BioDry: An Inexpensive,Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry “field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.     

Density dependent expression of a diatom retrotransposon

Retrotransposons are mobile genetic elements that encode for their own replication. Many studies have linked their expression to stress caused by environmental factors. Genome sequencing and EST libraries of the coastal diatom Phaeodactylum tricornutum indicate that this organism has an active copia-like retrotransposon (Blackbeard), which is variably expressed under different culture conditions. In this study, we induce physiologic stress in P. tricornutum and measure Blackbeard expression over time. However, we find the dominant pattern of Blackbeard expression is related to cell culture density, not short-term physiologic stress. Density dependent expression of a retrotransposon in a diatom provides significant insight into the biogeography of diatom genome mutation. We suggest the shallow coastal ocean, where diatom densities are high, may be the geographic locus for generating genomic diversity in diatom lineages.     

Natural Microbial Community Compositions Compared by a Back-Propagating Neural Network and Cluster Analysis of 5S rRNA

The community compositions of free-living and particle-associated bacteria in the Chesapeake Bay estuary were analyzed by comparing banding patterns of stable low-molecular-weight RNA (SLMW RNA) which include 5S rRNA and tRNA molecules. By analyzing images of autoradiographs of SLMW RNAs on polyacrylamide gels, band intensities of 5S rRNA were converted to binary format for transmission to a back-propagating neural network (NN). The NN was trained to relate binary input to sample stations, collection times, positions in the water column, and sample types (e.g., particle-associated versus free-living communities). Dendrograms produced by using Euclidean distance and average and Ward's linkage methods on data of three independently trained NNs yielded the following results. (i) Community compositions of Chesapeake Bay water samples varied both seasonally and spatially. (ii) Although there was no difference in the compositions of free-living and particle-associated bacteria in the summer, these community types differed significantly in the winter. (iii) In the summer, most bay samples had a common 121-nucleotide 5S rRNA molecule. Although this band occurred in the top water of midbay samples, it did not occur in particle-associated communities of bottom-water samples. (iv) Regardless of the season, midbay samples had the greatest variety of 5S rRNA sizes. The utility of NNs for interpreting complex banding patterns in electrophoresis gels was demonstrated.     

Similarities in Restriction Fragment Patterns of Mitochondrial DNAs of PhytophthoraSpecies Strongly Depend on the Restriction Enzyme Used Due to Heterogeneous Base Distribution and Sequence Conservation

Mitochondrial DNAs of six morphologically different Phytophthora species were digested with 15 restriction enzymes. The numbers of restriction fragments obtained differed considerably from those theoretically expected for random base distribution. Enzymes with relatively many G and C in their recognition sequences produced significantly larger numbers of fragments. Moreover, fragments generated by most of these enzymes were more often shared by two or more species than those from enzymes with more A and T in their recognition sequence. It is concluded that base distribution in mitochondrial DNA of Phytophthora is heterogeneous,AT-rich stretches occurring scattered over the mitochondrial genome and GC-rich regions present in conserved sequences, presumably genes. A practical consequence for taxonomic RFLP studies is that optimal enzymes can be selected, depending on the desired level of resolution.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Metacaspase involvement in programmed cell death of the marine cyanobacterium Trichodesmium

Metacaspases are cysteine specific proteases implicated in cell-signalling, stress acclimation and programmed cell death (PCD) pathways in plants, fungi, protozoa, bacteria and algae. We investigated metacaspase-like gene expression and biochemical activity in the bloom-forming, N₂-fixing, marine cyanobacterium Trichodesmium, which undergoes PCD under low iron and high-light stress. We examined these patterns with respect to in-silico analyses of protein domain architectures that revealed a diverse array of regulatory domains within Trichodesmium metacaspases-like (TeMC) proteins. Experimental manipulations of laboratory cultures and oceanic surface blooms of Trichodesmium from the South Pacific Ocean triggered PCD under Fe-limitation and high light along with enhanced TeMC activity and upregulated expression of diverse TeMC representatives containing different regulatory domains. Furthermore, TeMC activity was significantly and positively correlated with caspase-like activity, which has been routinely observed to increase with PCD induction in Trichodesmium. Although both TeMC and caspase-like activities were stimulated upon PCD induction, inhibitor treatments of these proteolytic activities provided further evidence of largely distinct substrate specificities, even though some inhibitory crossover was observed. Our findings are the first results linking metacaspase expression and activity in PCD induced mortality in Trichodesmium. Yet, the role/s and specific activities of these different proteins remain to be elucidated.