Protein phosphatase type 5 (PP5) belongs to the PPP-family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by hormones (i.e. glucocorticoids) or cellular stress (i.e. hypoxia, oxidative stress). Recently, studies conducted with siRNA and antisense oligonucleotides indicate that PP5 plays an important role in the regulation of stress-induced signaling cascades that influence both cell growth and the onset of apoptosis. Therefore, the identification of small molecule inhibitors of PP5 is desired for use in studies to further define the biological/pathological roles of PP5. Such inhibitors may also prove useful for development into novel antitumor agents. Here we describe methods to express and purify large amounts of biologically active PP5c, an inhibitor-titration based assay to determine the amount of PP5 in solution, and a fluorescent phosphatase assay that can be used to screen chemical libraries and natural extracts for the presence of catalytic inhibitors.

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genomebased phylogenies. IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-Woyke T, Elshahed M. 2019.Genomic characterization of candidate division LCP-89 reveals an atypical cell wall structure, microcompartment production, and dual respiratory and fermentative capacities. Appl Environ Microbiol 85:e00110-19. https://doi .on July 11, 2020 by guest http://aem.asm.org/ Downloaded from Ͻ50% completion and Ͻ10% contamination; MQD, medium-quality draft (SAG/MAG) with Ն50 completion and Ͻ10% contamination; HQD, high-quality draft (SAG/MAG) with Ͼ90% completion, Ͻ5% contamination, and the presence of rRNA opero...