Evolution: functional evolution of nuclear structure.

The evolution of the nucleus, the defining feature of eukaryotic cells, was long shrouded in speculation and mystery. There is now strong evidence that nuclear pore complexes (NPCs) and nuclear membranes coevolved with the endomembrane system, and that the last eukaryotic common ancestor (LECA) had fully functional NPCs. Recent studies have identified many components of the nuclear envelope in living Opisthokonts, the eukaryotic supergroup that includes fungi and metazoan animals. These components include diverse chromatin-binding membrane proteins, and membrane proteins with adhesive lumenal domains that may have contributed to the evolution of nuclear membrane architecture. Further discoveries about the nucleoskeleton suggest that the evolution of nuclear structure was tightly coupled to genome partitioning during mitosis

Mapping the protistan 'rare biosphere'

The use of cultivation-independent approaches to map microbial diversity, including recent work published in BMC Biology, has now shown that protists, like bacteria/archaea, are much more diverse than had been realized. Uncovering eukaryotic diversity may now be limited not by access to samples or cost but rather by the availability of full-length reference sequence data

Eight unique basal bodies in the multi-flagellated diplomonad Giardia lamblia

Giardia lamblia is an intestinal parasitic protist that causes significant acute and chronic diarrheal disease worldwide. Giardia belongs to the diplomonads, a group of protists in the supergroup Excavata. Diplomonads are characterized by eight motile flagella organized into four bilaterally symmetric pairs. Each of the eight Giardia axonemes has a long cytoplasmic region that extends from the centrally located basal body before exiting the cell body as a membrane-bound flagellum. Each basal body is thus unique in its cytological position and its association with different cytoskeletal features, including the ventral disc, axonemes, and extra-axonemal structures. Inheritance of these unique and complex cytoskeletal elements is maintained through basal body migration, duplication, maturation, and their subsequent association with specific spindle poles during cell division. Due to the complex composition and inheritance of specific basal bodies and their associated structures, Giardia may require novel basal body-associated proteins. Thus, protists such as Giardia may represent an undiscovered source of novel basal body-associated proteins. The development of new tools that make Giardia genetically tractable will enable the composition, structure, and function of the eight basal bodies to be more thoroughly explored

Robust and stable transcriptional repression in Giardia using CRISPRi.

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems

Draft Genome Sequence of the Caffeine-Degrading Methylotroph Methylorubrum populi Pinkel.

A pink-pigmented facultative methylotroph, Methylorubrum populi Pinkel, was isolated from compost by selective enrichment with caffeine (3,5,7-trimethylxanthine) as the sole carbon, nitrogen, and energy source. We report here its high-quality draft genome sequence, assembled in 35 contigs totaling 5,630,907 bp. We identified 5,681 protein-coding sequences, including those putatively involved in caffeine degradation

Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bhatnagar, S., Cowley, E. S., Kopf, S. H., Pérez Castro, S., Kearney, S., Dawson, S. C., Hanselmann, K., & Ruff, S. E. Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom. Environmental Microbiome, 15(1),(2020): 3, doi:10.1186/s40793-019-0348-0.Background: Lagoons are common along coastlines worldwide and are important for biogeochemical element cycling, coastal biodiversity, coastal erosion protection and blue carbon sequestration. These ecosystems are frequently disturbed by weather, tides, and human activities. Here, we investigated a shallow lagoon in New England. The brackish ecosystem releases hydrogen sulfide particularly upon physical disturbance, causing blooms of anoxygenic sulfur-oxidizing phototrophs. To study the habitat, microbial community structure, assembly and function we carried out in situ experiments investigating the bloom dynamics over time. Results: Phototrophic microbial mats and permanently or seasonally stratified water columns commonly contain multiple phototrophic lineages that coexist based on their light, oxygen and nutrient preferences. We describe similar coexistence patterns and ecological niches in estuarine planktonic blooms of phototrophs. The water column showed steep gradients of oxygen, pH, sulfate, sulfide, and salinity. The upper part of the bloom was dominated by aerobic phototrophic Cyanobacteria, the middle and lower parts by anoxygenic purple sulfur bacteria (Chromatiales) and green sulfur bacteria (Chlorobiales), respectively. We show stable coexistence of phototrophic lineages from five bacterial phyla and present metagenome-assembled genomes (MAGs) of two uncultured Chlorobaculum and Prosthecochloris species. In addition to genes involved in sulfur oxidation and photopigment biosynthesis the MAGs contained complete operons encoding for terminal oxidases. The metagenomes also contained numerous contigs affiliating with Microviridae viruses, potentially affecting Chlorobi. Our data suggest a short sulfur cycle within the bloom in which elemental sulfur produced by sulfide-oxidizing phototrophs is most likely reduced back to sulfide by Desulfuromonas sp. Conclusions: The release of sulfide creates a habitat selecting for anoxygenic sulfur-oxidizing phototrophs, which in turn create a niche for sulfur reducers. Strong syntrophism between these guilds apparently drives a short sulfur cycle that may explain the rapid development of the bloom. The fast growth and high biomass yield of Chlorobi-affiliated organisms implies that the studied lineages of green sulfur bacteria can thrive in hypoxic habitats. This oxygen tolerance is corroborated by oxidases found in MAGs of uncultured Chlorobi. The findings improve our understanding of the ecology and ecophysiology of anoxygenic phototrophs and their impact on the coupled biogeochemical cycles of sulfur and carbon.This work was carried out at the Microbial Diversity summer course at the Marine Biological Laboratory in Woods Hole, MA. The course was supported by grants from National Aeronautics and Space Administration, the US Department of Energy, the Simons Foundation, the Beckman Foundation, and the Agouron Institute. Additional funding for SER was provided by the Marine Biological Laboratory