Pan genome of the phytoplankton Emiliania underpins its global distribution.

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

Draft Genome Sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, Bacillus sp. Strain V59.32b, Bacillus sp. Strain MER_TA_151, and Paenibacillus sp. Strain MER_111, Isolated from Cleanrooms Where the Viking and Mars Exploration Rover Spacecraft Were Assembled.

We report the draft genome sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, and Bacillus sp. strain V59.32b, isolated from the Viking spacecraft assembly cleanroom, and Bacillus sp. strain MER_TA_151 and Paenibacillus sp. strain MER_111, isolated from the Mars Exploration Rover (MER) assembly cleanroom.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi.

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

Analysis of expressed sequence tags from calcifying cells of marine coccolithophorid (Emiliania huxleyi).

An expressed sequence tag (EST) approach was used to investigate gene expression in the unicelluar marine alga Emiliania huxleyi. We randomly selected 3000 EST sequences from a cDNA library of transcripts expressed under conditions promoting coccolithogenesis. Cluster analysis and contig assembly resulted in a unigene set of approximately 1523 ESTs. Only 36% of the unique sequences exhibited significant homology to sequences in GenBank. Of particular interest were the numerous transcripts with homology to sequences associated with sexual reproduction and calcium homeostasis in other unicellular and multicellular organisms. The majority of ESTs (64%) had little or no significant sequence homology to entries in GenBank, suggesting a potential for further novel gene discovery. The catalog of ESTs reported herein represents a significant increase in the limited sequence information currently available for E. huxleyi and should make the coccolithophorid more accessible to powerful genomics and postgenomics technologies.

Sequential ovulation and fertility of polyoestrus in American black bears (Ursus americanus).

American black bears (Ursus americanus) are seasonally polyoestrous and exhibit delayed implantation, which may allow equal and independent fertility of recurrent oestruses of a mating season. We postulated that the luteal inactivity during delayed implantation allows bears to have sequential ovulation during a polyoestrous mating season such that each oestrus of a polyoestrous female will have equivalent fertility, and pregnancy would not preclude subsequent ovulation and superfetation. Controlled mating experiments were conducted on semi-free-ranging female American black bears during three mating seasons, wherein females were bred by different male cohorts in each oestrus. Behavioural observation, vulva score ranking, genetic paternity analysis, gross morphology of ovaries and microscopic morphology of diapaused embryos were used to evaluate the fertility of each subsequent oestrus in polyoestrous females. Oestrus duration, number of successful mounts and median vulva scores were similar between first and subsequent oestruses of the season. Polyoestrus occurred in 81.3% of oestrous females, with a 9.7 ±â€…5.5 day (mean ±â€…SD) inter-oestrous interval. Sequential ovulation was documented in three polyoestrous females, including one that possessed both a corpus haemorrhagicum and a developed corpus luteum. Among polyoestrous dams, four of nine embryos were conceived in the first oestrus and five of nine in the second oestrus. These results indicate that each oestrus of polyoestrous females is capable of fertility, even if the female is already pregnant from a prior oestrus. Although superfetation was not directly observed in the present study, our results strongly suggest the potential of superfetation in the American black bear and provide novel insight into the complex behavioural and physiological breeding mechanisms of bears. Given that most endangered bear species share similar reproductive traits with American black bears, captive breeding programmes could take advantage of superfetation by mating females with different males at each subsequent oestrus of the season in order to increase the genetic diversity of captive endangered bears.

Identification and preliminary characterization of two cDNAs encoding unique carbonic anhydrases from the marine alga Emiliania huxleyi.

Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized (see the work of Quinn et al. in this issue [P. Quinn, R. M. Bowers, X. Zhang, T. M. Wahlund, M. A. Fanelli, D. Olszova, and B. A. Read, Appl. Environ. Microbiol. 72:5512-5526, 2006]). In this study, the cloning and preliminary characterization of two genetically distinct carbonic anhydrase cDNAs are described. Phylogenetic analysis indicated that these two genes belonged to the gamma (gamma-EhCA2) and delta (delta-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of delta-EhCA1 revealed that it encodes a protein of 702 amino acids (aa) (ca. 77.3 kDa), with a transmembrane N-terminal region of 373 aa and an in-frame C-terminal open reading frame of 329 aa that defines the CA region. The gamma-EhCA2 protein was 235 aa in length (ca. 24.9 kDa) and was successfully expressed in Escherichia coli BL21(DE3) and purified as an active recombinant CA. The expression levels of each transcript from quantitative reverse transcription-PCR experiments under bicarbonate limitation and over a 24-h time course suggest that these isozymes perform different functions in E. huxleyi.

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

Suppressive subtractive hybridization of and differences in gene expression content of calcifying and noncalcifying cultures of Emiliania huxleyi strain 1516.

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.