Genome sequences of 13 Bacillus anthracis Sterne bacteriophages isolated from soil in the western United States.

Bacillus anthracis, classified as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC), is the causative agent of anthrax in both humans and livestock. Herein, we report the full genome sequences of 13 bacteriophages that infect B. anthracis Sterne. These phages are grouped into four clusters and are similar to previously described Bacillus phages.

Intrinsically disordered regions are not sufficient to direct the compartmental localization of nucleolar proteins in the nucleus.

The nucleolus is a non-membrane bound organelle central to ribosome biogenesis. The nucleolus contains a mix of proteins and RNA and has 3 known nucleolar compartments: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The spatial organization of the nucleolus is influenced by the phase separation properties of nucleolar proteins, the presence of RNA, protein modification, and cellular activity. Many nucleolar proteins appear to concentrate within the borders of the compartments. We investigated whether the intrinsically disordered regions from several proteins provided the information needed to establish specific compartment localization using Xenopus laevis oocytes. For the proteins we tested, the disordered regions were not sufficient to direct specific domain localization and appear dispensable with respect to compartmentalization. Among the proteins that colocalize to the DFC are the quartet that comprise the box H/ACA pseudouridylation complex. In contrast to the insufficiency of IDRs to direct compartment localization, we found that the DFC accumulation of 2 box H/ACA proteins, Gar1 and Nhp2, was disrupted by mutations that were previously shown to reduce their ability to join the box H/ACA complex. Using a nanobody to introduce novel binding to a different DFC localized protein, we restored the localization of the mutated forms of Gar1 and Nhp2.

Component analysis of nucleolar protein compartments using Xenopus laevis oocytes.

The nucleolus is a multi-compartment, non-membrane-bound organelle within the nucleus. Nucleolar assembly is influenced by proteins capable of phase separation. Xenopus laevis oocytes contain hundreds of large nucleoli that provide experimental access for nucleoli that is unavailable in other systems. Here we detail methods to streamline the in vivo analysis of the compartmentalization of nucleolar proteins that are suspected of phase separation. The nucleolus is the main hub of ribosome biogenesis and here we present data supporting the division of proteins into nucleolar domains based on their function in ribosome biogenesis. We also describe the use of vital dyes such as Hoechst 33342 and Thioflavin T in nucleolar staining. Additionally, we quantify nucleolar morphology changes induced by heat shock and actinomycin D treatments. We suggest these approaches will be valuable in a variety of studies that seek to better understand the nucleolus, particularly those regarding phase separation. These approaches may also be instructive for other studies on phase separation, especially in the nucleus.

Conformational Flexibility of Ubiquitin-Modified and SUMO-Modified PCNA Shown by Full-Ensemble Hybrid Methods.

Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.