Bacterial Sources Research Articles

Field conditions for the synergistic increase of biomethane in the goaf of coal mines filled with corn straw

AbstractSynergistic fermentation of coal and corn straw is an effective tool to increase biomethane production. However, a large gap exists between the biomethane production conditions of corn straw filling coal mine goafs and laboratory experiments. In order to determine the effect of the field environment on synergistic biomethane production, biomethane production experiments with coal and corn straw were carried out under different conditions to find the key factors restricting the potential of biomethane production. The obtained results showed that various bacterial sources had significant influences on the biomethane production of coal and corn straw, and domesticated bacterial sources provided fermentation systems with more efficient biomethane production capacities than mine water sources. Biomethane production of coal and corn straw was relatively high under mixed conditions, but it was also promoted under unmixed conditions. Different inorganic minerals had different effects on synergistic biomethane production, which varied. For example, calcite, montmorillonite, and kaolin are common minerals in coal‐bearing strata that significantly enhance synergistic biomethane production of coal and corn straw. However, pyrite was found to significantly inhibit the synergistic biomethane production effect of coal and corn straw. Highly metamorphosed anthracite coal also presented biomethane production potential when stimulated by corn straw as a carbon source. The obtained results revealed the influences of different field conditions on the biomethane production of coal and corn straw and provided a reference for the field application of corn straw filling in coal mine goafs.

Genetic history of Cambridgeshire before and after the Black Death.

The extent of the devastation of the Black Death pandemic (1346-1353) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at the local scale. Here, we report 275 ancient genomes, including 109 with coverage >0.1×, from later medieval and postmedieval Cambridgeshire of individuals buried before and after the Black Death. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. While we detect long-term shifts in local genetic ancestry in Cambridgeshire, we find no evidence of major changes in genetic ancestry nor higher differentiation of immune loci between cohorts living before and after the Black Death.

Immunomodulating Enzymes from Streptococcus pyogenes-In Pathogenesis, as Biotechnological Tools, and as Biological Drugs.

Streptococcus pyogenes, or Group A Streptococcus, is an exclusively human pathogen that causes a wide variety of diseases ranging from mild throat and skin infections to severe invasive disease. The pathogenesis of S. pyogenes infection has been extensively studied, but the pathophysiology, especially of the more severe infections, is still somewhat elusive. One key feature of S. pyogenes is the expression of secreted, surface-associated, and intracellular enzymes that directly or indirectly affect both the innate and adaptive host immune systems. Undoubtedly, S. pyogenes is one of the major bacterial sources for immunomodulating enzymes. Major targets for these enzymes are immunoglobulins that are destroyed or modified through proteolysis or glycan hydrolysis. Furthermore, several enzymes degrade components of the complement system and a group of DNAses degrade host DNA in neutrophil extracellular traps. Additional types of enzymes interfere with cellular inflammatory and innate immunity responses. In this review, we attempt to give a broad overview of the functions of these enzymes and their roles in pathogenesis. For those enzymes where experimentally determined structures exist, the structural aspects of the enzymatic activity are further discussed. Lastly, we also discuss the emerging use of some of the enzymes as biotechnological tools as well as biological drugs and vaccines.

Short- to Medium-Term Corrosion of Mild Steel in Highly Calcareous Seawaters: Effects of Calcium Carbonate Concentration, Coupon Orientation, and Nutrient Addition

Mild steels exposed to marine immersion conditions in which carbonate solubilities are exceeded, leading to deposition of calcareous material, have been shown to exhibit much-reduced corrosion rates compared with corrosion under conditions in which there is no exceedance of solubility limits and therefore no calcareous deposition. However, the differences in corrosion including the potential effect of microbiologically influenced corrosion (MIC) in these conditions have not been examined systematically in the literature. The study reported herein involves short- to medium-term marine immersion exposures (up to 18 month) of mild steel coupons in natural seawater (control) and in environments using the same seawater source dosed with (1) CaCO3, acting as an inorganic particulate suspension representative of that encountered in seawater with elevated calcareous content; and (2) a nitrate salt, acting as an industrial pollutant and bacterial nutrient source. The results show the extent to which the deposition of particulate CaCO3 from suspension in seawater onto and into the rusts reduces the rate of corrosion of mild steel; a rate that was found to be dependent on the coupon orientation within the seawater. Coupons that were permanently buried beneath a cap of settled CaCO3 particles exhibited a 70% reduction in corrosion rate compared with the representative control coupons. The results also show that for these exposure durations and conditions, while iron-related bacteria and sulfate-reducing bacteria populations were identified in high concentrations (respectively ∼1 × 106 cfu/g and ∼1 × 102 to 4 × 106 cfu/g, depending on coupon orientation) within the rust products themselves after 18 month of exposure, there was no clear evidence of MIC. These findings indicate that short and medium-term (nonaccelerated) studies of MIC for mild steel are insufficient or even misleading for the prediction of long-term corrosion rates in these environments.

Accelerating protein-protein interaction screens with reduced AlphaFold-Multimer sampling.

Discovering new protein-protein interactions (PPIs) across entire proteomes offers vast potential for understanding novel protein functions and elucidate system properties within or between an organism. While recent advances in computational structural biology, particularly AlphaFold-Multimer, have facilitated this task, scaling for large-scale screenings remains a challenge, requiring significant computational resources. We evaluated the impact of reducing the number of models generated by AlphaFold-Multimer from five to one on the method's ability to distinguish true PPIs from false ones. Our evaluation was conducted on a dataset containing both intra- and inter-species PPIs, which included proteins from bacterial and eukaryotic sources. We demonstrate that reducing the sampling does not compromise the accuracy of the method, offering a faster, efficient, and environmentally friendly solution for PPI predictions. The code used in this article is available at https://github.com/MIDIfactory/AlphaFastPPi. Note that the same can be achieved using the latest version of AlphaPulldown available at https://github.com/KosinskiLab/AlphaPulldown.

Separation of Bacterial Extracellular Vesicles via High-Performance Anion Exchange Chromatography.

Bacterial extracellular vesicles (BEVs) have extraordinary biotechnological potential, but traditional purification methods lack desirable scalability and commonly co-isolate protein impurities, limiting clinical translation. Anion exchange chromatography (AEC) separates molecules based on differences in net charge and is widely used for industrial biomanufacturing of protein therapeutics. Recently, AEC has recently been applied for purification of EVs from both mammalian and bacterial sources. Since most bacteria produce BEVs with a negative surface membrane change, AEC can potentially be widely used for BEV purification. Here, we describe a method utilizing high-performance AEC (HPAEC) in tandem with size-based tangential flow filtration for improved BEV purification. We have previously found this method can reduce co-isolated protein impurities and potentiate anti-inflammatory bioactivity of probiotic BEVs. Thus, this method holds promise as a scalable alternative for improved BEV purification.

A critical review on intestinal mucosal barrier protection effects of dietary polysaccharides.

Studies have shown that dietary polysaccharides, which are widely present in natural foods, have an important impact on the intestinal mucosal barrier. Dietary polysaccharides can maintain the intestinal barrier function through multiple mechanisms. The intestinal barrier is composed of mechanical, chemical, immune, and biological barriers, and dietary polysaccharides, as a bioactive component, can promote and regulate these four barriers. Dietary polysaccharides can enhance the expression of tight junction proteins and mucins such as occludin-1 and zonula occludens-1 (ZO-1) between intestinal epithelial cells, inhibit inflammatory response and oxidative stress, increase the growth of beneficial bacteria, produce beneficial metabolites such as short chain fatty acids (SCFAs), and promote the proliferation and metabolism of immune cells. Given the critical role of the intestinal mucosal system in health and disease, the protective effects of dietary polysaccharides may be potentially valuable for the prevention and treatment of gut-related diseases. Therefore, it is of great significance to further study the mechanism and application prospects of the intestinal mucosal barrier derived from plant, animal, fungal and bacterial sources.

Dose Determination and Administration of Bacterial Extracellular Vesicles for In Vivo Preclinical Studies.

Essentially all bacteria secrete nano-sized (~20-200nm) bacterial extracellular vesicles (bEVs) loaded with proteins, lipids, glycans, and nucleic acids. bEVs facilitate interactions among cells of the same species, different microbial species, and even with cells of multicellular organisms in the context of colonization or infection. Their interactions with host organism immune cell receptors vary depending on the producing bacterial species and are now being harnessed for the development of bEVs as a potential immunotherapeutic platform. Both basic/mechanistic and preclinical therapeutic development studies are thus increasing in number and require implementation of methods for multiparametric analytical characterization as well as in vivo administration in preclinical animal models of disease. We summarize a variety of analytical methods that can be used to calculate bEV dose for preparations made from diverse bacterial sources (including sterility testing, total protein concentration, particle concentration, and lipopolysaccharide concentration). We also describe basic methodology for intravenous administration of bEV preparations via tail vein injection in laboratory mice. Throughout the description of methodology, we highlight potential pitfalls and alternatives to further equip the reader for troubleshooting should challenges arise. Robust and reproducible characterization is a prerequisite of bEV preparation quality control and consistent dosing during preclinical development. This will allow for more streamlined testing of candidate therapeutic bEVs within a given research laboratory, and furthermore facilitate reproducibility of findings across laboratories.

Optimization of cultural parameters for lipase production by indigenous stenotrophomonas sp. And pseudomonas aeruginosa isolated from lipid-rich environments

The present study aims to identify prospective lipase-producing bacteria and optimize different culture parameters to produce maximum lipase. A total of 37 lipase-positive bacteria were isolated from lipid-rich environments. Among them, strains S1N-2 and S10P-1 were found to have potential and were molecularly identified as Stenotrophomonas species ICMM10 and Pseudomonas aeruginosa BA7823, respectively. For maximum lipase production, the optimum pH was 7 and the temperature was 37ºC. Using these conditions, the isolates were grown on media with additional carbon and nitrogen sources along with different inoculum concentrations. The maximum amount of lipase produced by Stenotrophomonas sp. ICMM10 was 136.47 ± 1.63 U/ml, which is 5-fold higher than before. Pseudomonas aeruginosa BA7823 produced a maximum lipase of 96.33 ± 0.21 U/ml, which was about 9-fold higher than before. These results would provide useful information about lipase production by indigenous isolates, which may be employed as a potential bacterial lipase source of biotechnological interest. Bangladesh J. Bot. 52(4): 979-988, 2023 (December)

The Evaluation of the Effectiveness of Biomineralization Technology in Improving the Strength of Damaged Fiber-Reinforced LWAC.

Concrete cracks and local damage can affect the bond performance between concrete and steel bars, thereby reducing the durability of reinforced concrete structures. Compared with general concrete crack repair methods, biomineralization repair not only has effective bonding capabilities but is also particularly environmentally friendly. Therefore, this study aimed to apply biomineralization technology to repair damaged fiber-reinforced lightweight aggregate concrete (LWAC). Two groups of LWAC specimens were prepared. The experimental group used lightweight aggregates (LWAs) containing bacterial spores and nutrient sources, while the control group used LWAs without bacterial spores and nutrient sources. These specimens were first subjected to compression tests and pull-out tests, respectively, and thus were damaged. After the damaged specimen healed itself in different ways for 28 days, secondary compression and pull-out tests were conducted. The self-healing method of the control group involved placing the specimens in an incubator. The experimental group was divided into experimental group I and experimental group II according to the self-healing method. The self-healing method of experimental group I was the same as that of the control group. The self-healing method of experimental group II involved soaking the specimen in a mixed solution of urea and calcium acetate for two days, and then taking it out and placing it in an incubator for two days, with a cycle of four days. The test results show that in terms of the relative bond strength ratio, the experimental group II increased by 17.9% compared with the control group. Moreover, the precipitate formed at the cracks in the sample was confirmed to be calcium carbonate with the EDS and XRD analysis results, which improved the compressive strength and bond strength after self-healing. This indicates that the biomineralization self-healing method used in experimental group II is more effective.

Quantifying and mapping digital dermatitis–associated bacteria in lesion and nonlesion body sites and dairy farm environment

The source of infection of digital dermatitis (DD), an infectious lameness condition, is still uncertain. In this cross-sectional study, we aimed to identify potential reservoirs of DD bacteria in dairy cattle body sites with different stages of the disease and farm environments. We collected skin swabs from 85 dairy cows from 5 herds, 3 with and 2 without DD, from foot, hock, and udder cleft skin (with lesions or not), saliva, urine, and feces. We also obtained environmental samples. Real-time quantitative PCR targeted Treponema phagedenis, Treponema medium, Treponema pedis, Porphyromonas levii, Bacteroides pyogenes, Fusobacterium necrophorum, and Fusobacterium mortiferum. Digital dermatitis-associated Treponema spp. were exclusively detected in DD-affected herds in DD-foot and other skin lesions, healthy skin, saliva, and environmental samples. In contrast, the non-Treponema spp. were found in samples from both DD-negative and affected herds. As expected, DD lesions had higher bacterial loads than healthy skin. Interestingly, similar counts were observed in udder cleft lesions, indicating a potential opportunistic behavior on compromised skin. None of the targeted species were detected in fecal samples, but P. levii, B. pyogenes, and F. necrophorum were detected in urine. All 7 species were detected in saliva, although in low quantities. No associations were observed between the presence of each bacterial species in DD lesions and urine; however, there was an association between the presence of DD-Treponema spp. in lesions and saliva, hock, and udder skin. Feces and urine do not seem to be a DD bacteria primary source, but saliva and other skin lesions may play a role. Longitudinal studies would improve our understanding of DD-associated bacteria's transient or persistent presence in these sites. Investigating the sources of DD-associated bacteria will guide future interventions to minimize bacterial shedding and transmission, ultimately more effectively reducing bacterial load, transmission, and sources of infection in dairy herds.

Replacement of Dietary Fish Protein with Bacterial Protein Results in Decreased Adiposity Coupled with Liver Gene Expression Changes in Female Danio rerio

BackgroundEffective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. ObjectiveOur objective was to evaluate a bacterial-based single-cell protein (SCP) for the production of open-source standardized diets with defined health characteristics for the zebrafish research community. MethodsWe completed a 16-wk feeding trial using juvenile D. rerio 31 d postfertilization (10 tanks per diet and 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient [standard reference (SR) diet] or a novel bacterial SCP source [bacterial protein (BP) diet]. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio with confirmatory rtPCR) were performed for each diet treatment. ResultsD. rerio fed the BP diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments, suggesting normal physiological function. Genes differentially expressed in female D. rerio fed the BP diet compared with females fed the SR diet were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. ConclusionProtein source substantially affected body growth metrics and composition as well as gene expression. These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Polyoxazoline-Conjugated l-Asparaginase: An Antibody-Production-Free Therapeutic Agent for Acute Lymphoblastic Leukemia.

l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.

Bacterial profile of pork from production to retail based on high-throughput sequencing

Pork is a common vehicle for foodborne pathogens, including Salmonella spp. and Yersinia enterocolitica. Cross-contamination can occur at any stage of the pork production chain, from farm to market. In the present study, high-throughput sequencing was used to characterize bacterial profiles and track their changes along the whole supply chain. Tracked meat samples (pig on the farm, carcass in the slaughterhouse, unprocessed carcass and processed meat in the processing plant, and fresh pork at the local retail stores) and their associated environmental samples (e.g., water, floor, feed, feces, and workers' gloves) were collected from sequential stages (n = 96) and subjected to 16S rRNA metataxonomic analyses. At the farm, a total of 652 genera and 146 exclusive genera were identified in animal and environmental samples (pig, drain, floor, fan, and feces). Based on beta diversity analysis, it was demonstrated that the microbial composition of animal samples collected at the same processing step is similar to that of environmental samples (e.g., drain, fan, feces, feed, floor, gloves, knives, tables, and water). All animal and environmental samples from the slaughterhouse were dominated by Acinetobacter (55.37 %). At the processing plant, belly meat and neck meat samples were dominated by Psychrobacter (55.49 %). At the retail level, key bacterial players, which are potential problematic bacteria and important members with a high relative abundance in the samples, included Acinetobacter (8.13 %), Pseudomonas (6.27 %), and Staphylococcus (2.13 %). In addition, the number of confirmed genera varied by more than twice that identified in the processing plant. Source tracking was performed to identify bacterial contamination routes in pork processing. Animal samples, including the processing plant's carcass, the pig from the farm, and the unwashed carcass from the slaughterhouse (77.45 %), along with the processing plant's gloves (5.71 %), were the primary bacterial sources in the final product. The present study provides in-depth knowledge about the bacterial players and contamination points within the pork production chain. Effective control measures are needed to control pathogens and major pollutants at each stage of pork production to improve food safety.

Highly biocidal poly(vinyl alcohol)-hydantoin/starch hybrid gels: A “Trojan Horse” for Bacillus subtilis

HypothesisPoly (vinyl alcohol) (PVA) cryogels can be functionalized with n-Halamines to confer biocidal features useful for their application as wound-dressing tools. Their efficacy can be boosted by stably embedding a polymeric bacterial food source (e.g., starch) in the gel matrix. The bioavailability of the food source lures bacteria inside the gel network via chemotactic mechanisms, promoting their contact with the biocidal functionalities and their consequent inactivation. ExperimentsThe synthesis of a novel hydantoin-functionalized PVA (H-PVA-hyd) is proposed. The newly synthesized H-PVA-hyd polymer was introduced in the formulation of H-PVA-based cryogels. To promote the cryogelation of the systems we exploited phase-separation mechanisms employing either a PVA carrying residual acetate groups (L-PVA) or starch as phase-segregating components. The permanence of the biocidal functionality after swelling was investigated via proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FT-IR) microscopy. The activated H-PVA-hyd cryogels have been tested against bacteria with amylolytic activity (Bacillus subtilis) and the outcomes were analyzed by direct observation via confocal laser scanning microscopy (CLSM). FindingsThe cryogels containing starch resulted in being the most effective (up to 90% bacterial killing), despite carrying a lower amount of hydantoin groups than their starch-free counterparts, suggesting that their improved efficacy relies on a “Trojan Horse” type of mechanism.

Branched GDGT source shift identification allows improved reconstruction of an 8,000-year warming trend on Sumatra

Branched Glycerol Dialkyl Glycerol Tetraethers (brGDGTs) in sedimentary archives are increasingly used for paleotemperature reconstructions due to their strong correlation with mean annual air temperature. However, environmental factors can influence the brGDGT producing bacterial community, potentially affecting the brGDGT-temperature relationship and introducing fundamental errors in reconstructions. Here we assess the reliability of the methylation index of brGDGTs (MBT′5ME) in sediments as a paleotemperature proxy by tracking provenance differences based on brGDGT fractional abundances in a short lake core, two peat cores and surface soils on Sumatra (n = 333 in total). Then, we attempt to reconstruct the Holocene paleotemperatures on Sumatra using the two peat cores. Our results indicate distinct brGDGT and H-shaped brGDGT (H-GDGT) compositions in soil, lake and peat environments, suggesting production by different bacterial communities. We introduce a new index, the isomerization of H-GDGTs (IRH) that can distinguish between these environments. In an 11,000-year long peat core from Diatas, we find that brGDGT composition changes are dominated by bacterial community shifts rather than temperature changes. In contrast, a core from the nearby Padang peatland can be robustly used for a brGDGT-based paleotemperature reconstruction since there are no signs of past environmental or brGDGT source shifts. The results from Padang indicate a gradual warming trend over the past 8,000 years, consistent with climate model simulations and nearby sea surface temperature reconstructions. However, current MBT′5ME calibrations yield larger warming trends compared to simulations and other proxy studies, suggesting the need for tropical and/or peat-specific brGDGT temperature calibrations. Our findings demonstrate the importance of assessing environmental shifts and bacterial source community changes when employing brGDGT paleothermometry. The methodological framework outlined in this study can be used in future research for reliable down-core brGDGT temperature reconstructions. Our proxy reconstruction over the past 8,000 years offers novel insights into the Holocene temperature evolution from a region with low climate seasonality.

Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11.

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.

Aridification alters the diversity of airborne bacteria in drylands of China

Dryland ecosystems, which vary sensitively to drought owing to climate change and human activities, frequently cause large-scale aeolian dispersal of terrestrial-mineral and biological particles. To date, very few studies have focused on the atmosphere-land exchange of bacteria in drylands. To understand the bacterial transport from the terrestrial environment to the atmosphere over drylands, soil and aerosol samples were collected at 19 sites in arid and semi-arid regions across drylands of China in the summer of 2017. The 16S rRNA gene sequencing revealed that the bacterial diversity in the atmosphere was significantly lower than that in terrestrial ecosystems for all the collected samples. Moreover, bacterial diversity in the atmosphere increases in arid regions compared to semi-arid regions, while those of the terrestrial environment were maintained regardless of the region. Additionally, the terrestrial bacterial sources contributed to the airborne bacteria more significantly in arid regions than those in semi-arid regions (accounting for 10.0 ± 1.1% and 3.5 ± 1.1%, respectively). The arid conditions tended to enhance the atmospheric emissions of Chloroflexi, while the members of Bacteroidetes were dominant in both the atmospheres of the semi-arid and arid regions. The differences in bacterial community structures between the terrestrial and atmospheric ecosystems are thought to depend on the ground surface characteristics such as vegetation and terrestrial substrates. Meteorological drought may alter terrestrial characteristics, which regulate airborne bacterial dispersal from dryland to its downwind regions, suggesting that more attention should be paid to the bacterial roles in the biology-health-climate process in the future.

Land use and rainfall influences on bacterial levels and sources in stormwater ponds.

Urban stormwater runoff is a known source of microbial contamination of stormwater ponds. However, less is known about the influences of land use and rainfall on microbial quality over time in these receiving waters. In this study, two fecal indicator bacteria (FIB), namely Escherichia coli and thermotolerant coliforms, were monitored in three stormwater ponds in Calgary, Alberta, Canada. The stormwater ponds were selected due to their potential as water sources for beneficial uses such as irrigation, which requires lower water quality than drinking water, thereby alleviating the pressure on the city's potable water demands. The selected stormwater ponds vary in size and shape, contribution catchment size, and percentages of several primary land use types. Microbial source tracking for human, dog, seagull, Canada goose, ruminant, and muskrat was also conducted to determine sources of bacterial contamination in the stormwater ponds. Sampling was conducted near the pond surface and adjacent to the shoreline, specifically near the outfalls that discharge stormwater runoff into the ponds and the inlets that convey water out of the ponds. Overall, the FIB concentrations in the vicinity of pond outfalls were significantly or relatively higher than those near pond inlets. The contamination in the McCall Lake and the Country Hills stormwater ponds showed higher amounts of human markers (40 to 60%) compared to the Inverness stormwater pond (< 20%), which coincided with their higher FIB concentration medians. The results revealed that stormwater drained from catchments with a higher percentage of commercial land use was more contaminated than those with primary residential land use, while the impacts of residential development on the FIB levels in the Inverness stormwater pond were not obvious. Furthermore, FIB concentrations in the ponds increased in response to both rain events and inter-event dry periods, with human-specific markers being predominant despite the high levels of animal markers during inter-event dry periods. Human-origin sources might be among the main microbial loading contributors in the pond catchments in general. All these findings can inform the development or improvement of measures for mitigating microbial pollution, strategies for reusing stormwater, and maintenance programs.

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing.

Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.