* School of Biological Sciences, University of Sydney, Australia and Sydney University Biological Informatics and Technology Centre (SUBIT), University of Sydney, NSW, Australia; Department of Biology, Macquarie University, NSW, Australia; and
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
Correspondence: E-mail: alark{at}mail.usyd.edu.au.
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Abstract |
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Key Words: prochlorophyte chlorophyll a/b protein Acaryochloris marina Prochloron didemni lateral genes transfer chlorophyll-d light-harvesting protein complexes
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Introduction |
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The origin and phylogenetic position of Pcb light-harvesting protein complexes relative to other members of the six-helix Chlbinding protein family (e.g., IsiA, and CP43 and CP47 of photosystem II) is of considerable interest. Prochlorothrix and Prochlorococcus have been shown to have multiple pcb genes (van der Staay, Yurkova, and Green 1998; Garczarek et al. 2000), and the products of individual genes have been shown to be associated specifically with either photosystem I or photosystem II in Prochlorococcus sp. MIT9313 (Bibby et al. 2003a). We have characterized multiple pcb genes from A. marina and Prochloron didemni and demonstrate a single origin of pcb genes for Chl bcontaining and Chl dcontaining organisms, together with an ancient duplication of the genes. Given the positions of these organisms in 16S rRNA trees, this strongly suggests a widespread lateral transfer of the genes for these light-harvesting systems.
The Acaryochloris marina and Prochloron didemni pcb sequences reported here have GenBank accession numbers AY552463and AY 552464, respectively.
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Materials and Methods |
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A. marina pcb Probes and Gene Sequences
The general degenerate primers for pcb genes 5'-ATGCARACNTAYGGNAAYCC-3' (primer 3144) and 5'-ATRTGCCADATNCCNCC-3' (primer 3145) (La Roche et al. 1996) were used for amplification by the polymerase chain reaction (PCR) by 35 cycles of 94°C, 1 min; 52°C, 1 min; and 72°C, 3 min for extension. A 516-bp PCR product was obtained from genomic DNA of A. marina, which was shown by BlastX sequence analysis to contain part of a pcb gene. This fragment, designated A12, was labeled with fluorescein using the Renaissance Random Primer Fluorescein labeling kit New England Nuclear, Boston, Mass. according to the manufacturer's instructions and used to screen a Sau3A genomic library of A. marina established in bacteriophage EMBL3 (Promega). Several independent hybridizing clones were isolated. Inserts from three of them were subcloned into plasmid pGEM-3Z (Promega) and sequenced. Two of these clones contained the same insert. A 4,700-bp sequence was obtained from the two different clones, which both contained an identical overlapping region of 600 bp.
The fluorescein-labeled 516-bp fragment (A12) was also used to probe Southern blots of genomic DNA of A. marina digested with a range of restriction enzymes to determine the number of copies of pcb genes. The resultant DNA digests were electrophoresed in 0.7% (w/v) agarose gels in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) containing 0.3 mg·mL1 ethidium bromide and then transferred to Immobilon-Ny+ membrane (Millipore, Australia) by capillary action in 20xSSC buffer overnight. DNA was fixed onto the membrane by UV cross-linking. Hybridization used 10 mL of A12 probe and was carried out overnight at 58°C to 60°C. Hybridization, stringency wash procedures, and chemiluminescence detection were performed using the Renaissance Random Primer Fluorescein Labeling Kit following the manufacturer's instructions.
Prochloron didemni DNA Isolation, Library Construction, and Sequence Determination
Prochloron didemni cells were expelled by gently squeezing Lissoclinum patella colonies, which had been slashed with a razor blade. Cells were filtered through Miracloth into 1M Tris-HCl pH 8.0 then washed once in buffer and DNA extracted as described above for A. marina. The DNA was further purified by centrifugation through a CsCl gradient. Fifty milligrams of purified DNA was subjected to a brief exposure to BglII and, after phenol-chloroform and chloroform purification, was ligated into EMBL3 DNA cut with BamHI (Promega). Plaques from the resulting Prochloron didemni library were screened with a 984-bp PCR product generated from the cloned sequence reported previously (La Roche et al. 1996) using primers 3144 and 3321 (5'-GGNYYTNTGGCAYGC-3'). DNA from a library clone hybridizing to the probe was digested with SalI and showed the insert to be approximately 13 kbp. After digestion with SalI/HindIII, fragments were cloned in pUC18. Colonies containing recombinant plasmids were screened as before, and a single colony giving a positive signal was picked and sequenced. Additional sequence data were obtained by sequencing of a PCR product generated using a forward primer based on the sequence already obtained (5'-AAAGGATCCGGTATTGCGGATGGCGGC-3') and a reverse primer (5'-AAAGGATCCGCDCGRGTWGCRTGCCAHACRTGDCC-3' based on the conserved motif GHVWHATRA of Prochlorothrix hollandica pcb sequences.
Identification of A. marina pcb Gene Products Using Mass Spectral Analysis
Chl dbinding protein complexes were isolated by nondenaturing electrophoresis followed by denaturing SDS-PAGE to obtain approximately 34-kDa Pcb-type polypeptides (Chen, Quinnell, and Larkum 2002a, 2002b), which were excised from the stained gel matrix (Wang, Sun, and Chitnis 2000) and analyzed by mass spectral analysis at the Department of Chemistry and Biochemistry, Arizona State University. The Pcb-type protein sequences predicted from DNA sequence data were analyzed for their trypsin cleavage sites using the program Peptide Cutter (http://kr.expasy.org/tools/peptidecutter/), which predicts the mass weight map of cut fragments (Barrett, Rawlings, and Woessner 1998).
Phylogenetic Analysis
Phylogenetic analysis was performed on protein sequences aligned using ClustalW (Thompson, Higgins, and Gibson 1994), followed by manual refinement. MEGA version 2 (Kumar, Tamura, and Nei 1994) was used to construct Neighbor-Joining (distance) and maximum-parsimony trees and bootstrapped 1,000 times. ProML, SeqBoot, and Consense from the PHYLIP program (Felsenstein 1989) were used to infer and assemble bootstrapped maximum-likelihood trees (256 replicates), using the JTT model of amino acid substitution and also incorporating the best-tree estimated by minimum ln-likelihood. The sequences of isiA, pcb and psbC genes used in the analyses were obtained from GenBank and http://www.jgi.doe.gov/JGI_microbial/html/ and are listed in table 1.
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Results |
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Phylogenetic Tree Reconstruction
The sequence alignment is provided as Supplementary Material online. The phylogenetic analyses were performed using the region of 308 residues with excluded the gaps; that is, the loop between helix 5 and helix 6 of CP43. The relatedness of the Pcb proteins to each other, and to cyanobacterial IsiA proteins and to a variety of CP43 proteins was studied by phylogenetic tree reconstruction from the aligned amino acid sequences using maximum-likelihood (fig. 2), parsimony and distance-matrix methods (fig. 3). The bootstrap supports for the branches on these trees is shown in the figures and generally show good support for the tree topology. The phylogenetic tree in figure 2 shows the unrooted relationship among 35 proteins. The PcbA/C of A. marina group with the other Pcb proteins with reliable bootstrap support, although with extended edge lengths (fig. 2).
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Discussion |
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The Evolution of pcb Genes
There is a strong similarity in the arrangement of pcb genes in A. marina and Prochloron didemni, with duplicated genes in tandem in both organisms. This is also similar to the arrangement in Prochlorothrix hollandica, although the latter contains three pcb genes (fig. 1C), pcbA/B/C, with the pcbC gene strongly divergent from the other two pcb genes (van der Staay, Yurkova, and Green 1998). The Acaryochloris pcbA and pcbC genes are most similar to the pcbA/B and pcbC genes, respectively, of Prochlorothrix, and the same is true for the Prochloron genes. All three pcbC genes share a 24 amino acid deletion compared with the others between amino acids 296 and 297 in the numbering of van der Staay Yurkova, and Green (1998). This would suggest that the pcb genes of all these organisms are derived from a series of duplications, as originally suggested for Prochlorothrix hollandica (van der Staay, Yurkova, and Green 1998) (fig. 1C). A range of pcb gene organization is seen in Prochlorococcus isolates, with a single gene in an isolate adapted to high light intensity, two genes in an isolate adapted to moderate light, and multiple genes in a strain adapted to low light (Garczarek et al. 2001).
The relatedness of the Pcb proteins to each other, to cyanobacterial IsiA proteins, and to a variety of CP43 proteins is shown in figure 2 using maximum-likelihood and in figure 3 using parismony and distance-matrix methods. It is particularly noteworthy that all the PcbA/B sequences group together to the exclusion of IsiA and CP43 (psbC) sequences in contrast to other suggestions (Rüdiger 2002). The same is true for the PcbC sequences. This indicates a single origin of the pcbA/B genes and of the pcbC genes, whether binding Chl b or Chl d, rather than multiple origins (from IsiA or CP43 sequences). Rooting the trees using CP43 (which seems justified, given the integral role of this protein in photosystem II), suggests an evolution of the pcb genes as shown in figure 4. An ancestral gene for a Chl-binding protein (probably derived by duplication of a gene for CP43) was duplicated to give a pcbC gene and a second gene that became specialized as an isiA gene in some lineages and in others as a single pcbA/B gene. (The latter may have duplicated again to generate tandem pcbA/B genes.) In many of the pcbC-isiA lineages, the pcbC gene was lost, but this was not the case with all of them. We propose that the Pcb proteins were (and remain) able to bind a range of different Chls, according to what was available. It is not clear whether IsiA proteins can do likewise. This scheme could also account for the unexpected existence of pcbC homologs adjacent to isiA as seen in cyanobacteria such as Fischerella muscicola PCC73103 (Geiss et al. 2001). In some lineages, the pcbA/B genes proliferated further, to allow organisms to exploit niches with different light levels, as seen with Prochlorococcus strains today (Garczarek et al. 2001). If the inferred order of divergence of the gene families were incorrect, one could postulate a simple divergence of a Chl-binding protein into IsiA or Pcb families, followed by duplication and divergence within the Pcb family.
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The prochlorophytes are widely separated from each other and scattered among the phycobilisome-containing and IsiA-containing cyanobacteria in phylogenetic trees based on 16S rRNA sequences (Urbach, Roberson, and Chishholm 1992). The prochlorophytes are also widely separated from A. marina in 16S rRNA trees (Miyashita et al. 2003). This appears to conflict with the inference of a single origin of pcbA/B and pcbC sequences. The simplest way to account for this observation is to invoke lateral gene transfer of pcb genes into isiA-containing lineages, generally (but perhaps not invariably) replacing the need for phycobilisomes. This model would also readily explain the apparent existence of an isiA gene in the pcb-containing Prochloron didemni (La Roche et al. 1996) and the existence of phycobiliprotein genes in some prochlorophytes, if these genes were not completely lost. Lateral gene transfer among prokaryotes is well documented (Nesbo, Boucher, and Doolittle 2001). In addition, the existence of a phycoerythrin-containing cyanobacterium Synechocystis trididemni in didemnid ascidians (also the host organisms for Prochloron didemni) that groups very closely with Prochloron didemni in 16S and rbcL trees (Shimada et al. 2003) adds support to the proposal of lateral transfer. It is interesting that prochlorophytes contain a homologous Chl aoxygenase sequence (Tomitani et al. 1999), suggesting that this too may have been transferred laterally. It is not clear at what stage the ability to produce Chl d was developed, but it is clear that oxygenic photosynthetic bacteria are relatively flexible in the use of different chlorophyll types for light harvesting (Satoh et al. 2001). Noting that some prochlorophytes also contain a Chl clike pigment (Larkum et al. 1994), we suggest that the use of a particular light-harvesting Chl by an organism is merely a consequence of which Chl(s) they are able to synthesize. In this regard, it is worth noting that a wide variety of light-harvesting proteins can be reconstituted in vitro with chlorophylls, which are not present in the native complexes (Grabowski, Cunningham, and Gantt 2002; Scheer 2003). In addition, reconstitutions of plant light-harvesting complex (LHCII) demonstrate that some chlorophyll-binding sites can accept either chlorophyll a or b (Bassi et al. 1999), and it is probable that this also occurs in vivo. Of particular relevance to the flexibility and opportunism (Green 2001) for binding exotic chlorophylls that may become available, is the demonstration that engineering chlorophyll a oxygenase together with an LHCII gene into Synechocystis PC6803 results in significant chlorophyll b incorporation into CP43 and CP47 (Xu, Vavilin, and Vermaas 2001).
It is possible to propose a different model in which the ancestor to all oxygenic photosynthetic bacteria contained both PcbA/B/C and IsiA/phycobilisome systems. (This arrangement is not shown in figure 4 but could be derived by duplication of the chlorophyll-binding protein gene to generate both a pcbA/B and an isiA gene.) Although this avoids the need to propose lateral transfer, it would require the development of a mixture of light-harvesting systems followed by the elimination of one or other independently in a large number of lineages, which seems less plausible.
These results, therefore, show that the Pcb system used by Chl b-containing and Chl d-containing prokaryotes evolved once and that the distribution of different light-harvesting systems in 16S rRNA trees can most easily be explained by lateral gene transfer. This model can also account for the fact that the two forms of Prochlorococccus, the deep water form (Dufresne et al. 2003) and the shallow water forms (Dufresne et al. 2003; Rocap et al. 2003), whose genomes have been sequenced, have little in common apart from the presence of pcb genes. It also explains other unexpected observations such as the existence of pcb genes in some cyanobacteria (Geiss et al. 2001).
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Acknowledgements |
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Footnotes |
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