Outer membrane vesicles can contribute to cellulose degradation in Teredinibacter turnerae, a cultivable intracellular endosymbiont of shipworms.

Teredinibacter turnerae is a cultivable cellulolytic Gammaproeteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose, and pectin and contribute to lignocellulose digestion in the shipworm gut. However, the mechanism by which symbiont-made enzymes are secreted by T. turnerae and subsequently transported to the site of lignocellulose digestion in the shipworm gut is incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce outer membrane vesicles (OMVs) that contain a variety of proteins identified by LC-MS/MS as carbohydrate-active enzymes with predicted activities against cellulose, hemicellulose, and pectin. Reducing sugar assays and zymography confirm that these OMVs retain cellulolytic activity, as evidenced by hydrolysis of CMC. Additionally, these OMVs were enriched with TonB-dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations suggest potential roles for OMVs in lignocellulose utilization by T. turnerae in the free-living state, in enzyme transport and host interaction during symbiotic association, and in commercial applications such as lignocellulosic biomass conversion.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

An aquatic environmental DNA filtration system to maximize recovery potential and promote filtration approach standardization.

Background: Aquatic environmental DNA (eDNA) has emerged as a promising approach to identify organisms in freshwater and marine environments. While the recovery of eDNA from water most commonly involves capture of biological debris on a filter matrix, practitioners are yet to converge on standardized approaches for filtration, particularly in the field. This lack of standardization has resulted in inconsistent handling of samples following collection, limiting interpretation of results across studies and burdening groups with inconvenient storage and transport logistics that may compromise eDNA integrity. Methods: A simple to assemble and low-cost ($350 USD) water filtration system is demonstrated that can be used in field and laboratory settings to reduce time between sample acquisition and eDNA filtration, maximizing eDNA sample recovery. Quantitative PCR is used to show the utility of the platform for laboratory and marine eDNA analysis. Results: The resulting eDNA collection system is easily transported in a rugged water-resistant case, operates for more than eight hours on a 12-volt lead-acid battery, and has an unobstructed filtration rate of 150.05 ± 7.01 mL/min and 151.70 ± 6.72 mL/min with 0.22 µm and 0.45 µm Sterivex filters, respectively. We show that immediate sample filtration increases eDNA recovery in the laboratory, and demonstrate collections in aquaria and marine environments. We anticipate that providing easy to obtain, open hardware designs for eDNA sample collection will increase standardization of aquatic eDNA collection methods and improve cross-study comparisons.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

Complete Genome Sequence of wAna, the Wolbachia Endosymbiont of Drosophila ananassae.

Here, we present the complete genome sequence of the Wolbachia endosymbiont wAna, isolated from Drosophila ananassae and derived from Oxford Nanopore and Illumina sequencing. We anticipate that this will aid in Wolbachia comparative genomics and the assembly of D. ananassae specifically in regions containing extensive lateral gene transfer events.