Genome-Scale Metabolic Network Reconstruction of Thermotoga sp. Strain RQ7

Thermotoga are anaerobic hyperthermophiles that have a deep lineage to the last universal ancestor and produce biological hydrogen gas accompanying cell growth. In recent years, systems level approaches have been used to elucidate their metabolic capacities, by integrating mathematical modeling and experimental results. To assist biochemical engineering studies of T. sp. strain RQ7, this work aims at building a metabolic model of the bacterium that quantitatively simulates its metabolism at the genome scale. The constructed model, RQ7_iJG408, consists of 408 genes, 692 reactions, and 538 metabolites. Constraint-based flux balance analyses were used to simulate cell growth in both complex and defined media. Quantitative comparison of the predicted and measured growth rates resulted in good agreements. This model serves as a foundation for an integrated biochemical description of T. sp. strain RQ7. It is a useful tool in designing growth media, identifying metabolic engineering strategies, and exploiting physiological potentials of this biotechnologically significant organism

Using native Lake Erie bacteria and their enzymes for degradation/removal of microcystin toxins from water

Microcystin-LR (MC-LR) is one of the most toxic and common cyanotoxins released by freshwater harmful algal blooms (HABs). While conventional water treatment practices can reduce/remove MC-LR from drinking water, bioremediation (i.e., biofilters) has additional advantages of being cost-effective and environmentally friendly. We previously isolated Lake Erie bacteria that degraded MC-LR into non-toxic fragments but the MC-LR degradation enzymes remain unknown. As such, the goal of this study was to use genomic sequencing and transcriptomics to identify MC-LR degrading genes/enzymes for water treatment. First, whole genome sequencing was performed on five MC-LR degrading bacteria, designated as ‘Group N,’ to predict potential MC-LR degrading genes/enzymes. Next, Group N bacteria were grown in sterile-filtered lake water, with or without MC-LR, and samples were collected every 3-4 days for either transcriptomic (RNA sequencing) or mass spectrometry analyses (MC-LR quantitation and fragment analysis). Bioinformatic analysis of genome sequences identified potential amidohydrolase, dipeptidase, leucyl aminopeptidase, peptidase, serine hydrolase, metallopeptidase, and serine hydrolase enzymes that could cleave MC-LR. Mass spectrometry analysis confirmed that Group N bacteria degraded cyclic MC-LR into non-toxic linear and tetrapeptide fragments. Finally, transcriptomic analysis is currently underway to identify genes upregulated in MC-LR containing cultures (compared to cultures without MC-LR). Putative MC-LR degrading enzymes will be recombinantly expressed/purified and MC-LR degradation confirmed by in vitro assays. These studies have the potential to reveal new methods to efficiently, safely, and cost-effectively remove MC-LR from water sources

Adapted laboratory evolution of Thermotoga sp. strain RQ7 under carbon starvation.

ObjectiveAdaptive laboratory evolution (ALE) is an effective approach to study the evolution behavior of bacterial cultures and to select for strains with desired metabolic features. In this study, we explored the possibility of evolving Thermotoga sp. strain RQ7 for cellulose-degrading abilities.ResultsWild type RQ7 strain was subject to a series of transfers over six and half years with cellulose filter paper as the main and eventually the sole carbon source. Each transfer was accompanied with the addition of 50 μg of Caldicellulosiruptor saccharolyticus DSM 8903 genomic DNA. A total of 331 transfers were completed. No cellulose degradation was observed with the RQ7 cultures. Thirty three (33) isolates from six time points were sampled and sequenced. Nineteen (19) of the 33 isolates were unique, and the rest were duplicated clones. None of the isolates acquired C. saccharolyticus DNA, but all accumulated small-scale mutations throughout their genomes. Sequence analyses revealed 35 mutations that were preserved throughout the generations and another 15 mutations emerged near the end of the study. Many of the affected genes participate in phosphate metabolism, substrate transport, stress response, sensory transduction, and gene regulation

Identification and Quantification of Degradation Products of Microcystins using High-Resolution UHPLC-Orbitrap-MS

Sharmila I. Thenuwara1, Jyotshana Gautam2, Samuel Simpson1, Johnna Birbeck3, Judy A. Westrick3, Jason F. Huntley2, Dragan Isailovic1 1Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606 2Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614 3Lumigen Instrumentation Center, Department of Chemistry, Wayne State University, Detroit, MI 48202 Microcystins (MCs) are heptapeptides produced by freshwater harmful algal blooms (HABs). Exposure to hepatotoxic MCs is a threat to humans and animals. Although conventional municipal water treatment processes can treat MC contaminated water, biodegradation of cyanotoxins using indigenous bacteria is cost-effective and environmentally friendly. We previously isolated and characterized five bacterial isolates from Lake Erie that degraded MC-LR into non-toxic fragments (Thees et al. 2018). Herein, degradation of MCs that are abundant in Lake Erie HABs, MC-LR and MC-RR, is investigated qualitatively and quantitatively using high-resolution LC-MS. UHPLC-Selected ion monitoring (SIM)-Orbitrap-MS analysis revealed two peaks at different retention times corresponding to m/z of a tetrapeptide degradation product. Fragmentation spectra of both peaks showed characteristic ADDA fragment with m/z 135.08. From the MS/MS spectrum, it was concluded that the peak with the shorter retention time than the substrate is linear tetrapeptide. These results indicate that the MC biodegradation mechanisms in Lake Erie bacteria may be distinct from those in other MC degrading bacteria. The enzymatic pathways and MC breakdown products are being investigated further

Probiotic Protects Kidneys Exposed to Microcystin-LR

Cyanobacterial Harmful Algal Blooms (CyanoHABs) occur when colonies of photosynthetic bacteria called cyanobacteria grow out of control, usually in warm, nutrient-rich, slow-moving waters. They are becoming increasingly prevalent around the world and release harmful toxins called cyanotoxins into bodies of water, which negatively affect human and ecological health. One such cyanotoxin is microcystin, with microcystin-leucine arginine (MC-LR) being the most widespread. Exposure to MC-LR inhibits serine and threonine protein phosphatase 1 and 2A in humans, causing a myriad of health problems. Fortunately, certain naturally occurring bacteria may be able to degrade MC-LR and reverse its effects. Mice were separated into five experimental groups based on three types of pre-treatments (control drinking water/vehicle, probiotic-supplemented drinking water, and heat-inactivated probiotic-supplemented drinking water) as well as two types of exposures (microcystin-LR and water/vehicle). RNA was extracted from kidneys for sequencing because MC-LR exacerbates kidney disease. Gene expression data were analyzed with 3 Pod Reports, an R package that produces a three-part report consisting of Gene Set Enrichment Analysis (GSEA), EnrichR, and integrative LINCS (iLINCS). MC-LR exposure was associated with upregulated cellular respiration and metabolism pathways and downregulated transcription pathways. Probiotic pre-treatment combined with MC-LR exposure was associated with upregulated lipoprotein particle pathways and downregulated respiration and ribosome pathways. Overall, the probiotic mixture corrected the transcriptional profile resulting from MC-LR exposure. Future high yield pathways that could be targeted for therapeutic benefit include VEGFR inhibitors and increased expression of renal kidney indicator genes such as EGFR

Cyanotoxin Degrading Lake Bacteria Significantly Alleviate Microcystin-LR Induced Hepatotoxicity in Both In Vitro and In Vivo Models

Our recent reports have shown that exposure to microcystin-LR (MC-LR) exacerbates the development of pre-existing liver and inflammatory bowel disease as well as alters gut microbiota that may significantly impact development of hepatotoxicity. We have isolated naturally occurring novel MC-LR degrading bacteria from Lake Erie, OH and hypothesized that they may alleviate MC-LR toxicity. qPCR analysis for markers of hepatotoxicity and inflammation in both in vivo and in vitro (using human Hep3B hepatocytes) settings showed significant downregulation in their expression in presence of MC degrading bacteria compared to the untreated groups. LC-MS analysis of the 24-hour urine samples in an in vivo setting with age matched Balb/c female mice that were pre-treated with the bacteria prior to 500 μg/kg MC-LR exposure for 24 hrs revealed significant reduction in urine MC-LR levels of mice pre-treated with MC-LR degrading bacteria as compared to the control group. Analysis of genes related to MC-LR induced apoptosis, DNA damage, ER stress, and fatty acid metabolism were also significantly downregulated in mice treated with MC degrading bacteria compared to control mice exposed to the toxin alone. These results suggest a potential novel therapeutic approach that can be developed for MC-LR induced toxicity