Integration of microbiome and Koch's postulates to reveal multiple bacterial pathogens of whitish muscle syndrome in mud crab, Scylla paramamosain.

BACKGROUND: For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch's postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch's postulates to determinate causative agents of multiple pathogens-one disease. RESULTS: Microbiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch's postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently. CONCLUSIONS: This study fully demonstrated that our research strategy that integrates the microbiome and Koch's postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease. Video Abstract.

Virome Analysis Provides an Insight into the Viral Community of Chinese Mitten Crab Eriocheir sinensis.

Recent advances in viromics have led to the discovery of a great diversity of RNA viruses and the identification of a large number of viral pathogens. A systematic exploration of viruses in Chinese mitten crab (Eriocheir sinensis), one of the most important aquatic commercial species, is still lacking. Here, we characterized the RNA viromes of asymptomatic, milky disease (MD)-affected, and hepatopancreatic necrosis syndrome (HPNS)-affected Chinese mitten crabs collected from 3 regions in China. In total, we identified 31 RNA viruses belonging to 11 orders, 22 of which were first reported here. By comparing viral composition between samples, we observed high variation in viral communities across regions, with most of the viral species being region-specific. We proposed to establish several novel viral families or genera based on the phylogenetic relationships and genome structures of viruses discovered in this study, expanding our knowledge of viral diversity in brachyuran crustaceans. IMPORTANCE High-throughput sequencing and meta-transcriptomic analysis provide us with an efficient tool to discover unknown viruses and explore the composition of viral communities in specific species. In this study, we investigated viromes in asymptomatic and diseased Chinese mitten crabs collected from three distant locations. We observed high regional variation in the composition of viral species, highlighting the importance of multi-location sampling. In addition, we classified several novel and ICTV-unclassified viruses based on their genome structures and phylogenetic relationships, providing a new perspective on current viral taxa.

The microbiome and its association with antibiotic resistance genes in the hadal biosphere at the Yap Trench.

The hadal biosphere, the deepest part of the ocean, is known as the least-explored aquatic environment and hosts taxonomically diverse microbial communities. However, the microbiome and its association with antibiotic resistance genes (ARGs) in the hadal ecosystem remain unknown. Here, we profiled the microbiome diversity and ARG occurrence in seawater and sediments of the Yap Trench (YT) using metagenomic sequencing. Within the prokaryote (bacteria and archaea) lineages, the main components of bacteria were Gammaproteobacteria (77.76 %), Firmicutes (8.36 %), and Alphaproteobacteria (2.25 %), whereas the major components of archaea were Nitrososphaeria (6.51 %), Nanoarchaeia (0.42 %), and Thermoplasmata (0.25 %), respectively. Taxonomy of viral contigs showed that the classified viral communities in YT seawater and sediments were dominated by Podoviridae (45.96 %), Siphoviridae (29.41 %), and Myoviridae (24.63 %). A large majority of viral contigs remained uncharacterized and exhibited endemicity. A total of 48 ARGs encoding resistance to 12 antibiotic classes were identified and their hosts were bacteria and viruses. Novel ARG subtypes mexFYTV-1, mexFYTV-2, mexFYTV-3, vanRYTV-1, vanSYTV-1 (carried by unclassified viruses), and bacAYTB-1 (carried by phylum Firmicutes) were detected in seawater samples. Overall, our findings imply that the hadal environment of the YT is a repository of viral and ARG diversity.

Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply.

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbat S189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.