A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system.

CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.

Structural characterization and prebiotic activity of Bletilla striata polysaccharide prepared by one-step fermentation with Bacillus Licheniformis BJ2022.

Bletilla striata polysaccharide (BP) is one of the main active ingredients in Orchidaceae plant Bletilla striata. BP has a high molecular weight, high viscosity, and complex diffusion, which is not conducive to the absorption and utilization of the human body. For the first time, we produced fermented Bletilla striata polysaccharide (FBP) with a low polymerization degree using Bacillus licheniformis BJ2022 one-step fermentation. FBP was a neutral polysaccharide with the molecular weight of 6790 Da. It was composed of glucose and mannose at a molar ratio of 1:2.7. The glycosidic bonds of FBP were composed of ß-1,4-linked mannose, ß-1,4-linked glucose and ß-1,6-linked mannose according to methylation and NMR analysis. Compared with BP, FBP has a lower viscosity and higher solubility. The scanning electron microscopy results showed that the surface of FBP was porous and honeycomb-like. The rheology properties of FBP solution were close to non-Newtonian fluid. Using in vitro fermentation, we proved that FBP could regulate human gut microbiota and significantly increase the content of Bifidobacterium and Bacteroides. Our results suggested that Bacillus licheniformis fermentation significantly improved the physical and prebiotic properties of FBP. This study provides a new strategy for developing and utilizing Bletilla striata resources in China.

Enrofloxacin-induced transfer of multiple-antibiotic resistance genes and emergence of novel resistant bacteria in red swamp crayfish guts and pond sediments.

Antibiotic resistance genes (ARGs) can be transferred from environmental microbes to human pathogens, thus leading to bacterial infection treatment failures. The aquaculture polluted by over-used antibiotics is considered as a notorious reservoir of ARGs. However, the origin, diachronic changes, and mobility of ARGs under antibiotic exposure in aquaculture systems remain elusive. Our findings showed that enrofloxacin application also increased the relative abundance of various ARGs in addition to quinolone-resistance genes and induced ARG dissemination in crayfish gut and sediment bacteria. Further investigation indicated that the transposase-mediated recombination was the major driver of horizontal gene transfer (HGT) of ARGs under antibiotic stress. Notably, enrofloxacin application also induced the generation of some metagenome-assembled genomes (MAGs) carrying multiple ARGs, which were identified as novel species. Additionally, Enterobacteriaceae constituted a mobile ARG pool in aquaculture. Therefore, aquaculture provides potential wide environmental pathways for generation and spread of antibiotic resistance. Our findings of ARG temporal variations and dissemination pattern in aquaculture with artificial use of antibiotics are critical to the management of antibiotic resistance, which is of great ecosystem and health implications.

Postbiotics from Pichia kudriavzevii promote intestinal health performance through regulation of Limosilactobacillus reuteri in weaned piglets.

Postbiotics are attractive as alternatives to antibiotics for use against post-weaning diarrhea. However, their beneficial mechanisms are largely unknown. In the current study, we first demonstrated that supplementation with 0.5% Pichia kudriavzevii FZ12 postbiotics in the diet significantly reduced diarrhea incidence, promoted growth performance, improved gut health performance, and significantly enriched beneficial bacteria, particularly Lactobacillus spp., in the intestines of weaned piglets. Importantly, we identified a heat- and proteinase K-sensitive component, cytochrome c, of the postbiotics that significantly promoted the growth and biofilm formation of Limosilactobacillus reuteri FP13. We demonstrated the importance of P. kudriavzevii FZ12 postbiotics in improving the intestinal health of a model animal and revealed that cytochrome c is one of the important components of yeast postbiotics. These findings may provide new insights into microbe-postbiotics interplay that can be applied to guidelines for dietary modulation to alleviate weaning-induced diarrhea.

Effects of chlorogenic acid on growth, metabolism, antioxidation, immunity, and intestinal flora of crucian carp (Carassius auratus).

In recent years, with the harm caused by the abuse of antibiotics and the increasing demand for green and healthy food, people gradually began to look for antibiotic alternatives for aquaculture. As a Chinese herbal medicine, leaf extract chlorogenic acid (CGA) of Eucommia ulmoides Oliver can improve animal immunity and antioxidant capacity and can improve animal production performance. In this study, crucian carp (Carassius auratus) was fed with complete feed containing 200 mg/kg CGA for 60 days to evaluate the antioxidant, immuno-enhancement, and regulation of intestinal microbial activities of CGA. In comparison to the control, the growth performance indexes of CGA-added fish were significantly increased, including final body weight, weight gain rate, and specific growth rate (P < 0.01), while the feed conversion rate was significantly decreased (P < 0.01). Intestinal digestive enzyme activity significantly increased (P < 0.01); the contents of triglyceride in the liver (P < 0.01) and muscle (P > 0.05) decreased; and the expression of lipid metabolism-related genes in the liver was promoted. Additionally, the non-specific immune enzyme activities of intestinal and liver tissues were increased, but the expression level of the adenylate-activated protein kinase gene involved in energy metabolism was not affected. The antioxidant capacity of intestinal, muscle, and liver tissues was improved. Otherwise, CGA enhanced the relative abundance of intestinal microbes, Fusobacteria and Firmicutes and degraded the relative abundance of Proteobacteria. In general, our data showed that supplementation with CGA in dietary had a positive effect on Carassius auratus growth, immunity, and balance of the bacteria in the intestine. Our findings suggest that it is of great significance to develop and use CGA as a natural non-toxic compound in green and eco-friendly feed additives.

Developmental, Dietary, and Geographical Impacts on Gut Microbiota of Red Swamp Crayfish (Procambarus clarkii).

Red swamp crayfish (Procambarus clarkii) breeding is an important economic mainstay in Hubei province, China. However, information on the gut microbiota of the red swamp crayfish is limited. To address this issue, the effect of developmental stage, diet (fermented or non-fermented feed), and geographical location on the gut microbiota composition in the crayfish was studied via high-throughput 16S rRNA gene sequencing. The results revealed that the dominant phyla in the gut of the crayfish were Proteobacteria, Bacteroidetes,Firmicutes, Tenericutes, and RsaHF231. The alpha diversity showed a declining trend during development, and a highly comparable gut microbiota clustering was identified in a development-dependent manner. The results also revealed that development, followed by diet, is a better key driver for crayfish gut microbiota patterns than geographical location. Notably, the relative abundance of Bacteroidetes was significantly higher in the gut of the crayfish fed with fermented feed than those fed with non-fermented feed, suggesting the fermented feed can be important for the functions (e.g., polysaccharide degradation) of the gut microbiota. In summary, our results revealed the factors shaping gut microbiota of the crayfish and the importance of the fermented feed in crayfish breeding.

A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.