp. WH7803, Almo03 and RS9912); (d) PEI-B (as in *Synechococcus* spp. WH8102, CC9605 and Oli31).
<figure><img src="image_4.png" /><figcaption>**Figure 3**
Absorption (continuous line) and fluorescence (dotted line) properties of the isolated PEII complexes. (a) PEII-A (as in *Synechococcus* sp. WH7803); (b) PEII-B (as in *Synechococcus* sp. RCC307); (c) PEII-C (as in *Synechococcus* spp. CC9605 and WH8102). Type IV chromatic adapters have a PEII-B under white or green light and a PEII-C under blue light [34].</figcaption></figure>

at least shoulders) around 495 nm and 550 nm, due to the two chromophores they bind, and a maximal fluorescence emission around 565 nm. PEII-A (Figure 3a) is found only in *Synechococcus* pigment type 3a, including WH7803 [18], Almo03 and RS9912 (this study). Its molar PUB:PEB is most likely 1:5, although the cysteinyl site to which the sole PUB chromophore is bound (either α-75 or β-50/61) has not yet been ascertained. PEII-B (Figure 3b) is found in RCC307 (Table 1) and in all white light-grown chromatic adapters that have been screened thus far, including WH8020 [18], M11.1, M16.17 [34] and RS9916 (this study). Its molar PUB:PEB is 2:4. PEII-C (Figure 3c) is found in *Synechococcus* pigment type 3c, including WH8103 [18], WH8102 [19], Oli31 and CC9605 (this study) as well as in the blue light-grown chromatic adapters [34]. The molar PUB:PEB of this PEII has been shown to be 4:2 [18].

**Comparative analysis of the phycobilisome gene regions**

After careful annotation, we compared PBS gene complement (Additional data file 1) and organization in the 11 different genomes. One remarkable trait of marine *Synechococcus* is that most of the PBS genes are gathered into a few gene clus-

ters [19,37]. As in several other cyanobacteria, a first small cluster groups together four AP core genes, in the order *apcE-A-B-C*, while two other core genes, *apcD* and *apcF* (encoding the minor α-B and β-18 AP subunits, respectively) have no PBS gene in their close vicinity. Most of the PBS rod genes are located in a much larger cluster, the size of which increases with the complexity of the rod structure from approximately 9-10 Kbp in pigment type 1 up to 27-28.5 Kbp in chromatic adapters (Figure 4). Interestingly, the gene organization in this region is very similar for strains belonging to a given pigment type. It is also similar between the chromatic adapters and the medium PUB:PEB strain RCC307.

In most genomes, the 5'-end of the PBS rod gene region starts with a short gene of unknown function (*unk1*). In RCC307, however, the *unk1* ortholog is found elsewhere in the genome. The 3'-end of the region consists of a well conserved gene predicted to encode a low molecular weight phosphotyrosine phosphatase. In the blue-green, PE-lacking strains, the rest of the region is mainly composed of two identical *cpcB-A* operators encoding the C-PC α- and β-subunits and of genes encoding three rod linkers, one rod-core linker and two types of phycobilin lyases (CpcT and CpcE/F; see below). Both
<figure><img src="image_5.png" /><figcaption>**Figure 4**</figcaption></figure>

Comparison of PBS rod gene regions of the different pigment types of marine *Synechococcus*. Rectangles above and below the lines have a length proportional to the size of ORFs and correspond to the forward and the reverse strand, respectively. In several genomes, the sense of the rod region was inverted so that the regions all appear in the same direction. For the group formed by the chromatic adapters and RCC307, a few variations can be found with regard to the region shown here, which corresponds to BL107. First, the lyase-encoding gene(s) located near the 3'-end can either be a *rpcE-F* operon or *rpcG*, a *pecEF*-like fusion gene (see text). Second, the gene organization at the 5'-end can vary: *unk1* is located elsewhere in the genome of RCC307 and the gene following *unk2* is either the lyase gene *cpcT* in RS9916 and RCC307, *unk3* in BL107 and CC9902, or none of these in CC9311. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).
RS9917 and WH5701 have an additional *cpcB* gene copy outside the PBS rod gene region but, surprisingly, no additional *cpcA*.

A part of the PC gene cluster found in the blue-green strains (*cpcCI-D-B-A-CII*) is replaced in the fuchsia pink strain WH7805 by a set of 19 genes, likely involved in the synthesis and regulation of a PEI-like complex (Figure 2). The *pebA* and *pebB* genes, located at the 3'-end of this insertion, are known to be involved in the synthesis of PEB chromophores [38].

This PE region can also be found in all PEII-containing strains, but it is interrupted by an additional sub-region containing 5 to 9 genes, between the PE regulator *cpeR* [39] and the putative lyase gene *cpeY* in WH7803 (or *cpeZ* in the other strains). This inserted sub-region includes genes encoding the PEII α- and β-subunits, two phycobilin lyases, one linker polypeptide and two or three uncharacterized proteins.

In addition, all PEII-containing strains have, upstream of *cpcGII*, an ortholog of *aplA*. Its product, AplA, which shows homology to the AP α-subunit (ApcA), was recently described in *Fremyella diplosiphon* as belonging to a new class of cyanobacterial photosensors of unknown function [40].

In the following sections, we have analyzed more specifically the phyletic profile (that is, the different patterns of occurrence of orthologs in the set of *Synechococcus* genomes) and characteristics of three gene categories: genes encoding linker polypeptides (Table 2), genes encoding putative phycobilin lyases (Table 3) and genes of unknown function specifically located in the PBS rod gene region and, therefore, potentially involved in PBS metabolism or regulation (Table 4).

## Phycobilisome linker polypeptides

The core-membrane linker $L_{CM}$, encoded by *apcE*, possesses three predicted repeat (or linker-like) domains in all marine

<caption>**Table 2**</caption>

**Presence or absence of genes encoding linker polypeptides in the different marine Synechococcus genomes**

<table><thead><tr><th rowspan="2">Strain</th><th rowspan="2">Pigment type</th><th colspan="2">Allophycocyanin</th><th colspan="3">Phycocyanin</th><th colspan="2">Phycoerythrin I</th><th colspan="6">Phycoerythrin II</th></tr><tr><th>apcC (L<sub>C</sub>)</th><th>apcE (L<sub>CM</sub>)</th><th>cpcC (L<sub>R</sub>)</th><th>cpcD (L<sub>R</sub>)</th><th>cpcG (L<sub>RC</sub>)</th><th>cpeC (L<sub>R</sub>)</th><th>cpeE (L<sub>R</sub>)</th><th>mpeD<sup>*</sup> (L<sub>R</sub>)<sup>†</sup></th><th>mpeC (L<sub>R</sub>)<sup>†</sup></th><th>mpeE (L<sub>R</sub>)<sup>†</sup></th><th>mpeF (L<sub>R</sub>)<sup>†</sup></th><th>mpeG (L<sub>R</sub>)<sup>†</sup></th></tr></thead><tbody><tr><td>WH5701</td><td>I</td><td>CC</td><td>CC<sup>‡</sup></td><td>RC (cpcCI<sup>‡</sup>) RC (cpcCII<sup>‡</sup>) NC (cpcCIII<sup>‡§</sup>)</td><td>RC</td><td>GC (cpcGII)<sup>‡</sup> RC (cpcGIII)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>RS9917</td><td>I</td><td>CC</td><td>CC</td><td>RC (cpcCI) RC (cpcCII)</td><td>RC</td><td>GC (cpcGII) RC (cpcGIII)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>WH7805</td><td>2</td><td>CC</td><td>CC<sup>‡</sup></td><td>-</td><td>-</td><td>GC (cpcGII)<sup>‡</sup> RC (cpcGIII)</td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>WH7803</td><td>3a</td><td>CC</td><td>CC<sup>‡</sup></td><td>-</td><td>-</td><td>GC (cpcGII)<sup>‡</sup> RC (cpcGIII)</td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>-</td><td>RC<sup>‡</sup></td><td>-</td><td>-</td></tr><tr><td>RCC307</td><td>3b</td><td>CC</td><td>CC<sup>‡</sup></td><td>-</td><td>-</td><td>GC (cpcGII)<sup>‡</sup> RC (cpcGIII)</td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>NC<sup>‡</sup></td><td>-</td><td>NC</td></tr><tr><td>CC9311</td><td>3d (CA)</td><td>CC</td><td>CC</td><td>-</td><td>-</td><td>GC (cpcGII) RC (cpcGIII) NC (cpcGIII)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>-</td></tr><tr><td>CC9902</td><td>3d (CA)</td><td>CC</td><td>CC</td><td>-</td><td>-</td><td>GC (cpcGII) RC (cpcGIII) NC (cpcGIII)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>NC</td><td>-</td></tr><tr><td>BL107</td><td>3d (CA)</td><td>CC</td><td>CC</td><td>-</td><td>-</td><td>GC (cpcGII) RC (cpcGIII) NC (cpcGIII)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>NC</td><td>-</td></tr><tr><td>RS9916</td><td>3d (CA)</td><td>CC</td><td>CC<sup>‡</sup></td><td>-</td><td>-</td><td>GC (cpcGII)<sup>‡</sup> RC (cpcGIII)</td><td>RC<sup>‡†</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>RC<sup>‡</sup></td><td>-</td><td>NC</td></tr><tr><td>CC9605</td><td>3c</td><td>CC</td><td>CC</td><td>-</td><td>-</td><td>GC (cpcGII) RC (cpcGIII) NC (cpcGIII)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>-</td><td>-</td></tr><tr><td>WH8102</td><td>3c</td><td>CC</td><td>CC</td><td>-</td><td>-</td><td>GC (cpcGII) RC (cpcGIII)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>-</td><td>-</td></tr></tbody></table>

CC, gene located within the PBS core gene cluster; GC, gene located within a cluster comprising the *cpcGII* and *cpcS* genes; NC, gene unlinked to other PBS genes; RC, gene located within the PBS rod gene cluster. Novel gene names proposed in this study are underlined. The linker polypeptide compositions of *Synechococcus* spp. WH5701, WH7803, WH7805 and RS9916 were checked by mass spectrometry after cutting the bands out of the LiDS-PAGE gel shown in Figure 5. For annotating paralogs that originated from recent gene duplications and have no obvious differential functional specializations (one-function paralog family), we chose the genetic nomenclature used by Berlyn [77] for *Escherichia coli* K-12. *MpeD* is a chimeric protein made of two linker domains associated with PEI and PEII, respectively [19]. †Linkers chromophorylated with PUB. ‡Linkers that have been identified by mass spectrometry. §The product of this *cpcC* gene has an extended carboxyl terminus showing strong homology to CpcD. ¶CpcC and MpeC co-eluted in RS9916, explaining the darker band observed at approximately 36 kDa apparent molecular weight in Figure 5. CA, type IV chromatic adapter; L<sub>C</sub>, core linker; L<sub>CM</sub>, core-membrane linker; L<sub>R</sub>, rod linker; L<sub>RC</sub>, rod-core linker.
<caption>**Table 3**</caption>

Presence or absence of genes encoding putative phycobilin lyases in the different Synechococcus genomes

<table><thead><tr><th rowspan="2">Strain</th><th rowspan="2">Pigment type</th><th colspan="5">Phycocyanin</th><th colspan="8">Phycoerythrin I and/or II</th></tr><tr><th>cpcEF operon</th><th>rpcEF operon</th><th>rpcG*</th><th>cpcS†</th><th>cpcT‡</th><th>rpcT§</th><th>cpeS</th><th>cpeT</th><th>cpeU¶</th><th>cpeY</th><th>cpeZ</th><th>mpeV</th><th>mpeU</th><th>mpeY¶</th><th>mpeZ¶</th></tr></thead><tbody><tr><td>WH5701</td><td>I</td><td>RC</td><td>-</td><td>-</td><td>GC#</td><td>RC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>RS9917</td><td>I</td><td>RC</td><td>-</td><td>-</td><td>GC</td><td>RC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>WH7805</td><td>2</td><td>-</td><td>RC</td><td>-</td><td>GC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>-</td><td>-</td><td>-</td></tr><tr><td>WH7803</td><td>3a</td><td>-</td><td>RC</td><td>-</td><td>GC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>-</td><td>RC</td><td>-</td></tr><tr><td>RCC307</td><td>3b</td><td>-</td><td>RC</td><td>-</td><td>GC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td></tr><tr><td>CC9902</td><td>3d (CA)</td><td>-</td><td>RC</td><td>-</td><td>GC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td></tr><tr><td>CC9311</td><td>3d (CA)</td><td>-</td><td>RC</td><td>-</td><td>GC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td></tr><tr><td>BL107</td><td>3d (CA)</td><td>-</td><td>-</td><td>RC</td><td>GC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td></tr><tr><td>RS9916</td><td>3d (CA)</td><td>-</td><td>-</td><td>RC</td><td>GC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td></tr><tr><td>CC9605</td><td>3c</td><td>-</td><td>-</td><td>RC</td><td>GC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td></tr><tr><td>WH8102</td><td>3c</td><td>-</td><td>-</td><td>RC</td><td>GC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>-</td></tr></tbody></table>

GC, gene located within a cluster comprising the *cpcG1* and *cpcS* genes; NC, gene unlinked to other PBS genes; RC, gene located within the PBS rod gene cluster. Novel gene names proposed in this study are underlined. \*pecE/F-like fusion gene [19]. †Ortholog of *Nostoc* sp. PCC 7120 'cpeS1' gene [51,52] that we propose to rename *cpcS* (see text). ‡Ortholog of a *Synechococcus* sp. PCC 7002 *cpcT* gene [50]. §Novel *cpcT* paralog found downstream of *rpcA*. ¶Novel *cpcS* paralog found upstream of *pebA*. \*These two novel, closely related genes are both paralogs of *cpeY*. #Gene split into two different reading frames in this strain. CA, type IV chromatic adapter.

*Synechococcus* except strains CC9311 and RS9916, in which L<sub>CM</sub> has four such domains. RCC307 has the shortest L<sub>CM</sub> sequence (953 amino acids) compared to the other strains due to shorter Arm2 and Arm3 regions (see [15,41] for details on L<sub>CM</sub> domains). Besides the PC-associated linker genes found in the rod gene region of both blue-green strains (Figure 4), WH5701 has a third *cpcC* homolog (*cpcCIII*) located elsewhere in the genome that potentially encodes a chimeric protein since it has an extended carboxyl terminus showing strong similarity to CpcD. None of the PF-containing strains possesses *cpcC* and *cpcD* homologs. In all marine *Synechococcus*

genomes, the rod-core linker gene *cpcGII* is found in the PBS rod region whereas *cpcG1* is found outside this cluster. A third *cpcG* gene copy, which we refer to as *cpcGIII*, is present elsewhere in the genomes of BL107, CC9902, CC9311 and CC9605.

The total number of putative PE-associated linker genes varies from zero in the blue-green strains to six in the group constituted by the chromatic adapters and RCC307 (Table 2 and Figure 4). The location of the *mpeE* linker gene appears more variable than the other PEII genes, as it can be found

<caption>**Table 4**</caption>

Presence or absence of genes encoding conserved hypothetical genes located in the phycobilisome rod gene region

<table><thead><tr><th>Strain</th><th>Pigment type</th><th>unk1</th><th>unk2</th><th>unk3</th><th>unk4</th><th>unk5</th><th>unk6</th><th>unk7</th><th>unk8</th><th>unk9</th><th>unk10</th><th>unk11</th><th>unk12</th><th>unk13</th></tr></thead><tbody><tr><td>WH5701</td><td>I</td><td>RC</td><td>RC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>RS9917</td><td>I</td><td>RC</td><td>RC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>WH7805</td><td>2</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>WH7803</td><td>3a</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td><td>RC (fused/inversed)</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>RCC307</td><td>3b</td><td>NC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>-</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>CC9311</td><td>3d (CA)</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>CC9902</td><td>3d (CA)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>BL107</td><td>3d (CA)</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>RS9916</td><td>3d (CA)</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>NC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>CC9605</td><td>3c</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td></tr><tr><td>WH8102</td><td>3c</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td><td>RC</td></tr></tbody></table>

RC, gene located within the PBS rod gene cluster. In some strains, homologs of these genes are found elsewhere in the genomes (NC). CA, type IV chromatic adapter.
either in the PBS rod gene region (for example, upstream of *cpcGII* in CC9311 or downstream of *cpcGII* in RS9916) or a few genes upstream of this region (in RCC307, BL107 and CC9902) or even in a totally different location of the genome (in CC9605).

Surprisingly, the PEII-lacking strain WH7805 possesses a homolog of *mpeD*, a gene known to encode a chimeric protein made of a PEII-associated linker (amino terminus) and a PEI-associated CpeD-like linker (carboxyl terminus) [19]. However, closer examination of the amino-terminal part of this protein in WH7805 reveals a relatively low similarity with other MpeD sequences and a notable deletion of the region corresponding to amino acids 43-59 in *Synechococcus* sp. WH8102 [19] that is conserved in all other MpeD sequences (Additional data file 2). This region includes two cysteinyl residues involved in linking a PUB chromophore via a Δ2,3 double bond, a type of chromophorylation typical of PEII-associated linker polypeptides [21]. *Synechococcus* sp. WH7803 also lacks the *mpeC* gene, which encodes the distal PEII-associated linker polypeptide in other strains [19,21]. Finally, both chromatic adapters and RCC307 have, outside

the PBS core region, an additional gene potentially encoding a PEII-associated linker (Table 3). In phylogenetic trees made with all PEII linkers (Additional data file 3), these sequences are both related to the amino terminus of MpeD but are split between two distinct gene clusters, one gathering BL107, CC9311 and CC9902, which we propose to name MpeF, and the other gathering RS9916 and RCC307, which we propose to name MpeG.

In order to compare further the linker composition of marine *Synechococcus* strains and determine whether they are all present in the PBSs, we performed a lithium dodecyl sulphate (LiDS)-PAGE analysis of intact PBSs. The Coomassie stained gel shown in Figure 5 displays the PBS proteins of two to three strains per pigment type. For WH7803 and RCC307, a Tris-tricine running buffer provided a better separation of the linker polypeptides than Tris-glycine (Figure 5, right). For strains WH5701, WH7805, WH7803, RCC307 and RS9916, all linker polypeptide bands (except ApcC and CpcD, which are not detectable under these electrophoresis conditions) were cut out from the gel and then identified by mass spectrometry (Table 2). In all five strains, the upper band proved

<figure><img src="image_6.png" /><figcaption>**Figure 5**
Coomassie blue stained LiDS polyacrylamide gradient (10-20%) gel of PBS linkers run using a Tris-glycine buffer system (left). A Tris-tricine buffer (right) gave higher band resolution for RCC307 and WH7803. Green dots indicate linker polypeptides fluorescing green under UV light due to the presence of a PUB chromophore. Colored stars indicate the pigment type of each strain (see Figure 1 for color code). FNR: ferredoxin:NADP+ oxidoreductase.</figcaption></figure>
to be the core-membrane linker L<sub>CM</sub>, often accompanied by
its degradation product L<sub>CM</sub>', making a band of lower appar-
ent molecular weight. As expected, RS9916, which has an
extended *apcE* gene sequence, possesses the L<sub>CM</sub> band of low-
est electrophoretic mobility. Although the rod-core linker
CpcGI was systematically present in all four strains, no
CpcGII was detected by mass spectrometry, suggesting either
that the *cpcGII* gene is expressed at a much lower level than
*cpcGII* or that CpcGII is not present in the PBS fraction of
these strains. It is worth noting though that we previously
observed CpcGII (co-migrating with CpcGI) in a PBS fraction
from *Synechococcus* sp. WH8102 [19]. Interestingly, we iden-
tified all three predicted PC rod linkers in WH5701, including
the CpcCD-like protein, which is not found in the RS9917
genome. Furthermore, all PEII linkers predicted in WH7803,
RCC307 and RS9916 were detected by mass spectrometry,
except the products of the *mpeF* gene of RS9916 and of the
*mpeG* gene of RCC307 (Table 1). This suggests that either
these two potential linker genes are not expressed in our
standard culture conditions or their products are
undetectable on Coomassie-stained LiDS-PAGE gels due to
some inherent biochemical properties.

Lyases, lyase-isomerases and related genes

Four types of phycobilin lyases, enzymes involved in the
chromophorylation of phycobiliproteins, have been charac-
terized so far. One of these, the heterodimeric CpcE/F com-
plex, reversibly ligates a PCB molecule to Cys-84 of the α-
subunit of C-PC [42,43]. Two genes with strong homology to
the characterized *cpcE* and *cpcF* genes of *Synechococcus* spp.
PCC 7942 [44] and PCC 7002 [45] are found near the 3'-end
of the rod gene region in 7 out of the 11 marine *Synechococcus*
genomes. We have called these *cpcE-F* in the two C-PC-con-
taining strains (RS9917 and WH5701) and *rpcE-F* in
WH7803, CC9311 and CC9902, in agreement with the
nomenclature proposed by Wilbanks and Glazer [37]. Indeed,
*Synechococcus* sp. WH7803 (as well as WH8020 and
WH8103) possesses a R-PCII type PC that has a PEB at α-84
[16]. Though we have called these genes *rpcE/F* in strains
WH7805 and RCC307 as well (Additional data file 1), it is
worth noting that in phylogenetic trees made with concate-
nated CpeE-F or RpcE-F protein sequences using Gloeo-
bacter violaceus as an outgroup, these two strains cluster
with RS9917 and WH5701, with only moderate bootstrap
support (Additional data file 4). Both CpeE/F and RpcE/F
lyases from marine *Synechococcus* possess all sites described
by Zhao and coworkers [46] to be important for the activity of
CpeE/F in *Fischerella* sp. PCC 7603 (a.k.a. *Mastidocladus
laminosus*), so they cannot be differentiated on this basis. In
the four other *Synechococcus* genomes, including the high
PUB:PEB strains WH8102 and CC9605 and the chromatic
adapters BL107 and RS9916, these two lyase genes are
replaced by a single fusion gene that we propose to call *rpcG*
(Table 3). The amino- and carboxy-terminal parts of the *rpcG*
gene product show strong homology to the PecE and PecF of
*Fischerella* sp., respectively, the two subunits of a PCB lyase-

isomerase, which binds a PCB to Cys84 of the phycoerythro-
cyanin α-subunit and concomitantly isomerizes it into phyco-
violobilin [47,48]. A conserved motif 'NHCQGN' shown to be
crucial for the isomerase activity of *Fischerella* PecF is
present in the carboxyl terminus of the four marine *Syne-
chococcus* RpcG sequences (for example, positions 361-366 of
SYNW2005 in WH8102). This suggests that RpcG is also a
phycobilin lyase-isomerase, although several other sites
defined as potentially important for the activity of the PecE/F
enzyme in *Fischerella* sp. [49] are not conserved in those
sequences.

An ortholog of *cpcT*, shown in *Synechococcus* sp. PCC 7002 to encode a lyase catalyzing the binding of PCB at Cys153 of the C-PC β-subunit [50], is found in WH5701, RS9917, WH7805, RCC307 and RS9916 (Table 3). This gene belongs to a family of three paralogs, including *cpeT*, first described in the PE gene cluster of *F. diplosiphon* [39] and located at a similar position in all PE-containing marine *Synechococcus* (Figure 4). An uncharacterized gene located near the 5'-end of the PBS rod gene cluster of all PE-containing strains except RCC307 also belongs to this family. We propose to name this gene *rpcT*, since it is present in the PC-specific gene region of WH7803, which possesses R-PCII. Thus, most marine *Synechococcus* strains possess either *cpcT* or *rpcT*. Surprisingly, the RS9916 strain possesses both genes, confirming their paralogous nature (Additional data file 5).

Marine *Synechococcus* possess another family of three paralogous lyase genes. One of them encodes a lyase that was first characterized in *Nostoc* sp. PCC 7120 as catalyzing the binding of PCB at β-84 of both C-PC and phycoerythrocyanin [51]. More recently, this enzyme was shown to have an even larger spectrum of activity, since it is also able *in vitro* to bind PCB at Cys84 of all AP subunits (that is, ApcA, B, D and F) from *Nostoc* sp. as well as PEB at Cys84 of both α- and β-PE subunits (that is, CpeA and B) from *F. diplosiphon* [52]. Surprisingly, Zhao and co-workers have called this lyase 'CpeS1' though there is no PE in PCC 7120 and its best hit in the marine *Synechococcus* protein databases is not the product of the *cpeS* gene (located immediately upstream of *cpeT* in the PE gene sub-region; Figure 4), but the product of a gene found in tandem with *cpcGI* in all *Synechococcus* strains, including blue-green, PE-lacking strains. So, we suggest to rename it *cpcS* (Table 3, Figure 4 and Additional data file 6). Surprisingly, the *cpcS* gene is split into two different reading frames in WH5701. This is likely a sequencing error, because absence of chromophorylation at Cys84 in all AP and in β-PC subunits would likely render the energy transfer through these phycobiliproteins very poorly efficient. An uncharacterized gene located upstream of the *pebA-B* operon (Figure 4) constitutes the third member of this family of paralogous lyase genes (Additional data file 6), and we propose to name it *cpeU*.
PE-containing *Synechococcus* possess several genes in the PEI or PEII gene sub-regions that encode proteins showing homology to other types of lyases, likely involved in binding phycobilins to one or both PEs. These lyases include CpeY and CpeZ, which in *F. diplosiphon* were presumed to be subunits of a heterodimeric lyase, binding PEB to PE α- or β-subunits [53], but the precise site specificity of this enzyme is hitherto unknown. The *mpeU* and *mpeV* genes, which were first observed in *Synechococcus* sp. WH8020 by Wilbanks and Glazer [37], likely encode two additional lyases. These paralogous genes are both present in the chromatic adapters and in RCC307, whereas WH7803 and WH7805 have only *mpeV* and the high PUB:PEB strains only *mpeU* (Table 3). Finally, we found two novel, paralogous lyase genes, again closely related to one another and more distantly related to *cpeY*. We propose to name these genes *mpeY* and *mpeZ*. Contrary to CpeY and CpeZ, the products of these putative lyase genes likely do not form heterodimers, given their distinct phyletic profiles (Table 3). Indeed, *mpeY* is found in the PEII-specific sub-region of all PEII-containing strains (Figure 4) whereas *mpeZ* is found only in the genomes of the chromatic adapters and of RCC307, outside the PBS gene clusters.

## Conserved hypothetical genes located in the phycobilisome gene region

Table 4 reports the phyletic profile of 13 conserved hypothetical genes associated with the PBS rod region of all (or a majority of) strains. Many of them are seemingly specific to marine *Synechococcus* while some are found in other cyanobacterial genera, including *Prochlorococcus* and/or *Gloeo-bacter*. It is worth noting though that there are still very few genomes of phycoerythrin-containing cyanobacteria in current databases and it is likely that homologs will be found in those as they become available. In this study, we have given these genes the provisional names *unk1-13*, until a more complete characterization is performed.

As already mentioned, the *unk1* gene is located upstream of the PBS rod region in all strains except RCC307, in which *unk1* is located elsewhere in the genome. Another unknown gene (*unk2*) immediately follows *unk1* in most PE-containing strains (in RCC307, it is the first gene of the PBS rod gene region). The *unk2* gene is found three genes downstream of *unk1* in the two blue-green strains. The predicted Unk2 protein sequence generally shows a fairly large variability among the different *Synechococcus* strains, although the BL107 and CC9902 sequences are very closely related (91% identity at the amino acid level). Both Unk1 and Unk2 are short proteins with no recognizable motifs. The *unk3* gene is associated with the PBS rod region in only four out of the eleven genomes and encodes a protein with six putative transmembrane helices. It is therefore probably not directly related to PBS structure. The *unk4* gene is present upstream of *aplA* in all PE-containing strains and directly upstream of *cpcGII* in WH7805, which lacks *aplA*. The *unk5* gene, generally located downstream of *cpcGII*, has the same phyletic profile as *unk4* (Table

4) and its product possesses pentapeptide repeat motifs. Though very short (57-61 amino acids), the Unk6 protein is very well conserved among the PE-containing *Synechococcus*. A cluster of three consecutive short and conserved hypothetical genes (*unk7-9*) is found only in PEII-containing strains. Localization of these genes in a PEII-specialized sub-region strongly suggests that they are involved in some still unknown function specifically related to PEII. The predicted proteins Unk7 and Unk8 both possess a motif of unknown function (PF07862) also found in the product of a gene located in the *nif* cluster of several cyanobacteria as well as in the nitrogen-fixing proteobacterium *Azotobacter vinelandii* [54]. Surprisingly, in WH7803, *unk7* and *unk8* are fused and reversed with regard to *unk9*. This suggests that these genes encode two subunits of the same heterodimeric complex. In the high PUB:PEB strains WH8102 and CC9605, the PEII-specialized region ends with *unk10*, which is strongly conserved between these strains (90% identity at the amino acid level). Homologs of *unk10* are also found in the genomes of the chromatic adapters and in RCC307 but outside the PBS rod gene region and have only about 49% identity with sequences of the high PUB:PEB strains. Located in the PEI-specific region, the translated *unk11* gene is very variable in length and sequence (especially the 3'-end) among marine *Synechococcus* strains. In contrast, the neighboring gene *unk12* displays low sequence variability between strains. Finally, the *unk13* gene, though strongly conserved, was not correctly modeled in WH8102, in which a wrong open reading frame (ORF; SYNW2018) was predicted in a different reading frame. The *unk12* gene was previously known and was called *orf140* in WH8020 by Wilbanks and Glazer [37], who sequenced the 3'-end of the PBS rod gene region from *mpeB* to the phosphatase. By remodeling this region, we confirmed that the *unk11* and *unk13* genes are also present in this strain and were incorrectly assigned by these authors. Though partial, the organization and gene content of this region in WH8020 [37] is clearly similar to that of chromatic adapters (Figure 4), and this is confirmed by the ability of this strain to chromatically adapt (Table 1; see also [33]).

## Phylogeny of phycobilisome genes

Both PBS gene complement and organization in the genome are very similar for strains belonging to a given pigment type, independent of their position in phylogenetic trees based, for instance, on the 16S rRNA gene [23,34]. Thus, we wondered whether the phylogeny of PBS genes could differ from the core genome phylogeny. To answer this question, we built phylogenetic trees based on concatenated protein sequences of each phycobiliprotein type and compared them with reference trees made with all concatenated ribosomal proteins, which are good representatives of the core genome (Figure 6). Concatenation generally allows building phylogenies that are more robust when sequences are strongly conserved, as is the case for phycobiliproteins. Still, maximum parsimony (MP) analyses generally provided more variable results than maximum likelihood (ML) and neighbor joining (NJ) analyses due
to a relatively low number of informative sites. Whenever
possible, we used the primitive, PE-containing, freshwater
cyanobacterium *Gloeobacter violaceus* as an outgroup to root
our trees, in order to better understand evolution of PBSs
within the marine *Synechococcus* group.

The phylogenetic trees obtained with concatenated proteins
encoding the AP components (ApcA-B-C-D-F; Figure 6b)
share many characteristics with those based on ribosomal
proteins (Figure 6a). In both cases, RCC307 and WH5701 are
isolated on two long branches well apart from all other
strains. Furthermore, WH7803 and WH7805 on the one
hand, and CC9902 and BL107 on the other, appear closely
related to one another. The only variable positions are those
of the closely related strains RS9916 and RS9917, which clus-
ter on the same branch as WH7803, WH7805 and CC9311 in
the ribosomal tree, and at the base of the branch bearing
BL107, CC9902, CC9605 and WH8102 in the AP tree, but
with relatively low bootstrap support in the second case.

The phylogenetic trees of concatenated PC α- and β-subunits
(CpcGII was not included because mass spectrometry analy-
ses suggested it may not be part of the PBS; Table 2) show a
number of differences relative to the AP tree, including the
fact that the two blue-green strains group together (with high
bootstrap support) apart from all others (Figure 6c). This is
consistent with the fact that they both have C-PC (binding
only PCB), whereas all other strains have a PC form binding
both PCB and PEB. The relative positions of WH7805 and
RCC307 varied between phylogenetic methods. WH7805 is
known to contain R-PCIII [17] and this is probably the case
for RCC307 as well, based on their similar PC lyase gene con-
tent, including *cpcS*, *cpcT* and *rpcE-F* (Table 3). All strains
containing R-PCII (or possibly another, unidentified PC
form, for those strains possessing *rpcG*; Table 3) formed a
well-supported cluster with both ML and NJ methods, though
the relative positions of CC9311 and RS9916 were variable
within this cluster.

The phylogeny obtained for the concatenated PEI proteins
CpeA-B-Y-Z - addition of Unk12 did not significantly alter the
tree topologies, but gave lower bootstrap support (data not
shown) - fits well with the pigment types, as defined in Table
1. Indeed, the two high PUB:PEB strains group together, well
apart from the other PE-containing strains. RCC307 is found
at the base of a cluster formed by chromatic adapters (Figure
6d), consistent with the fact that all these strains share a sim-
ilar PBS gene complement and organization. Likewise, strains
WH7803 and WH7805 group together, consistent with the
similar organization of their PEI-like region, with *cpeZ* being
located downstream of *mpeV* instead of upstream of *cpeY* as
in all other PE-containing strains (Figure 4).

Phylogenetic trees obtained with the concatenated PEII pro-
teins MpeA-B-Y and Unk7-9 - inclusion of Unk7-9 does not
alter the tree topologies obtained with the sole MpeA-B-Y

sequences but provides better bootstrap support - are shown
without an outgroup, since this phycobiliprotein form is not
found in freshwater cyanobacteria. Still, these trees are glo-
bally similar to those obtained with PEI proteins, with three
main clusters, one gathering the medium PUB:PEB strain
RCC307 and the chromatic adapters, one gathering the two
high PUB:PEB strains, whereas the low PUB:PEB strain
WH7803 clusters apart from all others.

Discussion

Comparative genomics reveal novel genes involved in
phycobilisome metabolism

We have identified and compared a number of genes poten-
tially involved in the synthesis and chromophorylation of
PBSs in a variety of sequenced marine *Synechococcus* strains
spanning all PBS pigment types known so far in this group.
Strains displaying different pigment types have different gene
complements with a considerable increase in complexity
from type 1 (WH5701 and RS9917) to type 3d (chromatic
adapters). Synthesis of rods entirely composed of PC, as
found in the first type, requires at least 15 genes. This includes
two *cpcB-A* operons encoding C-PC α- and β-subunits, two
rod-core linker genes (*cpcGI* and *cpcGII*), two *cpcC* and one
*cpcD* rod linker genes (in WH5701, an additional *cpcC* gene,
*cpcCIII*, was in fact found to be a *cpcC/D* gene chimera), four
genes encoding three different lyases (CpcE/F, CpcS and
CpcT) and the PCB biosynthesis gene *pcyA*, which encodes
the PCB:ferredoxin oxidoreductase [55]. Whether *unk1* and
*unk2*, usually found at or near the 5'-end of the PBS rod gene
region (Figure 4), are also involved in PC metabolism awaits
experimental checking. An additional *cpcB* gene, absent from
other blue-green cyanobacteria such as *Synechococcus* sp.
PCC 7942 or *Synechocystis* sp. PCC 6803, is found unlinked
to other PBS genes in both WH5701 and RS9917. While the
three *cpcB* copies are almost identical in RS9917, the isolated
copy is somehow divergent from the other two in WH5701.
This may indicate a recent change in function. All PEII-con-
taining strains possess an AP-like gene encoding a protein
derived from a phycobiliprotein, the homolog of which, *apA*,
was shown in *F. diplosiphon* to encode a photoreceptor not
linked to the PBS [40]. So it is possible that the additional
CpcB found in the blue-green strains might have a similar
function though, contrary to AplA, this protein appears to
have retained the ability to interact with the α-PC subunit.
Indeed, amino acids involved in maintaining these interac-
tions [56] are conserved in all CpcB copies.

By comparing the PBS gene complement of strain WH7805
with that of blue-green strains, it appears that the occurrence
of a single PEI-like PE type in the rod necessitates at least 19
genes. This includes one set of PE α- and β-subunit encoding
genes, three linker genes (*cpeC*, *cpeE* and a *mpeD*-like gene),
six putative lyase genes, two genes involved in PEB synthesis
(*pebA* and *pebB*) and a number of novel genes of yet unknown
function, including *unk5*, *6, 12, 13* and perhaps *unk11*. Indeed,
<figure><img src="image_7.png" /><figcaption>(b) – Allophycocyanin
(ApcA-B-C-D-F)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>(c) – Phycocyanin
(CpcA-B or RpcA-B)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>(d) – Phycoerythrin I
(CpeA-B-Y-Z)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>(e) – Phycoerythrin II
(MpeY-A-B-unk7-8-9)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 6** (see legend on next page)</figcaption></figure>
<caption>**Figure 6** (see previous page)</caption>

ML trees made with concatenated amino acid sequences of (a) all 51 ribosomal proteins (6,754 amino acid positions), (b) the AP proteins ApcA-B-C-D-F (710 amino acid positions), (c) the PC proteins CpcA-B or[RpcA-B (332 amino acid positions), (d) the PEI proteins CpeA-B-Y-Z (943 amino acid positions) and (e) the PEII proteins MpeA-B-Y and Unk7-8-9 (1,007 amino acid positions). The first four trees are rooted with corresponding proteins from the primitive, freshwater cyanobacterium *Gloeobacter violaceus*, taken as an outgroup. The PEII tree is unrooted since these proteins are specific for marine *Synechococcus* spp. Numbers at internal branches correspond to bootstrap values for 1,000 replicate trees obtained with ML/NJ/MP methods. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).

all these *unk* genes are specific to PE-containing *Synechococ-
cus* and all but *unk11* are well conserved. Despite its tiny size,
explaining why it has often been missed by annotation
software, *unk6* is likely a true gene since it is also present in
all *Prochlorococcus* strains (data not shown). In both
*Prochlorococcus* and marine *Synechococcus* spp., *unk6* is
located upstream of the putative phycobilin lyase gene *cpeS*.

Acquisition of a second PE type, PEII, involves comparatively
few additional genes, from six in WH7803, including *unk7/8*
and *unk9*, up to twelve genes in type IV chromatic adapters
and RCC307 (*mpeA*, *B*, *C*, *D*, *E*, *F* or *G*, *U*, *Y*, *Z* and
*unkz*, *8*,
and *9*), among which the seven underlined genes are novel
PEII genes. The fact that PEII synthesis and regulation proc-
esses require fewer genes than for PEI implies that several
genes involved in these processes are common to both PE
forms. This obviously includes the PEB synthesis genes *pebA/*
*B*, but likely also a number of lyase genes.

Predicting lyase gene function

Examination of the number, phylogenetic relatedness and
phyletic profiles of all predicted lyase genes (Table 3) can give
us clues about the possible functional specificity of these
enzymes. The number of chromophore binding sites on the α-
and β-subunits of phycobiliproteins varies from seven in pig-
ment type 1 - that is, four in AP (ApcA, B, D and F subunits
have one each) and three in PC (one in CpcA/RpcA, two in
CpcB/RpcB) - up to eighteen in PEII-containing strains - that
is, four in AP, three in PC, five in PEI (two in CpeA, three in
CpeB) and six in PEII (three in MpeA, three in MpeB) -. Fur-
thermore, it is thought that type IV chromatic adapters can
have either PUB or PEB at two chromophore binding sites of
MpeA [34]. Finally, while the chromophorylation of LCM with
PCB is thought to be auto-catalyzed and, thus, likely does not
require any lyase activity [49], chromophorylation with PUB
of the two to four PEII rod linkers (Table 2) probably requires
one or several specific PUB lyases (or PEB lyase-isomerases).
By comparison, the number of predicted proteins showing
homology to known lyases varies from 3 in blue-green strains
up to 12-13 in RCC307 and chromatic adapters.

All three phycobilin lyases identified in the genomes of *Syne-
chococcus* spp. WH5701 and RS9917 (Table 3) have charac-
terized homologs in freshwater cyanobacteria. This reduced
set of lyases is most likely sufficient to catalyze the chromo-
phorylation with PCB of all AP and C-PC binding sites.
Indeed, the CpcS lyase (named 'CpeS1' by Zhao and cowork-
ers [51,52]) is active on almost all α-84 and β-84 cysteinyl res-

idues. The only exception is C-PC α-84, chromophorylation of
which is under the control of the heterodimeric lyase CpeE/F
[42,43]. Chromophorylation of the last cysteinyl residue, that
is, C-PC β-155, is catalyzed by another specific lyase, CpcT
[50]. A fairly large difference exists between the sequences
and active sites of the CpcE/F lyase, which binds PCB (a type
1 chromophore carrying a Δ3,3¹-ethylidene group and a single
bond between C-2 and C-3) to C-PC α-84, and those of the
lyase-isomerase PecE/F, which binds phycobiliviolin (a type
2 chromophore carrying a 3-vinyl group and a Δ2,3-double
bond) to the homolog position of α-phycoerythrocyanin
[47,48]. Thus, the replacement in four *Synechococcus* strains
(BL107, RS9916, CC9605 and WH8102) of *cpeE* and *cpeF*
genes by a fusion gene encoding a PecE/F-like protein (that
we have called RpcG) is quite significant and it is possible that
the PC synthesized by these strains binds a type 2 chromo-
phore at α-84. This interesting hypothesis suggests that a bet-
ter biochemical characterization of the PC found in these
strains is needed. Finally, in all PEII-containing *Synechococ-
cus* strains except RCC307 and RS9916, the *cpcT* gene is
absent (Table 3) and seemingly replaced by a gene of the same
family of paralogs, located in the PC-specific gene cluster
(Figure 4), that we have called rpcT. Given the presence of the
rpcT gene (and absence of *cpcT*) in *Synechococcus* sp.
WH7803 in which a PEB is bound at β-153 of R-PCII [16],
RpCT is a plausible candidate for catalyzing this specific
chromophorylation. Surprisingly, RS9916 possesses both
CpcT and RpcT paralogs, suggesting it may either bind PCB or
PEB at this site.

Predicting the function of lyase genes potentially involved in
bilin attachment to PEI and PEII is much more difficult than
for PC, given the larger number of binding sites on these phy-
cobiliproteins. The only lyase gene specific to all PEII-cont-
taining strains is *mpeY* (Table 3 and Figure 4). The PEII α-
subunit has one chromophore-binding cysteinyl residue that
has no homolog in its PEI counterpart, α-75. In WH8103 and
white light-grown WH8020, α-75 has been shown to bind a
PUB [18]. We hypothesize that MpeY could be a PUB lyase (or
a PEB lyase-isomerase) involved in the chromophorylation of
PEII α-75 with PUB. However, another specific feature of
PEII complexes is that they are held together with two to four
PUB-chromophorylated linkers (Table 3) so, alternatively,
*mpeY* might encode a lyase involved in the PUB chromopho-
rylation of one (or several) PEII rod linker(s).

The presence of two additional lyase genes in chromatic
adapters compared to strains exhibiting either pigment types
3a or 3c (Table 2) suggests that this more complex lyase com-
plement is required for type IV chromatic adaptation. Indeed,
this process is thought to consist of the reversible exchange of
both PEII α-83 and α-140 chromophores from PEB to PUB
[34], and not in the differential expression of several sets of
phycobiliprotein genes, like in type III chromatic adaptation
(see, for example, [57] for a review). The presence of only one
set of genes encoding PEI and PEII α- and β-subunits in all
genomes of chromatic adapters supports this hypothesis.
Because type IV chromatic adaptation implies the conversion
of a PEII-B into a PEII-C under blue light (and conversely
under white light; Table 1), it is reasonable to assume that
chromatic adapters need two more PUB lyases (or PEB lyase-
isomerase) than pigment type 3a strains, which permanently
have PEB at PEII α-83 and α-140, and two more PEB lyases
than pigment type 3c, which permanently have PUB at these
two positions. The phyletic pattern of the *mpeV* gene (Table
3) which, besides its occurrence in chromatic adapters, is also
present in WH7803 and WH7805 and absent in the high
PUB:PEB strains, suggests it could encode a PEB lyase. Con-
versely, *mpeU* has the reverse phyletic profile and, thus, could
encode a PUB lyase (or PEB lyase-isomerase). The specificity
of the putative lyase MpeZ is harder to interpret. Surprisingly,
the complex PBS gene set found in chromatic adapters is
shared by RCC307, which is the sole strain to have pigment
type 3b of all marine *Synechococcus* strains screened so far.
Indeed, we have determined that all strains except RCC307
described as having a PUB:PEB of approximately 0.7-0.8 by
Fuller *et al.* [23] are actually chromatic adapters. This
includes strain RCC61 (data not shown), which belongs to the
same phylogenetic clade as RCC307 (that is, clade X) [23].
Therefore, we suggest that RCC307 may have lost the ability
to chromatically adapt, perhaps due to a mutation in a
domain important for lyase activity or the inactivation or loss
of some regulatory gene(s) required for this process.

Predicted models of PBS structures

Most sequenced *Synechococcus* strains have typical PBS
cores with three AP cylinders. The presence of an additional
L<sub>CM</sub> domain in CC9311 and RS9916 suggests that their PBS
core may have two additional half-cylinders, as previously
observed in freshwater species such as *Nostoc* sp. PCC 7120
[58]. It is thought that up to eight rods can be bound to such
a PBS core (Figure 7). The presence of an extended L<sub>CM</sub> was
previously reported from another chromatic adapter, *Syne-
chococcus* sp. M16.17 [34] and one may wonder whether such
PBS cores might only occur in this pigment type. An answer
to this question awaits screening of *apcE* genes (or of the L<sub>CM</sub>
linker size on LiDS-PAGE gels) in a much wider range of
strains, as well as direct evidence from electron microscopic
images of isolated phycobilisomes.

The large diversity of PBS rod pigmentation observed so far
within the marine *Synechococcus* group rests on combina-
tions of at least three PC types (C-PC, R-PCII, R-PCIII), two
PEI types (PEI-A/A* and PEI-B) and three PEII types (PEII-

A through C) (Table 1). The number and nature of rod linker polypeptides present in the different *Synechococcus* strains can help predict the structure of their PBS rods. Given the striking similarity in pigmentation and gene complement between the freshwater strains *Synechococcus* sp. PCC 7942 or *Synechocystis* sp. PCC 6803 and *Synechococcus* sp. RS9917, the latter strain likely has a very similar PBS rod structure [59,60], that is, three C-PC hexamers (Figure 7a, left). Since *Synechococcus* sp. WH5701 has one more (CpcCD-like) rod linker than RS9917 (Table 2), it is possible that this strain has rods with one additional PC disc (Figure 7a, right).

Like all PE-containing strains, WH7805 lacks the CpcC and
CpcD rod linker polypeptides, the absence of which implies it
has only a single PC hexamer at the base of each PBS rod. This
PC can be of two types depending on strains, C-PC or R-PCIII
(Table 1 and Figure 7b). WH7805 has three PE linkers,
including a homolog of the long, chimeric rod linker MpeD
(Figures 4 and 5) instead of a shorter, CpeD-like linker, like in
*F. diplosiphon* [61]. Its amino-terminal moiety is very diver-
gent, however, and does not possess the ability to bind a PUB
chromophore, a characteristic common to all PEII rod linkers
(Additional data file 2). In the type III chromatic adapter *F.
diplosiphon* grown under green light, PBS rods are composed
of one PC and three PE hexamers [15,57]. Since they have a
MpeD-like rod linker equivalent to two typical rod linkers in
length, we suggest that *Synechococcus* pigment type 2 strains
might have one more PE disc in their rods than *F. diplosiphon*
(Figure 7b).

In a previous paper, we have proposed a model for the struc-
ture of PBS rods of the pigment type 3c strain WH8102, which
we have predicted to have six hexamers: one PC, two PEI and
three PEII [19]. The other high PUB:PEB strain CC9605
appears to have similar PBS rods (Figure 7d). Because it is
missing the (distal) linker gene *mpeC*, we assume that the
type 3a strain WH7803 has only two PEII hexamers (Figure
7c). Despite the presence of an additional, PEII rod linker
gene (*mpeF* or *mpeG*) in chromatic adapters and in RCC307,
we found no evidence by mass spectrometry of any such link-
ers in PBS preparations from RCC307 and RS9916 (Table 2).
So, it is very unlikely that these strains have more than three
PEII hexamers. Indeed, in this case, they would have a higher
whole cell PUB:PEB under blue light than WH8102 or
CC9605, whereas this ratio is similar or even lower in chrom-
atic adapters (Table 1). It is possible though that under
some specific culture conditions, *mpeF* or *mpeG* could be
expressed and that their products could then replace some
other PEII linker in the PBS rods.

New insights into PBS evolution

One major finding from our comparative analyses is that the
PBS rod gene complement is highly similar for strains having
the same pigmentation (Figure 4 and Tables 2, 3, 4), inde-
pendent of their position in 16S rRNA [23], 16S-23S rDNA
<figure><img src="image_12.png" /><figcaption>**Figure 7**</figcaption></figure>

Proposed models of PBS structure for the different *Synechococcus* pigment types and subtypes. PBS cores are generally composed of three cylinders, but in some chromatic adapters possessing an extended L~CM~, it is likely composed of two additional half cylinders (see, for example, [58]). In pigment type 1, rods are composed of C-PC only; in pigment type 2, rods are composed of either C-PC, or R-PCIII and a PEI-like phycobiliprotein; in pigment type 3, rods comprise R-PC and two PE types (PEI and PEII). Cells of the latter pigment type bind PEB and PUB at a low (3a), medium (3b), high (3c) or variable (3d or type IV chromatic adapter) ratio. Colored stars indicate the pigment type of each strain (see Figure 1 for color code).
internal transcribed spacer [24] or ribosomal protein phylog-
enies (Figure 6a), the latter being a proxy for the core genome
phylogeny. This is particularly striking for the two blue-green
strains RS9917 and WH5701 which, though belonging to dif-
ferent subclusters (5.1 and 5.2, respectively, according to
Herdman and co-workers [26]), have a similar gene set and
organization of their PBS rod gene region (Figure 4). Phyloge-
netic trees based on concatenated PC α- and β-subunit
sequences from all marine *Synechococcus* also group these
two strains together, well apart from all others, in contrast to
those obtained with concatenated AP protein sequences,
which are globally more consistent with the ribosomal pro-
tein phylogeny (Figure 6). Similarly, phylogenies based on
PEI and PEII proteins are congruent with the separation of
PE-containing strains into pigment types 2 and 3 and sub-
types 3a-d, as defined in Table 1. Indeed, they group all
chromatic adapters (subtype 3d) together (Figure 6d,e). Fur-
thermore, RCC307 (subtype 3b), which has a similar PBS
gene complement and organization, is always found at the
base of the chromatic adapter group in PE trees, whereas it
appears very distantly related to them in AP trees. Finally,
subtypes 3a and 3c strains are found on distinct branches in
PE trees and are well separated from chromatic adapters.

Altogether, these data suggest that the different components
of the PBS have evolved almost independently from each
other in the marine *Synechococcus* group. Indeed, the core of
the PBS has seemingly evolved together with the core
genome, suggesting that light energy transport from the PBS
core to photosystem II is an evolutionarily ancient and
conservative mechanism that has not allowed much pheno-
typic variability during the course of evolution. In contrast,
the rod components appear to have evolved through complex
episodes of gene duplication, lateral gene transfer and/or
gene loss. The latter hypothesis is consistent with recent data
from Haverkamp and co-workers [62] showing that phyloge-
nies based on the *cpcB-A* and *cpeB-A* gene sequences notably
differ from phylogeny based on 16S rRNA sequences for a
variety of *Synechococcus* strains. Acquisition of the first PE (a
PEI-like phycobiliprotein) dates back to before the separation
of the marine *Synechococcus/Prochlorococcus* branch from
other cyanobacteria and was likely accomplished by duplica-
tion and divergence of ancestral PC genes [63]. In contrast,
acquisition of PEII components must have occurred after the
differentiation of the marine *Synechococcus* lineage, by
duplication and divergence of some PEI genes. Thereafter,
transfer of the PEI and/or PEII rod gene cluster might have
occurred from one lineage of marine *Synechococcus* to
another, possibly by lateral transfer via natural transforma-
tion or viruses. The occurrence of photosynthetic genes is fre-
quent in cyanophages [64] and this may include PBS genes,
such as the putative lyase *cpeT* gene found in the S-PM2 and
Syn9 genomes [65,66] or the *pebA* and *pcyA* genes found in
the P-SSM2 and P-SSM4 genomes [67,68]. So far, only indi-
vidual photosynthetic genes have been found in such phage
genomes, not gene clusters. However, it is quite possible that,

in some rare cases, much larger genome chunks (for example,
covering the whole PEII sub-region) could be conveyed by
cyanophages between *Synechococcus* spp. cells belonging to
distinct lineages.

Conclusion

The dazzling colors of marine *Synechococcus* rely on the com-
bination of a few phycobiliprotein forms, which can be assem-
bled into a variety of PBS structures (Figure 7). The variable
part of these photosynthetic antennae (that is, PBS rods) is
encoded and regulated in large part by a specialized genomic
region, which includes a number of genes of unknown func-
tion, but rapid progress in elucidating these functions is
envisioned using a combination of genetic and biochemical
approaches. During the course of evolution, marine *Syne-
chococcus* appear to have acquired more and more sophisti-
cated light-harvesting complexes, from simple C-PC rods to
elaborate rod structures comprising three distinct phyco-
biliprotein types. As a further sophistication, some marine
*Synechococcus* strains are able to modulate their PBS absorp-
tion capacity to harvest efficiently a larger range of visible
light quality. In the present study, we show that these type IV
chromatic adapters are much more frequent in culture
collections than previously thought, and this might be the
case in nature as well, since the distribution of this pigment
type in the field is currently unknown. The large diversity of
PBS pigmentation found among marine *Synechococcus*, as
well as the likely occurrence, during evolution, of PBS gene
exchanges between lineages ensuring that this diversity is
maintained to some extent at the level of individual lineages,
have allowed members of this genus to thrive in almost every
possible illuminated marine environment. This may be one of
the key reasons explaining the ecological success of the *Syne-
chococcus* group in the marine environment.

**Materials and methods**

Synechococcus strains and culture conditions

For biochemical analyses, marine *Synechococcus* spp. strains Alm03, BL107, CC9605, Oli31, RCC307, RS9912, RS9916, RS9917, WH5701, WH7803, WH7805, WH8018 and WH8102 were grown in 8 l polycarbonate flasks (Nalgene, Rochester, NY, USA) in PCR-S11 medium [69] supplemented with 5 mM NaNO₃. Cultures were grown at 22°C under approximately 15 µmol photons m⁻² s⁻¹ white light (Sylvania daylight fluorescent bulbs). To determine their PUB:PEB ratio and their eventual ability to perform type IV chromatic adaptation [34], two to four duplicate 10 ml cultures of a number of strains possessing two PEs were grown in parallel under 15 µmol photons m⁻² s⁻¹ white or blue light, prior to spectrofluorometric analyses made during the exponential growth phase. Blue light was obtained by wrapping tube racks with blue filter sheet (filter no. 183 'moonlight', Lee Filters, Andover, England). The origin of strains has been described previously [23] except for RCC307 and BL107, which were
respectively isolated from the Mediterranean Sea in June 1999 at 6° 10'E, 39° 10'N at a depth of 15 m by F Partensky and in September 2000 at 13° 33'E, 41° 43'N at the very deep depth of 1,800 m by Laure Guillou (Roscoff, France). All these strains are available from the Roscoff Culture Collection (RCC), Roscoff, France.

## In vivo spectrometry

Room temperature excitation fluorescence spectra (with emission at 580 nm) of whole *Synechococcus* cells grown under standard light conditions were recorded with a spectrofluorimeter LS-50B (Perkin Elmer, Waltham, MA, USA), as previously described [32], in order to measure the PUB to PEB fluorescence excitation ratio. *In vivo* absorbance spectra of whole cells were also recorded from 400 to 750 nm with a double monochromator spectrophotometer (Hitachi U-3310) equipped with a head-on photomultiplier detector. Spectra were recorded with a 1 nm interval and 5 nm slit width and normalized at 439 nm (blue absorption peak).

## Phycobiliprotein purification and characterization

PE purification was carried out as described previously [19]. Briefly, after cell breakage in a cooled French press system, a soluble extract devoid of chlorophyll *a* was obtained by differential ultracentrifugation in a buffer containing 10 mM phosphate pH 7.2 and the protease inhibitors EDTA, phenylmethylsulfonyl fluoride, aminocaproic acid and benzamidine, each at 1 mM final concentration. The soluble protein extract was then loaded onto a 0-30% sucrose density gradient and run overnight at 130,000 × g at 12°C. Phycobiliproteins were separated from the different colored sucrose gradient fractions on 7% acrylamide isoelectric focusing gels containing ampholyte carriers pH 4-6.5 (Amersham Biosciences, Buckinghamshire, UK). PE bands were cut out of the gel and crushed with an electric grinder in 10 mM tricine buffer pH 7.8. Acrylamide remnants were eliminated by centrifugation. When necessary, samples were concentrated using 30 kDa cut-off membranes (Centricon, Millipore, Billerica, MA, USA). Absorption and fluorescence emission spectra were recorded and corrected as described earlier [19].

## Intact phycobilisome extraction

PBSs were isolated on discontinuous sucrose density gradients in 0.75 M phosphate buffer containing protease inhibitors by the classic sucrose density gradient method [19]. Colored bands were precipitated with 10% (v/v) trichloroacetic acid and resuspended in 3% (w/v) LiDS denaturation buffer. Electrophoresis was carried out overnight using a 10-20% continuous gradient LiDS-PAGE at low amperage (10 mA). After migration, the gel was immersed in 20 mM zinc acetate in order to enhance phycobiliprotein fluorescence, washed with water and visualized under UV light, then stained with Coomassie blue G250. For some selected strains for which genome sequence was available, bands of linker polypeptides were cut out of the gel and identified by mass spectrometry, using the facilities of the Unité de Recherche

Biochimie et Structure des Protéines', Jouy en Josas, France. Briefly, each gel sample was digested overnight at 37°C in 25 µl trypsin (at 8 µg ml⁻¹). Mass spectra were acquired with a MALDI-TOF (Applied Biosystems model Voyager DE super STR, Foster City, CA, USA) equipped with a nitrogen laser with an emission wavelength of 337 nm and run in reflectron mode with an extraction delay of 130 ns. The matrix used was α-cyano-4-hydroxycinnamic acid at 4 mg ml⁻¹. Internal calibration was performed with trypsin peptides (842.5090 and 2,211.1040 Da).

## Comparative genomics

Eleven genomes of marine *Synechococcus* spp. were used for this study: The WH8102 (NC_005070), CC9902 (NC_007513) and CC9605 (NC_007516) genomes have been sequenced by the Joint Genome Institute, the CC9311 (NC_008319) genome by The Institute for Genome Research (TIGR), the WH7803 (NC_009481) and RCC307 (NC_009482) genomes by Genoscope (Evry, France) at the request of a consortium of European scientists coordinated by F Partensky, the RS9916 (NZ_AUA00000000), RS9917 (NZ_AANP00000000), BL107 (NZ_AATZ00000000), WH7805 (NZ_AAK00000000) and WH5701 (NZ_AANO00000000) genomes by the J Craig Venter Institute in the framework of the Gordon and Betty Moore Foundation Marine Microbial Genome Sequencing Project at the request of an international consortium coordinated by DJ Scanlan.

Gene families from the 11 marine *Synechococcus* were delineated using BLAST [70] with an e-value of 10⁻¹² and the TribeMCL algorithm [71]. Families of orthologous genes either located in the PBS region and/or involved in PBS biosynthesis or regulation were extracted and manually annotated. Non-modeled genes, missed by ORF finding software, were added to the dataset. The corresponding protein sequences were aligned using ClustalW [72] with default parameters and their amino terminus was corrected (that is, extended or shortened) if needed.

## Phylogenetic analyses

Phylogenetic analyses were performed using a variable number of concatenated protein sequences depending on each phycobiliprotein type (see results), allowing the use of longer sequences to reduce the variance in the distance estimates [73]. These sequences were automatically aligned using ClustalW [72]. Alignments were then manually refined and all gaps and highly variable regions (if any) were removed. Phylogenetic trees were generated using three different reconstruction methods: NJ (with PHYLO_WIN [74]), ML (with PHYML v2.4.4 [75]) and MP (with PHYLO_WIN). ML analyses were performed using the Jones Taylor Thornton model and the variability of substitution rates across sites and invariables sites was estimated. Bootstrap values (1,000 replicates) were calculated for all three methods in order to estimate the relative confidence in monophyletic groups and
they were all reported on the ML tree used as a reference. Phylogenetic trees were edited using the MEGA4 software [76].

## Abbreviations

AP, allophycocyanin; L~C~, core linker; L~CM~, core-membrane linker; LiDS, lithium dodecyl sulphate; L~R~, rod linker; L~RC~, rod-core linker; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor joining; ORF, open reading frame; PBS, phycobilisome; PC, phycocyanin; PCB, phycocyanobilin; PE, phycoerythrin; PEB, phycoerythrobilin; PUB, phycourobilin.

## Authors' contributions

CS and FP conceived the study and wrote most of the paper. DJS and FP together coordinated sequencing and annotation of 7 out of the 11 *Synechococcus* genomes used in this study. FP did most comparative genomics analyses and drew genomic regions and phycobilisome models. AD performed the clustering of orthologous genes and set up a web site for annotation for all phycobilisome genes. He also performed the phylogenetic analysis of ribosomal proteins. LG did most other phylogenetic analyses and helped in writing the corresponding part of the manuscript. CS did phycobilisome extractions from selected *Synechococcus* strains, isoelectric focusing gels for purifying intact phycobiliproteins and performed spectrometric analyses. JCT performed LiDS-PAGE analyses of linker polypeptides, cut selected bands out of these gels and supervised mass spectrometry analyses. MO improved the quality of the genome sequence of several *Synechococcus* strains and performed spectrophotometric analyses on intact cells. NB participated in the annotation of phycobilisome genes and checked occurrence of some of them in several unsequenced *Synechococcus*. DJS improved the overall quality of the manuscript.

## Additional data files

The following additional data files are available with the online version of this paper. Additional data file 1 is a table listing genes involved in PBS metabolism or regulation in the 11 genomes of marine *Synechococcus*. Additional data file 2 is an amino acid alignment of the amino terminus of the MpeD linker polypeptide from all sequenced, PE-containing, marine *Synechococcus* spp. Additional data file 3 is an unrooted ML tree based on amino acid sequences of MpeD (amino terminus only) and all other PEII-associated rod linker polypeptides (216 amino acid positions). Additional data file 4 is a ML tree based on concatenated amino acid sequences of CpcE and CpcF homologs in all sequenced marine *Synechococcus* spp. (444 amino acid positions). Additional data file 5 is an unrooted ML tree of the CpcT-CpeT-RpcT protein family (183 amino acid positions). Additional data file 6 is an

unrooted ML tree of the CpcS-CpeS-CpeU protein family (163 amino acid positions).

## Acknowledgements

We thank Nicolas Arrouy for participating in preliminary experiments during his Masters thesis and Florence Le Gall and Priscillia Gourvil for help with strain culturing. We are grateful to David Kehoe for critically reading the manuscript. Alain Guillot is acknowledged for performing mass spectrometry analyses and Brian Palenik for providing strains CC9605 and CC9311. This work was funded by the French program ANR PhycoSyn, the NERC grant NE/C000536/1, the European Network of Excellence Marine Genomics Europe and its flagship program SynChips.

## References

1. Johnson PW, Sieburth JM: *Chroococcoid cyanobacteria in the sea: a ubiquitous and diverse phototrophic biomass.* *Limnol Oceanogr* 1979, **24**:928-935.

2. Waterbury JB, Watson SW, Guillard RRL, Brand LE: *Widespread occurrence of a unicellular, marine planktonic, cyanobacterium.* *Nature* 1979, **277**:293-294.

3. Gradinger R, Lenz J: *Seasonal occurrence of picocyanobacteria in the Greenland Sea and central Arctic Ocean.* *Polar Biol* 1995, **15**:447-452.

4. Partensky F, Blanchot J, Lantoine F, Neveux J, Marie D: *Vertical structure of picophytoplankton at different trophic sites of the tropical northeastern Atlantic Ocean.* *Deep-Sea Res I* 1996, **43**:1191-1213.

5. Zubkov MV, Sleigh MA, Tarran GA, Burkill PH, Leakey RJG: *Pico- planktonic community structure on an Atlantic transect from 50 degrees N to 50 degrees S.* *Deep-Sea Res I* 1998, **45**:1339-1355.

6. Waterbury JB, Watson SW, Valois FW, Franks DG: *Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus.* *Can Bull Fish Aquat Sci* 1986, **472**:71-120.

7. Olson RJ, Zettler ER, Armbrust EV, Chisholm SW: *Pigment, size and distribution of Synechococcus in the North Atlantic and Pacific oceans.* *Limnol Oceanogr* 1990, **35**:45-58.

8. Partensky F, Blanchot J, Vaulot D: *Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters: a review.* In *Marine Cyanobacteria Volume 19*. Edited by: Charpy L, Larkum A. Monaco: Musée Oceanographique; 1999:457-475.

9. Scanlan DJ, West NJ: *Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus.* *FEMS Microbiol Ecol* 2002, **40**:1-12.

10. Scanlan DJ: *Physiological diversity and niche adaptation in marine Synechococcus.* *Adv Microb Physiol* 2003, **47**:1-64.

11. Glazer AN, Williams RC, Yeh SW, Clark JH: *Kinetics of energy flow in the phycobilisome core.* *Science* 1985, **230**:1051-1053.

12. Glazer AN: *Light guides - directional energy transfer in a photosynthetic antenna.* *J Biol Chem* 1989, **264**:1-4.

13. Grossman AR, Schaefer MR, Chiang GG, Collier JL: *The responses of cyanobacteria to environmental conditions: light and nutrients.* In *The Molecular Biology of Cyanobacteria* Edited by: Bryant DA. Dordrecht: Kluwer Academic Publishers; 1994:677-692.

14. MacColl R: *Cyanobacterial phycobilisomes.* *J Struct Biol* 1998, **124**:311-334.

15. Sidler WA: *Phycobilisome and phycobiliprotein structure.* In *The Molecular Biology of Cyanobacteria* Edited by: Bryant DA. Netherlands: Kluwer Academic Publishers; 1994:139-216.

16. Ong LJ, Glazer AN: *R-phycocyanin II, a new phycocyanin occurring in marine *Synechococcus* species.* *J Biol Chem* 1987, **262**:6323-6327.

17. Ong LJ, Glazer AN: *Structural studies of phycobiliproteins in unicellular marine cyanobacteria.* In *Light-energy Transduction in Photosynthesis: Higher Plant and Bacterial Models* Edited by: Stevens SEJ, Bryant DA. Rockville, MD: American Society of Plant Physiologists; 1988:102-121.

18. Ong LJ, Glazer AN: *Phycoerythrin of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in *Synechococcus* spp. phycoerythrin.* *J Biol Chem* 1991, **266**:9515-9527.
19. Six C, Thomas JC, Thion L, Lemoine Y, Zal F, Partensky F: Two novel phycoerythrin-associated linker proteins in the marine cyanobacterium *Synechococcus* sp. strain WH8102. *J Bacteriol* 2005, 187:1685-1694.

20. Everroad RC, Wood AM: Comparative molecular evolution of newly discovered picocyanobacterial strains reveals a phylogenetically informative variable region of beta-phycerythrin. *J Phycol* 2006, 42:1300-1311.

21. Wilbanks SM, Glazer AN: Rod structure of a phycoerythrin II-containing phycobilisome. II. Complete sequence and bilin attachment site of a phycoerythrin γ subunit. *J Biol Chem* 1993, 268:1236-1241.

22. Urbach E, Scanlan DJ, Distel DL, Waterbury JB, Chisholm SW: Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of *Prochlorococcus* and *Synechococcus* (Cyanobacteria). *J Mol Evol* 1998, 46:188-201.

23. Fuller NJ, Marie D, Partensky F, Vaulot D, Post AF, Scanlan DJ: Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine *Synechococcus* clade throughout a stratified water column in the Red Sea. *Appl Environ Microbiol* 2003, 69:2430-2443.

24. Ahlgren NA, Rocap G: Culture isolation and culture-independent clone libraries reveal new marine *Synechococcus* ecotypes with distinctive light and N physiologies. *Appl Environ Microbiol* 2006, 72:7193-7204.

25. Chen F, Wang K, Kan J, Suzuki MT, Wommack KE: Diverse and unique picocyanobacteria in Chesapeake Bay, revealed by 16S-23S rRNA internal transcribed spacer sequences. *Appl Environ Microbiol* 2006, 72:2239-2243.

26. Herdman M, Castenholz RW, Waterbury JB, Rippka R: Form-genus XIII. *Synechococcus*. In Bergey's Manual of Systematic Bacteriology Volume 1. 2nd edition. Edited by: Boone DR, Castenholz RW. New York: Springer-Verlag; 2001:508-512.

27. Waterbury J, Rippka R: Order Chroococcales Wettstein Emend. Rippka et al., 1979. In Bergey's Manual of Systematic Bacteriology Edited by: Krieg N, Holt J. Baltimore: Williams and Wilkins; 1989:1728-1746.

28. Toledo G, Palenik B, Brahamsha B: Swimming marine *Synechococcus* strains with widely different photosynthetic pigment ratios form a monophyletic group. *Appl Environ Microbiol* 1999, 65:5247-5251.

29. Penno S, Lindell D, Post AF: Diversity of *Synechococcus* and *Prochlorococcus* populations determined from DNA sequences of the N-regulatory gene *ntcA*. *Environ Microbiol* 2006, 8:1200-1211.

30. Wood AM, Phinney DA, Yentsch CS: Water column transparency and the distribution of spectrally distinct forms of phycoerythrin-containing organisms. *Mar Ecol Prog Ser* 1998, 162:25-31.

31. Wood AM, Lipsen M, Coble P: Fluorescence-based characterization of phycoerythrin-containing cyanobacterial communities in the Arabian Sea during the Northeast and early Southwest Monsoon (1994-1995). *Deep-Sea Res II* 1999, 46:1769-1790.

32. Six C, Thomas J-C, Brahamsha B, Lemoine Y, Partensky F: Photophysiology of the marine cyanobacterium *Synechococcus* sp. WH a new model organism. *Aquat Microb Ecol* 2004, 35:17-29.

33. Palenik B: Chromatic adaptation in marine *Synechococcus* strains. *Appl Environ Microbiol* 2001, 67:991-994.

34. Everroad C, Six C, Partensky F, Thomas J-C, Holtzendorff J, Wood AM: Biochemical bases of type IV chromatic adaptation in marine *Synechococcus* spp. *J Bacteriol* 2006, 188:3345-3356.

35. Jones H, Ostrowski M, Scanlan DJ: A suppression subtractive hybridization approach reveals niche-specific genes that may be involved in predator avoidance in marine *Synechococcus* isolates. *Appl Environ Microbiol* 2006, 72:2730-2737.

36. Alberte RS, Wood AM, Kursar TA, Guillard RRL: Novel phycoerythrinins in marine *Synechococcus* spp. *Plant Physiol* 1984, 75:732-739.

37. Wilbanks SM, Glazer AN: Rod structure of a phycoerythrin II-containing phycobilisome. I. Organization and sequence of the gene cluster encoding the major phycobiliprotein rod components in the genome of marine *Synechococcus* sp. WH8020. *J Biol Chem* 1993, 268:1226-1235.

38. Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC: Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.

*Plant Cell* 2001, 13:965-978.

39. Cobley JG, Clark AC, Weerasurya S, Queseda FA, Xiao JY, Bandrapali N, D'Silva I, Thounaojam M, Oda JF, Sumiyoshi T, Chu MH: CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium *Fremyella diplosiphon* and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR). *Mol Microbiol* 2002, 44:1517-1531.

40. Montgomery BL, Casey ES, Grossman AR, Kehoe DM: ApA, a member of a new class of phycobiliproteins lacking a traditional role in photosynthetic light harvesting. *J Bacteriol* 2004, 186:7420-7428.

41. Capuano V, Braux AS, Tandeau de Marsac N, Houmad J: The "anchor polypeptide" of cyanobacterial phycobilisomes. *Molecular characterization of the Synechococcus* sp. PCC 6301 *apcE* gene. *J Biol Chem* 1991, 266:7239-7247.

42. Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA, Glazer AN: *Phycocyanin alpha-subunit phycocyanobilin lyase*. *Proc Natl Acad Sci USA* 1992, 89:7017-7021.

43. Fairchild CD, Glazer AN: Oligomeric structure, enzyme kinet-ics, and substrate specificity of the phycocyanin alpha subu-nit phycocyanobilin lyase. *J Biol Chem* 1994, 269:8686-8694.

44. Bhalerao RP, Lind LK, Gustafsson P: Cloning of the cpcE and cpcF genes from *Synechococcus* sp. PCC 6301 and their inactivation in *Synechococcus* sp. PCC 7942. *Plant Mol Biol* 1994, 26:313-326.

45. Swanson RV, Zhou J, Leary JA, Williams T, de Lorimier R, Bryant DA, Glazer AN: Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment. *J Biol Chem* 1992, 267:16146-16154.

46. Zhao KH, Wu D, Zhang L, Zhou M, Bohm S, Bubenzer C, Scheer H: Chromophore attachment in phycocyanin - functional amino acids of phycocyanobilin-alpha-phycocyanin lyase and evidence for chromophore binding. *FEBS J* 2006, 273:1262-1274.

47. Zhao KH, Deng MG, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B, Scheer H: Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon. *FEBS Lett* 2000, 469:9-13.

48. Storf M, Parbel A, Meyer M, Strohmann B, Scheer H, Deng MG, Zheng M, Zhou M, Zhao KH: Chromophore attachment to biliproteins: Specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-Cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin. *Biochemistry* 2001, 40:12444-12456.

49. Zhao KH, Ping S, Bohm S, Bo S, Ming Z, Bubenzer C, Scheer H: Reconstitution of phycobilisome core-membrane linker, L-CM, by autocatalytic chromophore binding to ApcE. *Biochim Biophys Acta* 2005, 1706:81-87.

50. Shen G, Saunée NA, Williams SR, Gallo EF, Schluchter WM, Bryant DA: Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta subunit of phycocyanin in *Synechococcus* sp. PCC 7002. *J Biol Chem* 2006, 281:17768-17778.

51. Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, Scheer H: Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cystein-beta8 phycobiliprotein lyase activity of CpeS-like protein from *Anabaena* sp. PCC7120. *J Biol Chem* 2006, 281:8573-8581.

52. Zhao K, Su P, Tu J, Wang X, Liu H, Plöscher M, Eichacker L, Yang B, Zhou M, Scheer H: Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins. *Proc Natl Acad Sci USA* 2007, 104:14300-14305.

53. Kahn K, Mazel D, Houmad J, Tandeau de Marsac N, Schaefer MR: A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis. *J Bacteriol* 1997, 179:998-1006.

54. Jacobson M, Brigle K, Bennett L, Setterquist R, Wilson M, Cash V, Beynon J, Newton W, Dean D: Physical and genetic map of the major nif gene cluster from *Azotobacter vinelandii*. *J Bacteriol* 1989, 171:1017-1027.

55. Hagiwara Y, Sugishima M, Takahashi Y, Fukuyama K: Crystal structure of phycocyanobilin: ferredoxin oxidoreductase in complex with biliverdin IX alpha, a key enzyme in the biosynthesis of phycocyanobilin. *Proc Natl Acad Sci USA* 2006, 103:27-32.
56. Bickel PJ, Kechris KJ, Spector PC, Wedemayer GJ, Glazer AN: Finding important sites in protein sequences. Proc Natl Acad Sci USA 2002, **99**:14764-14771.

57. Kehoe DM, Gutu A: **Responding to color: The regulation of complementary chromatic adaptation.** Ann Rev Plant Biol 2006, **57**:127-150.

58. Ducret A, Muller SA, Goldie KN, Hefti A, Sidler WA, Zuber H, Engel A: **Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena** sp. PCC 7120. *J Mol Biol* 1998, **278**:369-388.

59. Elmorjani K, Thomas J-C, Sebban P: Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803. Arch Microbiol 1986, **146**:186-191.

60. Glazer AN: Phycobilisome: a macromolecular complex optimized for light energy transfer. Biochim Biophys Acta 1984, **768**:29-51.

61. Federspiel NA, Grossman AR: Characterization of the light-regulated operon encoding the phycoerythrin-associated linker proteins from the cyanobacterium *Fremyella diplosiphon*. *J Bacteriol* 1990, **172**:4072-4081.

62. Haverkamp T, Acinas SG, Doeleman M, Stomp M, Huisman J, Stal LJ: Diversity and phylogeny of Baltic Sea picocyanobacteria inferred from their ITS and phycobiliprotein operons. Environ Microbiol 2007.

63. Apt KE, Collier JL, Grossman AR: Evolution of the phycobiliproteins. J Mol Biol 1995, **248**:79-96.

64. Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, Chisholm SW: Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proc Natl Acad Sci USA 2004, **101**:11013-11018.

65. Mann NH, Clokie MR, Millard A, Cook A, Wilson WH, Wheatley PJ, Letarov A, Krisch HM: The genome of S-PM2, a "photosynthetic" T4-type bacteriophage that infects marine Synechococcus strains. J Bacteriol 2005, **187**:3188-3200.

66. Weigele PR, Pope WH, Pedulla ML, Houtz JM, Smith AL, Conway JF, King J, Hatfull GF, Lawrence JG, Hendrix RW: Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus. Environ Microbiol 2007, **9**:1675-1695.

67. Clokie MRJ, Mann NH: Marine cyanophages and light. Environ Microbiol 2006, **8**:2074-2082.

68. Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW: Three *Prochlorococcus cyanophage genomes: Signature features and ecological interpretations*. PLOS Biol 2005, **3**:790-806.

69. Rippka R, Coursin T, Hess W; Lichtlé C, Scanlan DJ, Palinska KA, Ite-man I, Partensky F, Houmad J, Herdman M: *Prochlorococcus marinus* Chisholm *et al*. 1992 *subsp. pastoris* *subsp. nov*. strain PCC the first axenic chlorophyll *a*₂/*b*₂-containing cyanobacterium (*Oxyphotobacteria*). Intl J Syst Evol Microbiol 2000, **50**:1833-1847.

70. Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, **25**:3389-3402.

71. Enright AJ, van Dongen S, Ouzounis CA: An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res 2002, **30**:1575-1584.

72. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, **22**:4673-4680.

73. Gadagkar SR, Rosenberg MS, Kumar S: Inferring species phylogenies from multiple genes: concatenated sequence tree versus consensus gene tree. J Exp Zool B 2005, **304**:64-74.

74. Galtier N, Gouy M, Gautier C: SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 1996, **12**:543-548.

75. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, **52**:696-704.

76. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 2004, **5**:150-163.

77. Berlyn MK: Linkage map of Escherichia coli K-12, edition 10: the traditional map. Microbiol Mol Biol Rev 1998, **62**:814-984.