Bacterial Gene Research Articles

Effect of the Inoculum-to-Substrate Ratio on Putative Pathogens and Microbial Kinetics during the Batch Anaerobic Digestion of Simulated Food Waste.

The effects of the inoculum (anaerobic digestion effluent) to substrate (simulated food waste) ratio (ISR) 4.00 to 0.25 on putative pathogens and microbial kinetics during batch mesophilic anaerobic digestion were investigated. Red fluorescent protein labelled (RFPAKN132) Escherichia coli JM105 was introduced as a marker species, and together with the indigenous Clostridium sp., Enterococcus sp., Escherichia coli, and total coliforms were used to monitor pathogen death kinetics. Quantitative polymerase chain reaction was also used to estimate the bacterial, fungal, and methanogenic gene copies. All the ISRs eliminated E. coli and other coliforms (4 log10 CFU/mL), but ISR 0.25 achieved this within the shortest time (≤2 days), while ISR 1.00 initially supported pathogen proliferation. Up to 1.5 log10 CFU/mL of Clostridium was reduced by acidogenic conditions (ISR 0.25 and 0.50), while Enterococcus species were resistant to the digestion conditions. Fungal DNA was reduced (≥5 log10 copies/mL) and was undetectable in ISRs 4.00, 2.00, and 0.50 at the end of the incubation period. This study has demonstrated that ISR influenced the pH of the digesters during batch mesophilic anaerobic digestion, and that acidic and alkaline conditions achieved by the lower (0.50 and 0.25) and higher (4.00 and 2.00) ISRs, respectively, were critical to the sanitisation of waste.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants

BackgroundMicroorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning.ResultsHere, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter.ConclusionsOur study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs.9DpTXjuT--dysnV-FwKdBeVideo

Soil microbial gene abundance rather than diversity and network complexity predominantly determines soil multifunctionality in Tibetan alpine grasslands along a precipitation gradient

Abstract The relationship between biodiversity and ecosystem functioning has mainly focused on plant communities, with comparably little known about soil microbial‐driven ecosystem functions. Climate change severely threatens soil microbial roles, but how soil microbial communities determine soil multifunctionality under climate change is poorly understood. Here, we evaluated the effects of diverse bacterial and fungal properties, including microbial gene abundance, diversity and network complexity, on soil multifunctionality (nine soil functions) across a 3000 km transect along a natural precipitation gradient in Tibetan alpine grasslands. Variation partitioning analyses were performed to disentangle the relative importance of bacterial and fungal properties to the variation of soil multifunctionality. Moreover, structural equation modelling was adopted to explore the influencing pathways of precipitation‐induced changes in plant and edaphic factors to soil microbial properties and, consequently, soil multifunctionality. Soil multifunctionality was positively associated with bacterial and fungal gene abundance, diversity and network complexity. Microbial gene abundance was the more important driver influencing soil multifunctionality than microbial diversity and network complexity. In addition, microbial gene abundance was mainly determined by precipitation‐induced changes in soil pH. Meanwhile, the effects of bacterial properties on soil multifunctionality were much larger than those of fungi. Soil multifunctionality was closely associated with different bacterial (cellulolysis, ligninolysis, nitrogen reduction, denitrification and nitrate fixation etc.) and fungal (soil saprotrophs, arbuscular mycorrhizal and plant pathogens etc.) functional guilds, which exert vital regulations on an array of soil biogeochemical cycling processes. Our results provide the large‐scale evidence of the relative contribution of soil microbial gene abundance, diversity and network complexity to the variation of soil multifunctionality in alpine grasslands with changing precipitation, which is pivotal for understanding microbial roles in modulating and predicting soil multifunctionality under future precipitation changes. Read the free Plain Language Summary for this article on the Journal blog.

Effect of cover crop on soil fertility and bacterial diversity in a banana plantation in southwestern China

A cover crop can be an effective strategy to mitigate soil degradation in crops, such as banana. In this study, soil bacterial diversity, chemical properties, enzymatic activities, and bacterial nutrient-related functional genes were examined in the intercropping area of a banana plantation from 2018 to 2020 that had conventional tillage with bare soil (CT), natural weed cover (WC), which was primarily goosegrass (Eleusine indica (L.) Gaerth), or a cover crop of cultivated Siratro (Macroptilium atropurpureum (DC.) Urb.) (SC). By 2020, SC followed by WC and CT treatments were higher in soil organic matter, available nitrogen, total nitrogen and total phosphorus content as well as increased phosphatase, catalase, invertase and urease activities. Compared to CT and WC treatments, the co-occurrence network of SC treatment had higher modularity index indicating a more stable bacterial community. The composition of community was significantly different between the CT and SC treatments in 2019 and 2020, and the WC treatment in 2020. The relative abundance of fatty acids and lipid biosynthesis genes in the soil bacterial microbiome were significantly higher with SC compared to CT and WC in 2019 and 2020, as well as for carbon and nitrogen functional genes in 2020. However, the relative abundance of nucleoside and nucleoside biosynthesis genes were significantly lower with SC compared to CT and WC in 2020. SC treatment increased the relative abundance of Bradyrhizobium and MND1 related to nitrogen cycle and utilization compared to CT in 2019 and 2020. In general, the major benefits of a Siratro cover crop was increased soil organic matter, nitrogen content, and nitrogen fixing bacteria, as well as a more stable soil bacterial community, indicating that it provided multiple potential benefits to the soil of banana.

Random Amplified Polymorphic DNA (RAPD) Markers Protocol of Bacterial Isolates from Two selected General Hospitals Wastewater (HWW)

In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 103Cfu/ml to 24.0 x 103Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 103 and 18.0 x103 Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways

Comparative identification of WRKY transcription factors and transcriptional response to Ralstonia solanacearum in tomato

In order to study the responses of tomato (Solanum lycopersicum) WRKY TFs to bacterial wilt caused by Ralstonia solanacearum, the most up-to-date genomes and transcriptional profiles were used to identify WRKY TFs in control and infected inbred lines. In total, 85 tomato WRKY TFs were identified and categorized into groups I, IIa + b, IIc, IId + e, and III. These WRKYs, especially those from group IIe, were mainly distributed at chromosome ends and in clusters. More than 45 % and 70 % of tomato WRKYs exhibited intraspecific and interspecific synteny, respectively. Nearly 60 % of tomato WRKYs (mainly in groups I and IIc) formed 73 pairs of orthologs with WRKYs in Arabidopsis and pepper, with Ka/Ks less than 1. Sixteen tomato WRKYs (mainly in groups IIa + b and IIc) responded strongly to biotic stress, and 12 differentially expressed WRKYs (mainly in groups III and IIb) were identified. RT-qPCR revealed that tomato WRKYs could respond to bacterial wilt through positive (predominant) or negative regulation. In particular, the interaction between Solyc03g095770.3 (group III) and Solyc09g014990.4 (group I) may play an important role. In brief, WRKY TFs were comprehensively identified in tomato and several bacterial wilt responsive genes were screened.

Impact of dissolved oxygen levels on N2O emissions and metabolically active bacterial community in biological nitrogen removal

Biological nutrient removal (BNR) is an effective process for removing nitrogen from wastewater using microorganisms under different environmental conditions. However, these processes can result in nitrous oxide (N2O) emission. Dissolved oxygen (DO) levels shape bacterial communities and affect N2O emissions in BNR. Ammonia-oxidizing bacteria (AOB), the main N2O producers in the BNR process, are significantly affected by DO. The bacterial communities responding to DO at the DNA level in BNR have been elucidated. However, the metabolically active microbes are truly participant in the biodegradation process. Thus, it is worth exploring how metabolically active bulk and AOB microbial communities and their N2O production respond to the DO levels. In this study, N2O emission were measured in lab-scale sequencing batch reactors at varying DO levels. High-throughput sequencing of the metabolically active bacterial 16S rRNA and amoA genes was performed. The results indicate that increasing DO levels positively influence metabolically active bacterial biodiversity and AOB relative abundance without increasing N2O emissions. The carbon source significantly affected denitrification and nitrification in activated sludge. Optimal DO levels and adequate carbon sources can effectively reduce N2O accumulation. However, excessive aeration reduces the abundance of Zoogloea, leading to compromised sludge settling. This study revealed the effect of DO on N2O producing microorganisms based on cDNA levels, which provided an important basis for mitigating N2O emission in BNR.

The altered TBI fecal microbiome is stable and functionally distinct.

Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ T cell effector functions

Concentrations of the secondary bile acid, deoxycholic acid (DCA), are aberrantly elevated in colorectal cancer (CRC) patients, but the consequences remain poorly understood. Here, we screened a library of gut microbiota-derived metabolites and identified DCA as a negative regulator for CD8+ Tcell effector function. Mechanistically, DCA suppressed CD8+ Tcell responses by targeting plasma membrane Ca2+ ATPase (PMCA) to inhibit Ca2+-nuclear factor of activated T cells (NFAT)2 signaling. In CRC patients, CD8+ Tcell effector function negatively correlated with both DCA concentration and expression of a bacterial DCA biosynthetic gene. Bacteria harboring DCA biosynthetic genes suppressed CD8+ Tcells effector function and promoted tumor growth in mice. This effect was abolished by disrupting bile acid metabolism via bile acid chelation, genetic ablation of bacterial DCA biosynthetic pathway, or specific bacteriophage. Our study demonstrated causation between microbial DCA metabolism and anti-tumor CD8+ Tcell response in CRC, suggesting potential directions for anti-tumor therapy.

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria.

Functional genomics techniques, such as transposon insertion sequencing and RNA-sequencing, are key to studying relative differences in bacterial mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy numbers along the chromosome. This can lead to artifacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control. Further, relative differences in gene-wise read counts may result from alterations in chromosomal replication dynamics, rather than selection or direct gene regulation. We term this artifact "chromosomal location bias" and implement a principled statistical approach to correct it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated into statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analyzing a ciprofloxacin-treated transposon insertion sequencing data set in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress.IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

Comprehensive analysis of multiple cytokines to stratify uropathogenic Escherichia coli pathogenesis in mouse model of urinary tract infection

PurposeUrinary tract infection (UTI) is one of the most common human bacterial infections primarily caused by uropathogenic E. coli (UPEC). Empiric treatment in UTI cause emergence of multidrug resistance and limit treatment options. Understanding UTI at the molecular level with respect to the causative pathogen as well as subsequent host response pose an absolute necessity towards appropriate clinical management. This study aimed to investigate host cytokine response in mouse UTI model with respect to bacterial colonization and associated virulence gene expression upon infection. MethodMouse UTI model was established with two clinical UPEC isolates E. coli NP105 and E. coli P025. UPEC colonization in bladder and kidney was evaluated by bacterial culture (CFU/ml). Histopathology of the tissues were examined by hematoxylin and eosin staining. PCR and real time PCR were used to detect the incidence and expression of respective bacterial genes. Cytokine concentrations in tissues and sera were evaluated using ELISA. GraphPad prism version 8.0.2 was used for statistical interpretation. ResultHighest bacterial colonization was observed on 7th and 9th day post infection (p.i). in bladder and kidney of mouse infected with E. coli P025 and E. coli NP105 respectively with a distinct difference in relative expression of fimH and papC adhesin genes in vivo. IL-1β level in tissues and sera of E. coli NP105 and E. coli P025 infected mouse was significantly different but the IL-17A, GCSF, TGF-β levels were comparable. ConclusionThese findings show a role of IL1β to stratify pathogenicity of UPEC in mouse UTI model.

Bidirectional Enhancement of Nitrogen Removal by Indigenous Synergetic Microalgal-Bacterial Consortia in Harsh Low-C/N Wastewater.

Conventional microalgal-bacterial consortia have limited capacity to treat low-C/N wastewater due to carbon limitation and single nitrogen (N) removal mode. In this work, indigenous synergetic microalgal-bacterial consortia with high N removal performance and bidirectional interaction were successful in treating rare earth tailing wastewaters with low-C/N. Ammonia removal reached 0.89 mg N L-1 h-1, 1.84-fold more efficient than a common microalgal-bacterial system. Metagenomics-based metabolic reconstruction revealed bidirectional microalgal-bacterial interactions. The presence of microalgae increased the abundance of bacterial N-related genes by 1.5- to 57-fold. Similarly, the presence of bacteria increased the abundance of microalgal N assimilation by 2.5- to 15.8-fold. Furthermore, nine bacterial species were isolated, and the bidirectional promotion of N removal by the microalgal-bacterial system was verified. The mechanism of microalgal N assimilation enhanced by indole-3-acetic acid was revealed. In addition, the bidirectional mode of the system ensured the scavenging of toxic byproducts from nitrate metabolism to maintain the stability of the system. Collectively, the bidirectional enhancement system of synergetic microalgae-bacteria was established as an effective N removal strategy to broaden the stable application of this system for the effective treatment of low C/N ratio wastewater.

Genome mining of Escherichia coli WG5D from drinking water source: unraveling antibiotic resistance genes, virulence factors, and pathogenicity

BackgroundEscherichia coli, a ubiquitous inhabitant of the gut microbiota, has been recognized as an indicator of fecal contamination and a potential reservoir for antibiotic resistance genes. Its prevalence in drinking water sources raises concerns about the potential dissemination of antibiotic resistance within aquatic ecosystems and the subsequent impact on public health. The ability of E. coli to acquire and transfer resistance genes, coupled with the constant exposure to low levels of antibiotics in the environment, underscores the need for comprehensive surveillance and rigorous antimicrobial stewardship strategies to safeguard the quality and safety of drinking water supplies, ultimately mitigating the escalation of antibiotic resistance and its implications for human well-being.MethodsWG5D strain, isolated from a drinking water distribution source in North-West Province, South Africa, underwent genomic analysis following isolation on nutrient agar, anaerobic cultivation, and DNA extraction. Paired-end Illumina sequencing with a Nextera XT Library Preparation kit was performed. The assembly, annotation, and subsequent genomic analyses, including phylogenetic analysis using TYGS, pairwise comparisons, and determination of genes related to antimicrobial resistance and virulence, were carried out following standard protocols and tools, ensuring comprehensive insights into the strain’s genomic features.ResultsThis study explores the notable characteristics of E. coli strain WG5D. This strain stands out because it possesses multiple antibiotic resistance genes, encompassing tetracycline, cephalosporin, vancomycin, and aminoglycoside resistances. Additionally, virulence-associated genes indicate potential heightened pathogenicity, complemented by the identification of mobile genetic elements that underscore its adaptability. The intriguing possibility of bacteriophage involvement and factors contributing to pathogenicity further enriches our understanding. We identified E. coli WG5D as a potential human pathogen associated with a drinking water source in South Africa. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. It further identified WG5D as a potential human pathogen. The occurrence of E. coli WG5D raised the awareness of the potential pathogens and the carrying of antibiotic resistance in drinking water.ConclusionsThe findings of this study have highlighted the advantages of the genomic approach in identifying the bacterial species and antibiotic resistance genes of E. coli and its potential as a human pathogen.

Laboratory tools for the direct detection of bacterial respiratory infections and antimicrobial resistance: a scoping review.

Rapid laboratory tests are urgently required to inform antimicrobial use in food animals. Our objective was to synthesize knowledge on the direct application of long-read metagenomic sequencing to respiratory samples to detect bacterial pathogens and antimicrobial resistance genes (ARGs) compared to PCR, loop-mediated isothermal amplification, and recombinase polymerase amplification. Our scoping review protocol followed the Joanna Briggs Institute and PRISMA Scoping Review reporting guidelines. Included studies reported on the direct application of these methods to respiratory samples from animals or humans to detect bacterial pathogens ±ARGs and included turnaround time (TAT) and analytical sensitivity. We excluded studies not reporting these or that were focused exclusively on bioinformatics. We identified 5,636 unique articles from 5 databases. Two-reviewer screening excluded 3,964, 788, and 784 articles at 3 levels, leaving 100 articles (19 animal and 81 human), of which only 7 studied long-read sequencing (only 1 in animals). Thirty-two studies investigated ARGs (only one in animals). Reported TATs ranged from minutes to 2 d; steps did not always include sample collection to results, and analytical sensitivity varied by study. Our review reveals a knowledge gap in research for the direct detection of bacterial respiratory pathogens and ARGs in animals using long-read metagenomic sequencing. There is an opportunity to harness the rapid development in this space to detect multiple pathogens and ARGs on a single sequencing run. Long-read metagenomic sequencing tools show potential to address the urgent need for research into rapid tests to support antimicrobial stewardship in food animal production.

Warming enhances the negative effects of shrub removal on phosphorus mineralization potential

Shrubs have developed various mechanisms for soil phosphorus utilization. Shrub encroachment caused by climate warming alters organic phosphorus mineralization capability by promoting available phosphorus absorption and mediating root exudates. However, few studies have explored how warming regulates the effects of dominant shrubs on soil organic phosphorus mineralization capability. We provide insights into warming, dominant shrub removal, and their interactive effects on the soil organic phosphorus mineralization potential in the Qinghai-Tibetan Plateau. Real-time polymerase chain reaction was used to quantify the soil microbial phosphatase genes (phoC and phoD), which can characterize the soil organic phosphate mineralization potential. We found that warming had no significant effect on the soil organic phosphate-mineralized components (total phosphate, organic phosphate, and available phosphate), genes (phoC and phoD), or enzymes (acid and alkaline phosphatases). Shrub removal negatively influenced the organic phosphate-mineralized components and genes. It significantly decreased soil organic phosphate mineralization gene copy numbers only under warming conditions. Warming increased fungal richness and buffered the effects of shrub removal on bacterial richness and gene copy numbers. However, the change in the microbial community was not the main factor affecting organic phosphate mineralization. We found only phoC copy number had significant correlation to AP. Structural equation modelling revealed that shrub removal and the interaction between warming and shrub removal had a negative direct effect on phoC copy numbers. We concluded that warming increases the negative effect of shrub removal on phosphorus mineralization potential, providing a theoretical basis for shrub encroachment on soil phosphate mineralization under warming conditions.

Actinomycetota, a central constituent microbe during long-term exposure to diazinon, an organophosphorus insecticide

Microbial biodegradation is a primary pesticide remediation pathway. Despite diazinon is one of the most frequently used organophosphate insecticides worldwide, its effect on soil microbial community remains obscure. We hypothesize that diazinon exposure reshapes microbial community, among them increased microbes may play a crucial role in diazinon degradation. To investigate this, we collected soil from an organic farming environment, introduced diazinon, cultivated it in a greenhouse, and then assessed its effects on soil microbiomes at three distinct time points: 20, 40, and 270 days after treatment (DAT). Results from HPLC showed that the level of diazinon was gradually degraded by 98.8% at 270 DAT when compared with day zero, whereas 16S rRNA gene analysis exhibited a significant reduction in the bacterial diversity, especially at the early two time points, indicating that diazinon may exert selection pressure to the bacteria community. Here, the relative abundance of phylum Actinomycetota increased at 20 and 40 DATs. In addition, the bacterial functional gene profile employing PICRUSt2 prediction also revealed that diazinon exposure induced the genomic function related to xenobiotics biodegradation and metabolism in soil, such as CYB5B, hpaC, acrR, and ppkA. To validate if bacterial function is caused by increased relative abundance in diazinon enriched soil, further bacteria isolation resulted in obtaining 25 diazinon degradation strains out of 103 isolates. Notably, more than 70% (18 out of 25) isolates are identified as phylum Actinomycetota, which empirically confirms and correlates microbiome and PICRUSt2 results. In conclusion, this study provides comprehensive information from microbiome analysis to obtaining several bacteria isolates responsible for diazinon degradation, revealing that the phylum Actinomycetota is as a key taxon that facilitates microbial biodegradation in diazinon spoiled soil. This finding may assist in developing a strategy for microbial detoxification of diazinon, such as using an Actinomycetota rich synthetic community (SynCom).

Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

Iontolysis using an electrically excited solid source of aqueous copper ions compatible with bacterial gene quantification

Iontolysis was demonstrated as a feasible low-voltage and low-frequency technique for bacterial DNA extraction. This was achieved through mild electrical excitation (3 Vpp with a 0.5 VDC offset at 5 Hz sinusoidal signal) of submerged copper thin films serving as a solid source for releasing aqueous copper ions into the bacterial sample upon electrical stimulation. The technique achieved approximately 40 % of the lysing efficiency of bead beating after 5 min. The electrical power consumption per Escherichia coli K12 bacterial sample was approximately 25 mW, making it suitable for use with existing portable analyzers. Its compatibility with the existing intercalating fluorescent dye gene quantification technique was demonstrated with high linearity (R2 = 0.97). Notably, it suggested the minimal presence of interfering species that may have been generated from the iontolysis byproducts. This compatibility is essential for existing bacterial gene quantification techniques to leverage the low electrical power consumption, simplicity of design and manufacturing, and ease of storage and shipping of the proposed iontolysis.

Lacticaseibacillus pabuli sp. nov., isolated from fermented cattle feed.

Strain BSF-3MT is a Gram-stain-positive, non-flagellated, facultative anaerobic and rod-shaped bacterium that was isolated from fermented feed collected at a cattle farm in the Daejeon region of the Republic of Korea. It was studied using polyphasic taxonomic methods. Using 16S rRNA gene sequences and the resulting phylogenetic tree, the strain was primarily identified as a member of the genus Lacticaseibacillus. Strain BSF-3MT contained a chromosome of 2.5 Mbp and a plasmid of 33.4 kbp. The G+C content of genomic DNA was 51.3 mol%. Strain BSF-3MT had the highest ortho-average nucleotide identity value of 73.7 % with Lacticaseibacillus songhuajiangensis 7-19T, its closest relative in the phylogenetic tree based on the 16S rRNA gene sequences and the phylogenomic tree based on up-to-date bacterial core genes. Based on the results of a polyphasic taxonomic study, strain BSF-3MT represents a novel species in the genus Lacticaseibacillus, for which the name Lacticaseibacillus pabuli sp. nov. is proposed. The type strain is BSF-3MT (=KACC 23028T=NBRC 116014T).

Identifying environmental factors affecting the microbial community composition on outdoor structural timber

Timber wood is a building material with many positive properties. However, its susceptibility to microbial degradation is a major challenge for outdoor usage. Although many wood-degrading fungal species are known, knowledge on their prevalence and diversity causing damage to exterior structural timber is still limited. Here, we sampled 46 decaying pieces of wood from outdoor constructions in the area of Hamburg, Germany; extracted their DNA; and investigated their microbial community composition by PCR amplicon sequencing of the fungal ITS2 region and partial bacterial 16S rRNA genes. In order to establish a link between the microbial community structure and environmental factors, we analysed the influence of wood species, its C and N contents, the effect of wood-soil contact, and the importance of its immediate environment (city, forest, meadow, park, respectively). We found that fungal and bacterial community composition colonising exterior timber was similar to fungi commonly found in forest deadwood. Of all basidiomycetous sequences retrieved, some, indicative for Perenniporia meridionalis, Dacrymyces capitatus, and Dacrymyces stillatus, were more frequently associated with severe wood damage. Whilst the most important environmental factor shaping fungal and bacterial community composition was the wood species, the immediate environment was important for fungal species whilst, for the occurrence of bacterial taxa, soil contact had a high impact. No influence was tangible for variation of the C or N content. In conclusion, our study demonstrates that wood colonising fungal and bacterial communities are equally responsive in their composition to wood species, but respond differently to environmental factors.Key points• Perenniporia meridionalis and Dacrymyces are frequently associated with wood damage• Fungal community composition on timber is affected by its surrounding environment• Bacterial community composition on structural timber is affected by soil contact