Project description:Integrons are genetic elements that enable bacterial adaptation by collecting new genes encoded in integron cassettes (ICs) to create a reservoir of adaptive functions. These cassettes typically lack their own promoters and rely on the integron platform for their expression. Integrons, well-known for spreading antibiotic resistance genes in clinically relevant Gram-negative species, include Mobile Integrons (MIs), that transport over 170 resistance genes. In contrast, Sedentary Chromosomal Integrons (SCIs), ubiquitous in Vibrio species, are primarily found within bacterial chromosomes. However, their functions are not related to antimicrobial resistance and are largely unexplored. SCIs, typified by the Superintegron (SI) in Vibrio cholerae, represent ancient and highly variable regions in bacterial genomes. The SI is extensive, housing 179 integron cassettes, mostly with unknown functions. Although 19 cassettes encode toxin-antitoxin (TA) systems, which stabilize the array, the intricacies of the SI are challenging to study due to its size and unique integrase. To investigate the SI's impact on V. cholerae, we developed the SeqDelTA approach, enabling the gradual deletion of the SI. This deletion facilitates the use of standard genetic tools without SI interference. Our in-depth analysis of the resulting ∆SI strain, covering various aspects, demonstrated no significant alterations in V. cholerae's physiology. Despite their extended coevolution, SCIs appear to be genetically isolated from the host genome.
2023-11-23 | GSE247496 | GEO
Project description:Integrons gene cassettes in effluents: from integrons to cassettomics
Project description:Nucleic acids in wastewater provide a rich source of data for detection and surveillance of microbes. We have longitudinally collected 116 RNA samples from a wastewater treatment plant in Berlin/Germany, from March 2021 to July 2022, and 24 DNA samples from May to July 2022. We tracked human astroviruses, enteroviruses, noroviruses and adenoviruses over time to the level of strains or even individual nucleotide variations, showing how detailed human pathogens can be observed using wastewater. For respiratory pathogens, a broad enrichment panel enabled us to detect waves of RSV, influenza, or common cold coronaviruses in high agreement with clinical data. By applying a profile Hidden Markov Model-based search for novel viruses, we identified more than 100 thousand novel transcript assemblies likely not belonging to known virus species, thus substantially expanding our knowledge of virus diversity. Phylogenetic analysis is shown for bunyaviruses and parvoviruses. Finally, we identify Hundreds of novel protein sequences for CRISPR-associated proteins such as Transposase B, a class of small RNA-guided DNA editing enzymes. Taken together, we present a longitudinal and deep investigation into wastewater-derived genomic sequencing data that underlines the value of sewage surveillance for public health, planetary virome research, and biotechnological potential.
Project description:Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions for bacteria. ICs generally encode promoterless genes, whose expression relies on the PC promoter within the integron platform. Cassette arrays are assumed to be operon-like structures in which expression is dependent on the distance to the Pc. This is especially relevant in large sedentary chromosomal integrons (SCIs,) like the ones in Vibrio species. We have identified 29 gene-less cassettes in 4 Vibrio SCIs, and explored whether their function could be related to regulating the transcription of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 resistance cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Additionally, one cassette had an antisense promoter capable of reducing trimethoprim resistance through transcriptional interference. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in large SCIs and their clinical importance if captured by mobile integrons.
Project description:Bio-augmentation could be a promising strategy to improve processes for treatment and resource recovery from wastewater. In this study, the Gram-positive bacterium Bacillus subtilis was co-cultured with the microbial communities present in wastewater samples with high concentrations of nitrate or ammonium. Glucose supplementation (1%) was used to boost biomass growth in all wastewater samples. In anaerobic conditions, the indigenous microbial community bio-augmented with B. subtilis was able to rapidly remove nitrate from wastewater. In these conditions, B. subtilis overexpressed nitrogen assimilatory and respiratory genes including NasD, NasE, NarG, NarH, and NarI, which arguably accounted for the observed boost in denitrification. Next, we attempted to use the the ammonium- and nitrate-enriched wastewater samples bio-augmented with B. subtilis in the cathodic compartment of bioelectrochemical systems (BES) operated in anaerobic condition. B. subtilis only had low relative abundance in the microbial community, but bio-augmentation promoted the growth of Clostridium butyricum and C. beijerinckii, which became the dominant species. Both bio-augmentation with B. subtilis and electrical current from the cathode in the BES promoted butyrate production during fermentation of glucose. A concentration of 3.4 g/L butyrate was reached with a combination of cathodic current and bio-augmentation in ammonium-enriched wastewater. With nitrate-enriched wastewater, the BES effectively removed nitrate reaching 3.2 mg/L after 48 h. In addition, 3.9 g/L butyrate was produced. We propose that bio-augmentation of wastewater with B. subtilis in combination with bioelectrochemical processes could both boost denitrification in nitrate-containing wastewater and enable commercial production of butyrate from carbohydrate- containing wastewater, e.g. dairy industry discharges. These results suggest that B. subtilis bio-augmentation in our BES promotes simultaneous wastewater treatment and butyrate production.
Project description:Laboratory tests with marine flatfish were conducted to investigate associations among gene expression, higher biological responses and wastewater effluent exposure. Previous studies showed molecular responses such as elevated concentrations of plasma estradiol and vitellogenin in wild male hornyhead turbot (Pleuronichthys verticalis). In the present study, male hornyhead turbot were exposed to environmentally realistic (0.5%) and higher (5%) concentrations of chemically enhanced advanced-primary (PL) and full-secondary treated (HTP) effluents from two southern California wastewater treatment plants (WWTP). Hepatic gene expression was examined using a custom low-density microarray. <br><br>
Project description:The transcriptome analysis by the human DNA microarray was applied to evaluate the impacts of whole wastewater effluents from the membrane bioreactors (MBRs) and the activated sludge process (AS), on the biological processes of human hepatoma HepG2 cells. The three conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test) and chemical analysis of the domestic effluent standards were conducted in parallel since they are well-established methods with previous applications to wastewater. A significant variation of effluent quality was sdemonstrated among the tested effluents despite that all effluents met the 40 national effluent standards. The three conventional bioassays supported the result of the transcriptome analysis, indicating the comparable or even higher sensitivity of the new assay. The most superior effluent quality was found in the MBR operated at a relatively long sludge retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 M-NM-<m). In addition, functional analysis of the differentially expressed genes revealed that the effluents made various impacts on the cellular functions, suggesting the transcriptome analysis by DNA microarray as more comprehensive, rapid and sensitive tool to detect multiple impacts of the whole effluents. Moreover, the potential genetic markers were proposed to quantitatively evaluate the treatability of the wastewater effluents. In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2 exposed to the raw wastewater, effluents from three types of membrane bioreactors (MBRs), and the activated sludge process. Wastewater DNA microarray with 8795 human genes. MQ water was used as control. For duplicate, two dishes were prepared for each sample and individually treated in parallel.