Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains

Background: Heterokont algae form a monophyletic group within the stramenopile branch of the tree of life. These organisms display wide morphological diversity, ranging from minute unicells to massive, bladed forms. Surprisingly, chloroplast genome sequences are available only for diatoms, representing two (Coscinodiscophyceae and Bacillariophyceae) of approximately 18 classes of algae that comprise this taxonomic cluster. A universal challenge to chloroplast genome sequencing studies is the retrieval of highly purified DNA in quantities sufficient for analytical processing. To circumvent this problem, we have developed a simplified method for sequencing chloroplast genomes, using fosmids selected from a total cellular DNA library. The technique has been used to sequence chloroplast DNA of two Heterosigma akashiwo strains. This raphidophyte has served as a model system for studies of stramenopile chloroplast biogenesis and evolution. Results: H. akashiwo strain CCMP452 (West Atlantic) chloroplast DNA is 160,149 bp in size with a 21,822-bp inverted repeat, whereas NIES293 (West Pacific) chloroplast DNA is 159,370 bp in size and has an inverted repeat of 21,665 bp. The fosmid cloning technique reveals that both strains contain an isomeric chloroplast DNA population resulting from an inversion of their single copy domains. Both strains contain multiple small inverted and tandem repeats, non-randomly distributed within the genomes. Although both CCMP452 and NIES293 chloroplast DNAs contains 197 genes, multiple nucleotide polymorphisms are present in both coding and intergenic regions. Several protein-coding genes contain large, in-frame inserts relative to orthologous genes in other plastids. These inserts are maintained in mRNA products. Two genes of interest in H. akashiwo, not previously reported in any chloroplast genome, include tyrC, a tyrosine recombinase, which we hypothesize may be a result of a lateral gene transfer event, and an unidentified 456 amino acid protein, which we hypothesize serves as a G-protein-coupled receptor. The H. akashiwo chloroplast genomes share little synteny with other algal chloroplast genomes sequenced to date. Conclusion: The fosmid cloning technique eliminates chloroplast isolation, does not require chloroplast DNA purification, and reduces sequencing processing time. Application of this method has provided new insights into chloroplast genome architecture, gene content and evolution within the stramenopile cluster

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions

Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in <it>Heterosigma akashiwo </it>(Raphidophyceae)

<p>Abstract</p> <p>Background</p> <p>Maintenance of homeostasis requires that an organism perceive selected physical and chemical signals within an informationally dense environment. Functionally, an organism uses a variety of signal transduction arrays to amplify and convert these perceived signals into appropriate gene transcriptional responses. These changes in gene expression serve to modify selective metabolic processes and thus optimize reproductive success. Here we analyze a chloroplast-encoded His-to-Asp signal transduction circuit in the stramenopile <it>Heterosigma akashiwo </it>(Hada) Hada <it>ex </it>Y. Hara <it>et </it>Chihara [syn. <it>H. carterae </it>(Hulburt) F.J.R. Taylor]. The presence, structure and putative function of this protein pair are discussed in the context of their evolutionary homologues.</p> <p>Results</p> <p>Bioinformatic analysis of the <it>Heterosigma akashiwo </it>chloroplast genome sequence revealed the presence of a single two-component His-to-Asp (designated Tsg1/Trg1) pair in this stramenopile (golden-brown alga). These data represent the first documentation of a His-to-Asp array in stramenopiles and counter previous reports suggesting that such regulatory proteins are lacking in this taxonomic cluster. Comparison of the 43 kDa <it>H. akashiwo </it>Tsg1 with bacterial sensor kinases showed that the algal protein exhibits a moderately maintained PAS motif in the sensor kinase domain as well as highly conserved H, N, G₁and F motifs within the histidine kinase ATP binding site. Molecular modelling of the 27 kDa <it>H. akashiwo </it>Trg1 regulator protein was consistent with a winged helix-turn-helix identity – a class of proteins that is known to impact gene expression at the level of transcription. The occurrence of Trg1 protein in actively growing <it>H. akashiwo </it>cells was verified by Western analysis. The presence of a PhoB-like RNA polymerase loop in Trg1 and its homologues in the red-algal lineage support the hypothesis that Trg1 and its homologues interact with a sigma 70 (σ⁷⁰) subunit (encoded by <it>rpoD</it>) of a eubacterial type polymerase. Sequence analysis of <it>H. akashiwo rpoD </it>showed this nuclear-encoded gene has a well-defined 4.2 domain, a region that augments RNA polymerase interaction with transcriptional regulatory proteins and also serves in -35 promoter recognition. The presence/loss of the His-to-Asp pairs in primary and secondary chloroplast lineages is assessed.</p> <p>Conclusion</p> <p>His-to-Asp signal transduction components are found in most rhodophytic chloroplasts, as well as in their putative cyanobacterial progenitors. The evolutionary conservation of these proteins argues that they are important for the maintenance of chloroplast homeostasis. Our data suggest that chloroplast gene transcription may be impacted by the interaction of the His-to-Asp regulator protein (which is less frequently lost than the sensor protein) with the RNA polymerase σ⁷⁰subunit.</p

Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in (Raphidophyceae)-3

<p><b>Copyright information:</b></p><p>Taken from "Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in (Raphidophyceae)"</p><p>http://www.biomedcentral.com/1471-2148/7/70</p><p>BMC Evolutionary Biology 2007;7():70-70.</p><p>Published online 3 May 2007</p><p>PMCID:PMC1885438.</p><p></p>f OmpR (grey), PhoB (white), and the complete receiver-regulator structure from (blue) reveals important similarities. The predicted Trg1 model closely resembles that of OmpR, particularly in the putative DNA binding region (α3 helix). Notably, the predicted Trg1 structure for the putative RNA polymerase interaction site (α-αloop, red) more closely matches that of PhoB. The phosphorylation site is shown as a purple sphere

Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains-5

G: small inverted (red) and tandem (blue) repeats; Third ring: sequence comparison to the other genome, including SNPs (blue), small insertions (green), deletions (red) and regions of extremely poor alignment (orange); Fourth ring: Location and size of fosmid clones color coded according to their orientation: supports depicted isoform (green), supports alternate isoform (pink), uninformative (black); Fifth ring: location of inverted repeats, large and small single copy domains. Red bar depicts location of 8 kb region inverted in CCMP452 relative to NIES293; inner circle: GC content.<p><b>Copyright information:</b></p><p>Taken from "Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains"</p><p>http://www.biomedcentral.com/1471-2164/9/211</p><p>BMC Genomics 2008;9():211-211.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2410131.</p><p></p

Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains-3

<p><b>Copyright information:</b></p><p>Taken from "Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains"</p><p>http://www.biomedcentral.com/1471-2164/9/211</p><p>BMC Genomics 2008;9():211-211.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2410131.</p><p></p

Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains-4

45 to 50 kb. This DNA is used to generate a fosmid library which is selectively screened for cpDNA-containing clones, which are then sequenced, annotated and assembled.<p><b>Copyright information:</b></p><p>Taken from "Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains"</p><p>http://www.biomedcentral.com/1471-2164/9/211</p><p>BMC Genomics 2008;9():211-211.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2410131.</p><p></p

Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains-1

G: small inverted (red) and tandem (blue) repeats; Third ring: sequence comparison to the other genome, including SNPs (blue), small insertions (green), deletions (red) and regions of extremely poor alignment (orange); Fourth ring: Location and size of fosmid clones color coded according to their orientation: supports depicted isoform (green), supports alternate isoform (pink), uninformative (black); Fifth ring: location of inverted repeats, large and small single copy domains. Red bar depicts location of 8 kb region inverted in CCMP452 relative to NIES293; inner circle: GC content.<p><b>Copyright information:</b></p><p>Taken from "Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains"</p><p>http://www.biomedcentral.com/1471-2164/9/211</p><p>BMC Genomics 2008;9():211-211.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2410131.</p><p></p

Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains-0

45 to 50 kb. This DNA is used to generate a fosmid library which is selectively screened for cpDNA-containing clones, which are then sequenced, annotated and assembled.<p><b>Copyright information:</b></p><p>Taken from "Chloroplast genome sequencing analysis of CCMP452 (West Atlantic) and NIES293 (West Pacific) strains"</p><p>http://www.biomedcentral.com/1471-2164/9/211</p><p>BMC Genomics 2008;9():211-211.</p><p>Published online 8 May 2008</p><p>PMCID:PMC2410131.</p><p></p