Clostridioides difficile SpoVAD and SpoVAE interact and are required for DPA packaging into spores

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., dry heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is packaged into the spore core, thereby replacing the majority of water. DPA is synthesized by the mother cell and its packaging into the spore core is regulated by the spoVA operon that has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA packaging into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC / SpoVAD / SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA packaging into the spore

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation

The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called ‘lipid rafts’, in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.

Luciferase of the Japanese syllid polychaete Odontosyllis umdecimdonta

1AbstractOdontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.3HighlightsThe polychaete O. undecimdonta uses a luciferin-luciferase bioluminescence systemO. undecimdonta bioluminescence does not require additional cofactorsThe luciferase of the Japanese fireworm is 329 amino acids longRecombinant luciferase is not secreted when expressed in human cellsExogenous luciferin does not seem to penetrate cell membranes-only lysate luminescesThe luciferase transcript is supported by full-length cDNA reads with 5’ and 3’ UTR