Search

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (approximately 1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 microm filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration. nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. A Conductivity, Temperature, and Depth (CTD) sensor is also be used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first. Then, nutrients, chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is approximately 1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the four-part collection series described here.

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.

Archaeal histone-encoding genes have been identified in marine Crenarchaea . The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli , binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya .

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

In the nematode Caenorhabditis elegans six GABAergic motor neurons, known as DDs1,2, remodel their patterns of synaptic connectivity during larval development3. DD remodelling involves a complete reversal of the direction of information flow within nerve processes without marked changes in process morphology. We used a marker localized in vivo to DD presynaptic zones to analyse how the timing of DD remodelling is controlled. In wild-type animals, DDs remodel their synaptic outputs within a 3–5-hour period at the end of the first larval stage. We show that the heterochronic gene lin-14, which controls the timing of stage-specific cell lineages4,5, regulates the timing of DD synaptic output remodelling. In lin-14 loss-of-function mutants, DDs remodel precociously. The degree of precocious remodelling is correlated with the level of lin-14 activity. Expression of lin-14(+) in the DDs of lin-14-null mutants rescues the precocious remodelling, indicating that lin-14 can act cell-autonomously. Consistent with this hypothesis, LIN-14 protein levels decrease in the DDs before remodelling. Our observations reveal a role of heterochronic genes in non-dividing cells, and provide an example of cell-autonomous respecification of neuronal connectivity.

Retinoids have antiproliferative effects on epithelial cells and have been used as chemopreventive and chemotherapeutic agents for several human cancers. Retinoid/interferon combinations have demonstrated activity in advanced stage cervical cancer. The objective of this study was to quantify and localize the expression of RAR-beta 2, a retinoid inducible receptor, in normal cervix and cervical squamous cell carcinoma by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RT-PCR. Specimens where obtained from 11 patients enrolled in a clinical trial to test all-trans retinoic acid (tRA) in combination with interferon-alpha 2a (IFN-alpha 2a) in the treatment of metastatic or recurrent cervical carcinoma. Expression of RAR-beta 2 in cervical carcinoma and normal cervix was measured by quantitative RT-PCR. DNA competitors were used to estimate the relative expression level of RAR-beta 2. Expression of RAR-beta 2 was examined in normal cervix by in situ RT-PCR. Expression of RAR-beta 2 in cervical carcinoma ranged from 0.33 to 1.40 with a mean of 0.89+/-0.13 vs. 1.0+/-0.13 for normal cervix (NS) with RAR-beta 2 reduced to less than or equal to 65% in five cases. Irt situ RT-PCR identified RAR-beta 2 most prominently in basal and para-basal epithelial cell layers of normal exocervix; stromal expression was markedly decreased. This is the first report to localize expression of RAR-beta 2 mRNA in normal cervical epithelium and quantify expression in normal cervix and cervical squamous cell carcinoma. Because retinoid receptors are the proximate mediators of retinoid action on gene expression, alteration of their expression or function could result in cancer development.

Background Marine ecosystem function is largely determined by matter and energy transformations mediated by microbial community interaction networks. Viral infection modulates network properties through mortality, gene transfer and metabolic reprogramming. Results Here we explore the nature and extent of viral metabolic reprogramming throughout the Pacific Ocean depth continuum. We describe 35 marine viral gene families with potential to reprogram metabolic flux through central metabolic pathways recovered from Pacific Ocean waters. Four of these families have been previously reported but 31 are novel. These known and new carbon pathway auxiliary metabolic genes were recovered from a total of 22 viral metagenomes in which viral auxiliary metabolic genes were differentiated from low-level cellular DNA inputs based on small subunit ribosomal RNA gene content, taxonomy, fragment recruitment and genomic context information. Auxiliary metabolic gene distribution patterns reveal that marine viruses target overlapping, but relatively distinct pathways in sunlit and dark ocean waters to redirect host carbon flux towards energy production and viral genome replication under low nutrient, niche-differentiated conditions throughout the depth continuum. Conclusions Given half of ocean microbes are infected by viruses at any given time, these findings of broad viral metabolic reprogramming suggest the need for renewed consideration of viruses in global ocean carbon models.

A revolution is unfolding in microbial ecology where petabytes of ‘multi-omics’ data are produced using next generation sequencing and mass spectrometry platforms. This cornucopia of biological information has enormous potential to reveal the hidden metabolic powers of microbial communities in natural and engineered ecosystems. However, to realize this potential, the development of new technologies and interpretative frameworks grounded in ecological design principles are needed to overcome computational and analytical bottlenecks. Here we explore the relationship between microbial ecology and information science in the era of cloud-based computation. We consider microorganisms as individual information processing units implementing a distributed metabolic algorithm and describe developments in ecoinformatics and ubiquitous computing with the potential to eliminate bottlenecks and empower knowledge creation and translation.

Background Pairwise comparison of time series data for both local and time-lagged relationships is a computationally challenging problem relevant to many fields of inquiry. The Local Similarity Analysis (LSA) statistic identifies the existence of local and lagged relationships, but determining significance through a p-value has been algorithmically cumbersome due to an intensive permutation test, shuffling rows and columns and repeatedly calculating the statistic. Furthermore, this p-value is calculated with the assumption of normality -- a statistical luxury dissociated from most real world datasets. Results To improve the performance of LSA on big datasets, an asymptotic upper bound on the p-value calculation was derived without the assumption of normality. This change in the bound calculation markedly improved computational speed from O(pm 2 n) to O(m 2 n), where p is the number of permutations in a permutation test, m is the number of time series, and n is the length of each time series. The bounding process is implemented as a computationally efficient software package, FAST LSA, written in C and optimized for threading on multi-core computers, improving its practical computation time. We computationally compare our approach to previous implementations of LSA, demonstrate broad applicability by analyzing time series data from public health, microbial ecology, and social media, and visualize resulting networks using the Cytoscape software. Conclusions The FAST LSA software package expands the boundaries of LSA allowing analysis on datasets with millions of co-varying time series. Mapping metadata onto force-directed graphs derived from FAST LSA allows investigators to view correlated cliques and explore previously unrecognized network relationships. The software is freely available for download at: http://www.cmde.science.ubc.ca/hallam/fastLSA/.