Metagenomic analysis reveals the dissemination mechanisms and risks of resistance genes in plateau lakes.

Antibiotic resistance genes (ARGs) are emerging as environmental pollutants that can persist and disseminate in aquatic environments. Lakes, as important sources of freshwater, also serve as potential natural reservoirs of ARGs. In this study, we analyzed the distribution and potential risks of resistance genes in five typical freshwater lakes on the Yunnan-Guizhou Plateau. Our findings revealed that multidrug and MLS ARGs dominated in the studied lakes. Notably, while Lugu Lake exhibited higher abundance of ARGs, mobile genetic elements (MGEs), and metal resistance genes (MRGs), a greater resistome risk was observed in the eutrophic Xingyun Lake. The dissemination processes of ARGs and MRGs are primarily driven by microbial communities and the horizontal gene transfer via MGEs. Limnohabitans, Flavobacterium, and Acinetobacter were identified as key players in the dissemination of ARGs. Our study highlights the persistence of ARGs and provides valuable baseline data and risk assessment of ARGs in plateau freshwater lakes.

Comparative proteomics reveals the mechanism of cyclosporine production and mycelial growth in Tolypocladium inflatum affected by different carbon sources.

Cyclosporine A (CsA) is a secondary cyclopeptide metabolite produced by Tolypocladium inflatum that is widely used clinically as an immunosuppressant. CsA production and mycelial growth differed when T. inflatum was cultured in different carbon source media. During early fermentation, CsA was preferred to be produced in fructose medium, while the mycelium preferred to accumulate in sucrose medium. On the sixth day, the difference was most pronounced. In this study, high-throughput comparative proteomics methods were applied to analyze differences in protein expression of mycelial samples on day 6, revealing the proteins and mechanisms that positively regulate CsA production related to carbon metabolism. The differences included small molecule acid metabolism, lipid metabolism, organic catabolism, exocrine secretion, CsA substrate Bmt synthesis, and transcriptional regulation processes. The proteins involved in the regulation of mycelial growth related to carbon metabolism were also revealed and were associated with waste reoxidation processes or coenzyme metabolism, small molecule synthesis or metabolism, the stress response, genetic information or epigenetic changes, cell component assembly, cell wall integrity, membrane metabolism, vesicle transport, intramembrane localization, and the regulation of filamentous growth. This study provides a reliable reference for CsA production from high-efficiency fermentation. This study provides key information for obtaining more CsA high-yielding strains through metabolic engineering strategies.

Antibiotic resistance genes and their links with bacteria and environmental factors in three predominant freshwater aquaculture modes.

Rapid development of aquaculture industry and increasing demand of various inputs (especially antibiotics), are suspected to promote the occurrence and spread of ARGs in aquaculture related environments. However, the occurrences of ARGs under different freshwater aquaculture practices are rarely known. Here, we investigated the seasonal profiles of the main ARGs, intI1 and bacteria in waters from three kinds of predominant freshwater aquaculture practices around the Honghu Lake (China), as well as their co-occurrences and interrelationships with antibiotics, heavy metals and general water quality. The results indicate that quinolone resistance genes (qnrB), tetracycline resistance genes (tetB and tetX) and sulfonamide resistance genes (sul1 and sul2) were the top five predominant ARGs with seasonal variations of abundance. Fish ponds were of the highest absolute abundances of tested ARGs than the other two modes. Crayfish ponds and their adjacent ditches shared similar ARGs profile. Different subtypes of ARGs belonging to the same class of resistance were varied in abundances. Some bacteria were predicted to carry different ARGs, which indicating multi-antibiotic resistances. Moreover, the combined environmental factors (antibiotics, heavy metals and water quality) partially shaped the profiles of ARGs and bacteria composition. Overall, this study provides new comprehensive understanding on the characterization of ARGs contamination in different freshwater aquaculture practices from the perspectives of environmental chemistry, microbiology and ecology. The results would benefit the optimization of aquaculture practices toward environmental integrity and sustainability.

The Effects of DNA Extraction Kits and Primers on Prokaryotic and Eukaryotic Microbial Community in Freshwater Sediments.

DNA based sequencing technology has revolutionized the field of microbial ecology and environmental studies. However, biases can be introduced at all experimental steps and, thus, affect the interpretation of microbial community. So far, previous studies on the biases introduced from the key steps of DNA extraction and primer sets mainly focused on the bacterial communities in soil or sediment samples, while little is known about the effect on the eukaryotic microbial communities. Here, we studied the effects of three different DNA extraction kits on both prokaryotic and micro-eukaryotic communities by 16S and 18S rRNA gene amplicon sequencing, and further disentangled the influence of primer choice on the micro-eukaryotic communities. Our results showed that the FastDNA SPIN Kit for Soil and DNeasy PowerSoil Kit produced much higher DNA yield with good reproducibility, and observed more eukaryotic OTUs compared to the MinkaGene DNA extraction kit, but all three kits exhibited comparable ability in recovering bacterial alpha diversity. Of the two primer sets, both targeting the V4 region of the 18S rRNA gene, the TAR primer set detected higher number of unique OTUs than the EK primer set, while the EK primer set resulted in longer amplicons and better reproducibility between replicates. Based on our findings, we recommend using the DNeasy PowerSoil Kit with the EK primer set to capture the abundant micro-eukaryotic taxa from freshwater sediment samples. If a more complete picture of the eukaryotic microbial community is desired, the TAR primer set in combination with the FastDNA SPIN Kit is more efficient in this study.

Nitrite and nitrate reduction drive sediment microbial nitrogen cycling in a eutrophic lake.

The anaerobic microbial nitrogen (N) removal in lake sediments is one of the most important processes driving the nitrogen cycling in lake ecosystems. However, the N removal and its underlying mechanisms regulated by denitrifying and anaerobic ammonia oxidation (anammox) bacteria in lake sediments remain poorly understood. With the field sediments collected from different areas of Lake Donghu (a shallow eutrophic lake), we examined the denitrifying and anammox bacterial communities by sequencing the nirS/K and hzsB genes, respectively. The results indicated that denitrifiers in sediments were affiliated to nine clusters, which are involved in both heterotrophic and autotrophic denitrification. However, anammox bacteria were only dominated by Candidatus Brocadia. We found that NO3- and NO2- concentrations, as well as Nar enzyme activity were the key factors affecting denitrifying and anammox communities in this eutrophic lake. The enrichment experiments in bioreactors confirmed the divergence of denitrification and anammox rates with an additional complement of NO2-, especially under a condition low nitrate reductase activity. The coupled denitrification and anammox may play significant roles in N removal, and the availability of electronic acceptors (i.e., NO2- and NO3-) strongly influenced the N loss in lake sediments. Further path analysis indicated that NO2-, NO3- and some N-related enzymes were the key factors affecting microbial N removal in lake sediments. This study advances our understanding of the mechanisms driving the of denitrification and anammox in lake sediments, which also provides new insights into coupled denitrification-anammox N removal in eutrophic lake ecosystems.

Whole Genome Sequence of an Edible Mushroom Stropharia rugosoannulata (Daqiugaigu).

Stropharia rugosoannulata, also known as Daqiugaigu in China, is a well-known edible mushroom that has been widely cultivated in China in recent years. Many studies have focused on its nutrients, bioactive compounds, and lignin degradation capacity, although there are few molecular and genetic breeding studies due to the lack of genomic information. Here, we present the 47.9 Mb genome sequence of an S. rugosoannulata monokaryotic strain (A15), which has 20 contigs and an N50 of 3.64 Mb, which was obtained by a combination of Illumina and Nanopore sequencing platforms. Further analysis predicted 12,752 protein-coding genes, including 486 CAZyme-encoding genes. Phylogenetic analysis revealed a close evolutionary relationship between S. rugosoannulata and Hypholoma sublateritium, Psilocybe cyanescens, and Galerina marginata based on single-copy orthologous genes. Proteomic analysis revealed different protein expression profiles between the cap and the stipe of the S. rugosoannulata fruiting body. The proteins of the stipe associated with carbon metabolism, energy production, and stress-response-related biological processes had higher abundance, whereas proteins involved in fatty acid synthesis and mRNA splicing showed higher expression in the cap than in the stipe. The genome of S. rugosoannulata will provide valuable genetic resources not only for comparative genomic analyses and evolutionary studies among Basidiomycetes but also for alleviating the bottlenecks that restrict the molecular breeding of this edible mushroom.