Ecological Divergence of a Novel Group of Chloroflexus Strains Along a Geothermal Gradient

Environmental gradients are expected to promote the diversification and coexistence of ecological specialists adapted to local conditions. Consistent with this view, genera of phototrophic microorganisms in alkaline geothermal systems generally appear to consist of anciently divergent populations which have specialized on different temperature habitats. At White Creek (Lower Geyser Basin, Yellowstone National Park), however, a novel, 16S rRNA-defined lineage of the filamentous anoxygenic phototroph Chloroflexus (OTU 10, phylum Chloroflexi) occupies a much wider thermal niche than other 16S rRNA-defined groups of phototrophic bacteria. This suggests that Chloroflexus OTU 10 is either an ecological generalist or, alternatively, a group of cryptic thermal specialists which have recently diverged. To distinguish between these alternatives, we first isolated laboratory strains of Chloroflexus OTU 10 from along the White Creek temperature gradient. These strains are identical for partial gene sequences encoding the 16S rRNA and malonyl coenzyme A (CoA) reductase. However, strains isolated from upstream and downstream samples could be distinguished based on sequence variation at pcs, which encodes the propionyl-CoA synthase of the 3-hydroxypropionate pathway of carbon fixation used by the genus Chloroflexus. We next demonstrated that strains have diverged in temperature range for growth. Specifically, we obtained evidence for a positive correlation between thermal niche breadth and temperature optimum, with strains isolated from lower temperatures exhibiting greater thermal specialization than the most thermotolerant strain. The study has implications for our understanding of both the process of niche diversification of microorganisms and how diversity is organized in these hot spring communities

In Vitro Reconstitution of Argonaute poly(ADP-ribosyl)ation and Its Impact on microRNA Activity

MicroRNA-mediated gene regulation is an important component of cell biology, involved in nearly all cellular processes. MicroRNAs are ~22 nucleotide non-coding RNAs that recognize complimentary mRNA targets and prevent them from being translated. To achieve this, the microRNA (miRNA) must be accompanied by one or more proteins known collectively as the RNA-induced silencing complex (RISC). The central component of the RISC is one of four Argonaute proteins. One of these proteins, Ago2, has endonuclease activity. Guided by a miRNA, Ago2 can directly cleave the mRNA target without assistance from any other RISC members. MiRNA repression is reduced upon stress when all Argonaute members, including Ago2, are modified by poly(ADP-ribose). Similar reduction in microRNA repression occurs upon knockdown of poly(ADP-ribose) degrading enzyme PARG or, conversely, by overexpression of specific poly(ADP-ribose) polymerases including PARP-12 and PARP-13. Moreover, Ago2 has been shown to be associated with PARP-5a, PARP-12 and PARP-13 by co-immunoprecipitation and co-localization at stress granules. The aim of this project is to use recombinant proteins to ADP-ribosylate Ago2 in vitro to determine whether and how this modification affects Ago2-mediated RNA activity and if the modification can be reversed by PARG or another poly(ADP-ribose) hydrolase, ARH3. This thesis will describe the purification of ARH3 and the catalyctic domain of PARP-12 from E. coli. ARH3 is demonstrated to be an active glycohdrolase and PARP-12cat is demonstrated to be a mono(ADP-ribose) tranferase. Our results suggest that Ago2 can be mono(ADP-ribosyl)ated by PARP-12cat and may be poly(ADP-ribosyl)ated by PARP-5a in vitro. Finally, an Ago2 cleavage assay has been recapitulated and optimized. The work in this thesis project has established the necessary tools to test the hypothesis that mono(ADP-ribosyl)ation or poly(ADP-ribosyl)ation will repress Ago2 activity and whether its activity can be recovered with PARG or ARH3. Other future experiments may elucidate the role of PARP-13

In Vitro Reconstitution of Argonaute poly(ADP-ribosyl)ation and Its Impact on microRNA Activity

MicroRNA-mediated gene regulation is an important component of cell biology, involved in nearly all cellular processes. MicroRNAs are ~22 nucleotide non-coding RNAs that recognize complimentary mRNA targets and prevent them from being translated. To achieve this, the microRNA (miRNA) must be accompanied by one or more proteins known collectively as the RNA-induced silencing complex (RISC). The central component of the RISC is one of four Argonaute proteins. One of these proteins, Ago2, has endonuclease activity. Guided by a miRNA, Ago2 can directly cleave the mRNA target without assistance from any other RISC members. MiRNA repression is reduced upon stress when all Argonaute members, including Ago2, are modified by poly(ADP-ribose). Similar reduction in microRNA repression occurs upon knockdown of poly(ADP-ribose) degrading enzyme PARG or, conversely, by overexpression of specific poly(ADP-ribose) polymerases including PARP-12 and PARP-13. Moreover, Ago2 has been shown to be associated with PARP-5a, PARP-12 and PARP-13 by co-immunoprecipitation and co-localization at stress granules. The aim of this project is to use recombinant proteins to ADP-ribosylate Ago2 in vitro to determine whether and how this modification affects Ago2-mediated RNA activity and if the modification can be reversed by PARG or another poly(ADP-ribose) hydrolase, ARH3. This thesis will describe the purification of ARH3 and the catalyctic domain of PARP-12 from E. coli. ARH3 is demonstrated to be an active glycohdrolase and PARP-12cat is demonstrated to be a mono(ADP-ribose) tranferase. Our results suggest that Ago2 can be mono(ADP-ribosyl)ated by PARP-12cat and may be poly(ADP-ribosyl)ated by PARP-5a in vitro. Finally, an Ago2 cleavage assay has been recapitulated and optimized. The work in this thesis project has established the necessary tools to test the hypothesis that mono(ADP-ribosyl)ation or poly(ADP-ribosyl)ation will repress Ago2 activity and whether its activity can be recovered with PARG or ARH3. Other future experiments may elucidate the role of PARP-13