Motone, K. | bioRxiv Science

bioRxiv2023

Characterization of carotenoid-producing Muricauda sp. strains isolated from the reef-building coral Galaxea fascicularis and subtropical seawater

Bacterial symbionts in corals and coral-associated zooxanthellae are attracting increasing attention as potential probiotics. Some members of the family Flavobacteriaceae are zooxanthellae-associated bacteria, which are known to protect zooxanthellae from stresses such as heat and light by producing carotenoids that reduce reactive oxygen species production. This study characterized two Flavobacteriaceae bacteria, Muricauda sp. strains ORYM1 (NBRC115792) and ORYM2 (NBRC115793), isolated from the reef-building coral Galaxea fascicularis and its surrounding seawater in Okinawa, Japan, respectively. The Muricauda sp. strain ORYM2 was isolated from subtropical seawater, and carotenoid production was assessed using ORYM2 as well as ORYM1, which was previously isolated from G. fascicularis. De novo genome sequencing revealed that both strains contain complete sets of zeaxanthin biosynthesis genes, similar to those found in other Muricauda spp. Thin-layer chromatography and high-performance liquid chromatography analyses demonstrated that ORYM1 and ORYM2 produce several carotenoids. The bacterial strains and carotenoids identified in this study provide insights into the biological roles of zooxanthellae-associated bacteria in protecting zooxanthellae and reef-building corals from environmental stresses. Statements and declarationsCompeting interests: The authors declare no competing interests.

microbiology↗

bioRxiv2023

Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we achieve single-amino acid level sensitivity, enabling sequencing of combinations of amino acid substitutions across long protein strands. For greater sequencing accuracy, we demonstrate the ability to reread individual protein molecules, spanning hundreds of amino acids in length, multiple times, and explore the potential for high accuracy protein barcode sequencing. Further, we develop a biophysical model that can simulate raw nanopore signals a priori, based on amino acid volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine intact, folded protein domains for complete end-to-end analysis. These results provide proof-of-concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

biophysics↗