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

ABSTRACT: 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.

SUBMITTER: Soto AR

PROVIDER: S-EPMC1538761 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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

Soto Amelia R AR Zheng Hong H Shoemaker Dorinda D Rodriguez Jason J Read Betsy A BA Wahlund Thomas M TM

Applied and environmental microbiology 20060801 8

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 biotechn ...[more]

PMID: 16885304

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Project description:Emiliania huxleyi is a unicellular micro-alga that forms massive oceanic blooms and plays key roles in global biogeochemical cycles. Mounting studies demonstrate various stimulatory and inhibitory influences that bacteria have on the E. huxleyi physiology. To investigate these algal-bacterial interactions, laboratory co-cultures have been established by us and by others. Owing to these co-cultures, various mechanisms of algal-bacterial interactions have been revealed, many involving bacterial pathogenicity towards algae. However, co-cultures represent a significantly simplified system, lacking the complexity of bacterial communities. In order to investigate bacterial pathogenicity within an ecologically relevant context, it becomes imperative to enhance the microbial complexity of co-culture setups. Phaeobacter inhibens bacteria are known pathogens that cause the death of E. huxleyi algae in laboratory co-culture systems. The bacteria depend on algal exudates for growth, but when algae senesce, bacteria switch to a pathogenic state and induce algal death. Here we investigate whether P. inhibens bacteria can induce algal death in the presence of a complex bacterial community. We show that an E. huxleyi-associated bacterial community protects the alga from the pathogen, although the pathogen occurs within the community. To study how the bacterial community regulates pathogenicity, we reduced the complex bacterial community to a five-member synthetic community (syncom). The syncom is comprised of a single algal host and five isolated bacterial species, which represent major bacterial groups that are naturally associated with E. huxleyi. We discovered that a single bacterial species in the reduced community, Sulfitobacter pontiacus, protects the alga from the pathogen. We further found that algal protection from P. inhibens pathogenicity is a shared trait among several Sulfitobacter species. Algal protection by bacteria might be a common phenomenon with ecological significance, which is overlooked in reduced co-culture systems.

Project description:The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism. Keywords: Emiliania, gene expression, nutrients, SAGE, phosphate, nitrogen Emiliania huxleyi CCMP 1516 was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton, Bigelow Laboratories. Cultures were grown at 18°C on a 14 h:10 h light:dark cycle (140 µmol quanta m-2 s-1). Nitrogen and phosphate replete (Replete: 35 µM NO3- and 1.5 µM PO43-), low nitrogen (-N: 10 µM NO3-) and low phosphate (-P: 0 µM PO43-) cells were grown in f/50 medium without Si. Locally collected seawater was filtered (pore size, 0.2 µm) and autoclaved. Filter-sterilized inorganic nutrients, trace metals and vitamins (thiamin, biotin and B12) were added after autoclaving. The cells were grown in 8 L batch cultures. The growth of cultures was monitored daily by cell number counted on a hemacytometer, and by relative fluorescence using a Turner Designs AU Fluorometer. Replete cells were harvested in mid-log phase while –N and –P cells were harvested at the onset of stationary phase for SAGE analysis.

Dataset Information

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

Publications

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

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