Beschreibung: Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution November 1992

Beschreibung: Sequences of small subunit (Ss) and large subunit (Ls) ribosomal RNA genes (rDNA) from the marine dinoflagellates Alexandrium tamarense, A. catenella, A. fundyense, A. affine, A. minutum, A. lusitanicum and A. andersoni were compared to assess the organisms' relationships. Cultures represent isolates from North America, Western Europe, Thailand, Japan, Australia and the ballast water of several cargo vessels, and include both toxic and non-toxic strains. An emphasis was placed on the A. tamarense/catenella/fundyense "species complex," a group of morphotypically-similar organisms found in many regions of the world. Two distinct SsrRNA genes, termed the "A gene" and the "B gene," were found in a toxic A. fundyense isolated from eastern North America. The B gene is considered to be a pseudogene. A restriction fragment length polymorphism (RFLP) assay developed to detect the A and B genes revealed five distinct groups of Alexandrium isolates. Three subdivide the A. tamarense/catenella/fundyense complex, but do not correlate with morphospecies designations. The two remaining groups are associated with cultures that clearly differ morphologically from the A. tamarense/ catenella/fundyense group: the fourth group consists of A. affine isolates, and the fifth group is represented by A. minutum, A.lusitanicum and A. andersoni. The B gene was only found in A. tamarense/catenella/ fundyense, but not in all members of this species complex. The B gene is not uniformly distributed among global populations of Alexandrium. All A. tamarense/catenella/fundyense isolates from North America harbor this gene, but it has also been found in some A. tamarense from scattered locations in Japan, as well as in A. tamarense from the ballast water of one cargo vessel which was on a defined run from Japan to Australia. The B gene may be endemic to North American populations of A. tamarense/catenella/fundyense. It is possible that in the recent past North American A. tamarense were introduced to Japanese waters, and cysts of these organisms have been transported from Japan to Australia. A subset of isolates examined using the the RFLP assay were also compared by cloning and sequencing a fragment of their LsrDNA. Eight major classes of LsrDNA sequences, termed "ribotypes," were identified. Five ribotypes subdivide members of the A. tamarense/catenella!fundyense complex; all isolates containing the B gene cluster as one ribotype. The three remaining ribotypes are typified by: 1) A. affine; 2) A. minutum and A. lusitanicum; and, 3) A. andersoni. LsrDNAs from A. minutum and A. lusitanicum are indistinguishable. A. minutum!lusitanicum/andersoni may represent another Alexandrium species complex, analogous to the A. tamarense/catenella/fundyense group. An organisms' ability to produce toxin appears to be correlated with its LsrDNA phylogenetic lineage. Ribotypes ascribed by the LsrDNA sequences are in complete agreement with, and offer a finer-scale resolution of, groups defined by SsrDNA restriction patterns. The SsrDNA RFLP groups and LsrDNA ribotypes are useful species- and population-specific markers. Alexandrium tamarense/catenella/fundyense exist as geneticallydistinct "strains" (populations), not three genetically-distinct species: representatives collected from the same geographic region appear the most similar, regardless of morphotype, whereas those from geographicallyseparated populations are more divergent even when the same morphospecies are compared. Contrary to this general pattern, A. tamarense/catenella from Japan were found to be exceptionally heterogeneous. Ballast water samples show that viable cysts (resting spores) of toxigenic A. tamarense/catenella are being discharged into Australian ports from multiple, genetically-distinct source populations. The rDNA sequences were also used to test theories accounting for the evolution and global dispersal of A. tamarense/catenella/fundyense. Results suggest a monophyletic radiation of these organisms from a common ancestor that included, or gave rise to, multiple morphotypes. Populations appear to have diverged as a result of vicariance (geographic isolation). The co-occurrence of genetically-distinct strains of these organisms is an indication of dispersal. An example of this is seen in Japan where an introduction of North American A. tamarense appears likely. Determining the timing of dispersal events is problematic if based strictly on rDNA sequence similarities, since these molecules undergo change on a scale of millions of years.

Beschreibung: This work was supported in part by a grant from the National Science Foundation to D. M. Anderson (contract number OCE89- 11226), the Woods Hole Oceanographic Institution Ocean Ventures Fund and the Woods Hole Oceanographic Institution Education Office .

Beschreibung: © The Author(s), 2011. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited. The definitive version was published in Frontiers in Microbiology 2 (2011): 215, doi:10.3389/fmicb.2011.00215.

Beschreibung: Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC–MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although further study is warranted on additional marine microbes.

Beschreibung: This work was funded by the National Science Foundation Chemical and Biological Oceanography, Center for Microbial Oceanography Research and Education (C-MORE), and the Gordon and Betty Moore Foundation.