Discovery of a freeliving picocyanobacterium with a hybrid

Discovery of a free-living picocyanobacterium with a hybrid ribosome and chlorophyll d as the major light-harvesting pigment. S. Miller 1, 2, and A. M. Wood 1, 1 Center for Ecology and Evolution University of Oregon, Eugene OR, USA 2 Division of Biological Sciences, The University of Montana, Missoula MT 59812 ABSTRACT: In the course of a biotic inventory of the Salton Sea (California), we isolated a unicellular picocyanobacterium with unusual lime-green pigmentation (Fig. 1 A) due to the presence of chlorophyll d as the predominant lightharvesting pigment (Fig. 2). Fine structure reveals many peripheral thylakoids and no phycobilisomes (Fig. 3) although we find evidence of PE genes (Everroad, not shown). The strain is closely related to Acaryochloris marina, which was isolated as a symbiont from a colonial ascidian, and which is found in pristine oceanic waters. In contrast, the free-living form we discovered lives in a hypereutrophic high salinity environment. Together, the strains represent a new lineage within the cyanobacterial radiation (Fig. 4). The V 1 region of the ribosome contains an insert not found in any other cyanobacteria (Fig. 5) and which appears to have been acquired from a proteobacterial donor between 10 and 200 mya. BP (Fig. 5). These results extend the concept of a mosaic prokaryotic genome that can be fluid across great evolutionary distances to our most widely used phylogenetic marker. Acaryochloris 2 A 51 Cyanobacterial Cell Structure and Phylogenetic affinity 0. 5 m Figure 3. Transmission electron micrograph showing the thylakoid membranes of strain 2 A 51. Phycobilisomes were not observed. Photo by J. Selker and S. Augustine (UO). Acaryochloris 2 A 51 A Chlorophyll d- containing picocyanobacterium Chlorophyll d - The evidence A B Figure 4 (right above). The 16 s SSU gene of Acaryochloris 2 A 51 is 99. 1% identical to that of A. marina MBIC-1101; these strains group as sister taxa within the cyanobacterial radiation and are most closely related to the tide pool isolate Synechococcus IR-11. METHODS: Nearly complete 16 S sequences spanning E. coli positions 8 -1334 and 359 -1528 were obtained by PCR amplification. Sequences were aligned using CLUSTAL and phylogenies constructed using maximum likelihood (ML), maximum parsimony (MP), and neighbor joining methods with PAUP 4. 0. The likelihood model estimated all parameters assuming a general time reversible model of nucleotide substitution and rate heterogeneity among sites. Phylogenies were bootstrapped with either 100 (ML) or 1000 (MP, NJ) pseudoreplicates. Sequence Homology, Structure, and Nucelotide Usage All Show Hybrid Ribosome V 1 Spur Contains Apparently Functional Proteobacterial Sequence Figure 1 A. Late exponential phase cultures of Synechococcus (left), chlorophyll d isolate from the Salton Sea, 2 A 51 (center); Chlorella (right). 1 B. Absorption spectra of 90% methanol extracts from strain 2 A 51 and Acaryochloris marina; the peaks at 448 and 698 nm are characteristic of chlorophyll d. Gloeobacter PCC 7421 Synechococcus IR 11 Strain 2 A 51 Strain MBIC-11017 E. coli N. cryotolerans A. defragrans A. faecalis Alcaligenes LMG 5906 Bordetella 61717 Figure 5 A. (above). In both Acaryochloris strains, we found that the variable V 1 region of the 16 S s. SSU gene was 14 -18 nucleotides longer than in all other cyanobacterial sequences. This region was excluded from the phylogenetic analysis. This anomaly is unlikely to result from PCR artifact since it’s occurrence in the two sequences was confirmed by separate DNA extraction and amplification of the gene in two different laboratories (Wood, Blankenship). The V 1 sequence of the Acaryochloris strains, which are identical to each other, are also 100% identical to the V 1 sequences of two -proteobacterial clinical isolates, suggesting possible acquisition by lateral gene transfer. 5´ G G CA GC A C ° 3´ AGC G G UGG 5´ G A U C U U ° ° A G C U A G G C 3´ 5´ G G G U G C U U ° · A U C A C G G C ´ 3 5´ G G CA GC A C G A G C U ° · G U U C C U C G U A C G 3´ G G G A G C U · U C C U C G U G A A G C U · U U U C G U G A G A G C U · U U C U C G U G A G C U · U U C C U C G U Figure 5 B (above). Secondary structure models for the V 1 region were developed using homologous models available at the Comparative RNA website (http: //www. rna. icmb. utexas. edu). Color coding shows similarity of phylogenetic affinity from Fig. 4 (left) or structure (right). The spur of the two Acaryochloris strains bear little structural similarity to other cyanbacterial spurs, but is identical to that of two proteobacteria and very similar to several others. The DNA sequence encoding the loop is TTCG in most cyanobcteria and, with the exception of the Acaryochloris strains is never CTTG. The chl d strains and -proteobacteria also share several bulge motifs (see also Fig. 5 A) that are absent from other cyanobacteria. DATING THE GENE TRANSFER -- Probably occurred 10 -200 Ma ago, in contrast to 2200 MA ago, when the -proteobacteria and cyanobacteria last shared a common ancestor. Fig 2. Mass spectra of HPLC elution peaks. Panels A and B show chlorophylls a and d for strain 2 A 51, respectively. Panels C and D show chlorophylls a and d for Acaryochloris marina, respectively. We thank T. Olson and R. Blankenship for HPLC and GCMS analysis. The origin of the insert in the V 1 spur is constrained to the time period after these strains last had a common ancestor with Synechococcus IR 11, but prior to their divergence from each other. We used ML to implement a general time reversible nucleotide substitution model with among site heterogeneity, with and without an imposed clock. For the entire tree in Fig. 4, as well as every possible sub-tree defined by the internal nodes in the phylogeny. , pairs of clock and no-clock, models were compared to the likelihood ratio tests. While the tree as a whole does not obey a global molecular clock, sequence evolution of the Node B Clade does conform to a local clock (-2 XL = 0. 13, P=0. 72). We next estimated the timing of the LGT event using the fossil record calibrated 16 S SSU t. RNA clock for a group of bacterial endosymbionts of aphids (Buchnera spp. ), which gives a rate of 0. 01 -0. 02 nucleotide substitutions per site per 50 million years. This provides an estimate of the timing of the divergence between Node A and B at between 10 and 200 Ma ago. This stands in sharp contrast to the time, greater than 2200 Ma ago, that the cyanobacteria and b-proteobacteria last shared a common ancestor.