Phylogenetic Relationships Among Subsurface Microorganisms

This project involves the development of group specific 16S ribosomal RNA-targeted oligonucleotide hybridization probes for the rapid detection of specific types of subsurface organisms (e.g., groups of microbes that share certain physiological traits). Major accomplishments for the period of 6/91 to 12/1/91 are described. Nine new probes have been synthesized on the basis of published 16S rRNA sequence data from the Ribosomal Database Project. We have initiated rapid screening of many of the subsurface microbial isolates obtained from the P24 borehole at the Savannah River Site. To date, we have screened approximately 50% of the isolates from P24. We have optimized our {und in situ} hybridization technique, and have developed a cell blot hybridization technique to screen 96 samples on a single blot. This is much faster than reading 96 individual slides. Preliminary experiments have been carried out which indicate specific nutrients can be used to amplify rRNA only in those organisms capable of metabolizing those nutrients. 1 tab., 2 figs

NOVEL ASPECTS OF ULTRASTRUCTURE IN TWO SPECIES OF CYANOBACTERIA (AGMENELLUM QUADRUPLICATUM, MASTIGOCLADUS LAMINOSUS)

Novel aspects of morphology and ultrastructure were examined in two species of cyanobacteria. A complete, three-dimensional, ultrastructural reconstruction of the unicellular cyanobacterium Agmenellum quadruplicatum was carried out by means of high-voltage electron microscopy of thick sections and computer-aided reconstruction of serial thin sections. The photosynthetic thylakoid system consisted of 3-6 membrane sheets that traversed and were parallel to the long axis of the cell. These sheets were arranged as an anastomosing network of concentric shells. They coalesced and approached the cytoplasmic membrane at three peripheral loci along the entire length of the cell. The central cytoplasm of the cell was completely surrounded by thylakoids; this appeared to be a true form of compartmentalization in a prokaryotic organism. The thylakoid membranes clearly interconnected with the cytoplasmic membrane at several locations within the cell. Some of the various intracellular inclusion bodies were always peripherally located, while others were always centrally located. The detailed three-dimensional arrangement of subcellular features was remarkably consistent from one cell to another. The morphology and ultrastructure of the branching, filamentous (stigonematalean) cyanobacterium Mastigocladus laminosus were examined with conventional light and electron microscopy. The vegetative morphology and ultrastructure of M. laminosus were similar to those of Fischerella ambigua, the only stigonematalean cyanobacterium examined in detail to date. The ultrastructural characteristics of mature heterocysts in M. laminosus were distinctly different from those of other cyanobacteria; the former lacked certain extra wall layers, lacked cyanophycin-like plugs, contained large numbers of closely packed intracytoplasmic membranes, and contained a previously unreported type of inclusion body. The heterocyst differentiation process also differed from that seen in other cyanobacteria; the earliest events involved internal changes rather than external and bundles of stacked, lamellar membranes were formed. These results showed that heterocyst differentiation and ultrastructure in different genera of cyanobacteria vary more widely than has been thought to be the case. M. laminosus was also shown to undergo aging and morphogenetic processes analogous to some of those known to occur in eukaryotic organisms

Thermoclinic Assessment Of A Preliminary Circulation Model For Lake George In The Jefferson Project

The Jefferson Project is a collaboration between the Rensselaer Polytechnic Institute, IBM, and the FUND for Lake George aimed at understanding and managing complex factors (road salt, storm water runoff, invasive species) threatening Lake George, New York. Lake George is located about 80 km north of Albany in upstate New York and is known internationally for its water clarity. Understanding the hydrodynamics of the lake is fundamental for creation and maintenance of a research and monitoring program for the early detection of and response to adverse environmental and biological change. In this work a 3D circulation model of the lake is developed to better understand the hydro-environmental conditions of the lake; forcing is by a combination of local public survey data for the water budget and atmospheric data from the NWS (NOAA National Weather Service). The model is validated by a combination of water chemistry data collected by Darrin Fresh Water Institute (DFWI) over the last three decades, and known empirical relationships of the lake\u27s structural profile. Numerical simulations run over several years to capture the seasonal progression of thermocline depth throughout the lake, the south to north salt and surface thermal gradients and the timing of the spring and fall overturn events. Validation is by comparison with physical and chemical measurements collected over the last three decades. The study presents a novel combination of observational data, numerical modelling and empirical relationships to better understand and predict the lake circulation, and consequently the natural ecosystem

Crane fly semiochemical overrules plant control over cyanobiont in Azolla symbioses

Semiochemicals from insects that restrict plant symbiont dinitrogen fixation had not been known. Here we report on a the glycosylated triketide δ-lactone only found in Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla.Cornicinine was chemically synthesized, as well as its aglycone and diastereoisomer. Only the glycosylated trans-A form was active: 500 nM cornicinine in the growth medium turned the dinitrogen-fixing cyanobacterial filaments from Nostoc azollae inside the host leaf cavities into akinete-like cells. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not affect the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc. Chlorosis occurred in hosts on nitrogen with and devoid of cyanobiont. Cornicinine, therefore, targeted host mechanisms resulting in coordinate cyanobiont differentiation.Sequence profiling of messenger RNA from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. Leaf-cavity transcripts in ferns grown on cornicinine reflected activation of Cullin-RING ubiquitin-ligase pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype. Transcripts accumulating when akinetes are induced, in leaf cavities of ferns on cornicinine and in megasporocarps, were consistent with increased JA-oxidase, sulfate transport and exosome formation.The work begins to uncover molecular mechanisms of cyanobiont differentiation in a seed-free plant symbiosis important for wetland ecology or circular crop-production today, that once caused massive CO2 draw-down during the Eocene geological past.Significance Coordinated differentiation of host and filamentous cyanobacteria underlies the development of ecologically important symbioses; this includes the floating ferns Azolla which share their wetland habitat with Nephrotoma cornicina craneflies containing the glycosylated triketide δ-lactone semiochemical, cornicinine. Cornicinine overrules cyanobiont differentiation thus inhibiting symbiosis N2-fixation and sexual reproduction; its mode of action resembles plant elicitation as suggested by transcriptional profiling of cells lining the cyanobiont cavities using a new release of the fern host genome.Competing Interest StatementThe authors have declared no competing interest

Genome Erosion in a Nitrogen-Fixing Vertically Transmitted Endosymbiotic Multicellular Cyanobacterium

Background: An ancient cyanobacterial incorporation into a eukaryotic organism led to the evolution of plastids (chloroplasts) and subsequently to the origin of the plant kingdom. The underlying mechanism and the identities of the partners in this monophyletic event remain elusive. Methodology/Principal Findings: To shed light on this evolutionary process, we sequenced the genome of a cyanobacterium residing extracellularly in an endosymbiosis with a plant, the water-fern Azolla filiculoides Lam. This symbiosis was selected as it has characters which make it unique among extant cyanobacterial plant symbioses: the cyanobacterium lacks autonomous growth and is vertically transmitted between plant generations. Our results reveal features of evolutionary significance. The genome is in an eroding state, evidenced by a large proportion of pseudogenes (31.2%) and a high frequency of transposable elements (,600) scattered throughout the genome. Pseudogenization is found in genes such as the replication initiator dnaA and DNA repair genes, considered essential to free-living cyanobacteria. For some functional categories of genes pseudogenes are more prevalent than functional genes. Loss of function is apparent even within the ‘core’ gene categories of bacteria, such as genes involved in glycolysis and nutrient uptake. In contrast, serving as a critical source of nitrogen for the host, genes related to metabolic processes such as cell differentiation and nitrogen-fixation are well preserved. Conclusions/Significance: This is the first finding of genome degradation in a plant symbiont and phenotypically complex cyanobacterium and one of only a few extracellular endosymbionts described showing signs of reductive genome evolution. Our findings suggest an ongoing selective streamlining of this cyanobacterial genome which has resulted in an organism devoted to nitrogen fixation and devoid of autonomous growth. The cyanobacterial symbiont of Azolla can thus be considered at the initial phase of a transition from free-living organism to a nitrogen-fixing plant entity, a transition process which may mimic what drove the evolution of chloroplasts from a cyanobacterial ancestor