Microbiology


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Protocols in Current Issue
0 Q&A 172 Views Apr 20, 2025

Inteins are elements translated within host proteins and removed via a unique protein splicing reaction. In this process, the two peptide bonds flanking the intein are rearranged, releasing the intein and leaving a standard peptide bond in its place. Due to their ability to shuffle peptide bonds in a specific and controlled manner, inteins have proven valuable in protein engineering, leading to the development of numerous impactful technologies. In one application, intein-based biosensors link the activity of a host protein to intein excision. Recently, we developed a biosensor to measure protein stability in vivo, in which the removal of an intein-protein fusion is required for antibiotic resistance. In our protocol, cells expressing our biosensor are logarithmically diluted and spotted on agar plates containing increasing levels of antibiotics. Following incubation, quantitative survival curves can be generated. We also developed a dual protein stability sensor where both antibiotic resistance and fluorescence can be used as readouts and demonstrated that co-expression of the chaperonin GroEL can promote survival and fluorescence. Taken together, our novel intein-based biosensor adds to the available tools to measure protein stability within the cellular environment.

0 Q&A 173 Views Apr 20, 2025

Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example—one of the most challenging viruses for generating viral cDNA clones because of the factors listed above.

0 Q&A 148 Views Apr 20, 2025

Quiescence, the temporary and reversible exit from proliferative growth, is a fundamental biological process. Budding yeast is a preeminent model for studying cellular quiescence owing to its rich experimental toolboxes and evolutionary conservation across eukaryotic pathways and processes that control quiescence. Yeast quiescent cells are reported to be isolated by the continuous linear Percoll gradient method and identified by combining different features such as cell cycle, heat resistance, and cell morphology (single cell). Generally, 10–25 mL of Percoll isotonic solution is first obtained by mixing Percoll with NaCl in 12.5–30 mL centrifugal tubes. Then, the gradient is prepared at high speed for 15–60 min. Finally, approximately 2 × 109 cells are collected, overlaid onto the preformed gradient, and centrifuged to obtain distinct cell fractions. This method requires more reagents and samples and special centrifuges and centrifuge tubes. Besides the cost, it is less favorable for experiments that require high-throughput analyses with a small volume of sample each time. The protocol described here aims to solve those problems by combining the use of 2 mL centrifugal tubes with density marker beads. The protocol also focuses on how to optimize the buoyant density distribution of the density gradient solution such that the density bands better match those of different fraction cells. This will help fully separate quiescent and non-quiescent cells. The protocol can be easily adapted to a wide variety of unicellular microbes with different buoyancy density differentiation during cultivation, such as yeast and bacteria.

0 Q&A 343 Views Apr 20, 2025

Xylan is the main component of hemicellulose and consists of a complex heteropolysaccharide with a heterogeneous structure. This framework, in addition to the crystalline structure of cellulosic fibers and the rigidity of lignin, makes lignocellulosic biomass (LCB) highly recalcitrant to degradation. Xylanases are glycoside hydrolases that cleave the β-1,4-glycoside linkages in the xylan backbone and have attracted increasing attention due to their potential uses in various industrial sectors such as pulp and paper, baking, pharmaceuticals, and lignocellulosic biorefining. For decades, the measurement of xylanase activity was based on reducing sugar quantification methods like DNS or Nelson/Somogyi assays, with numerous limitations in terms of specificity and interference from other enzymatic activities. A better alternative is the colorimetric Azo-Xylan assay, which specifically measures the endo-1,4-β-D-xylanase activity. In this study, the Azo-Xylan protocol was adapted from the company Megazyme to determine the enzymatic activity of thermostable xylanases produced by microbial consortia (i.e., microbiomes), aiming to determine biochemical features such as temperature and pH optima, thermostability, and shelf life. This modified approach offers a rapid, cost-effective, and highly specific method for the determination of xylanase activity in complex mixtures, helping the development of a xylanase-based method for the hydrolysis of hard-degrading substrates in bio-based industries.

0 Q&A 113 Views Apr 20, 2025

The ability to efficiently screen plant pathogen effectors is crucial for understanding plant–pathogen interactions and developing disease-resistant crops. Traditional methods are often labor-intensive and time-consuming. Here, we present a robust, high-throughput screening assay using the tobacco mosaic virus–green fluorescent protein (TMV-GFP) vector system. The screening system combines the TMV-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity (both activation and suppression). The biological function of these effectors can be easily evaluated within six days by observing the GFP fluorescence signal using a UV lamp. This protocol significantly reduces the time required for screening and increases the throughput, making it suitable for large-scale studies. The method is versatile, cost-effective, and can be adapted to effectors with immune interference activity from various pathogens.

0 Q&A 128 Views Apr 20, 2025

In molecular diagnosis, DNA extraction kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Previous fast extraction protocols such as detergent-based ones allow fast DNA extraction for nucleic acid amplification tests (NAAT), mainly polymerase chain reaction (PCR). The use of NaOH (dense alkali) to rupture cells and nuclei and destabilize the conformation of DNases might alleviate shortages and costs while retaining enough robustness to treat complicated samples with minimal environmental and logistical footprint. Biological samples are hand-crushed using a pestle in 1.5 mL tubes with 360 μL of 0.2 M NaOH for 3–5 min and incubated at 75 °C for 10 min. For immediate use, 115.2 μL of 1 M Tris (pH 8) and 364.8 μL nuclease-free water are added, and the sample is vortexed for 10 s and spun at 10,000× g for 3 min; then, 700 μL is transferred to a clean microtube. Two serial dilutions follow, and all concentrations are used as templates for PCR. A refined, storable extract can be produced by adding 70 μL of HCl 1 M (instead of Tris-HCl) and one volume of cold isopropanol to the extract for standard precipitation. This method can increase throughput in emergencies by field deployment in resource-limited settings (RLS) or allow benchtop backup in cases of acquisition disruption or sample surge in established facilities. The crude extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing NAAT in resource-limited settings with low costs and waste footprint or during prolonged crises, where supply chain failures may occur. The refined version produces alcohol-precipitated nucleic acids, suitable for both immediate use and for storage or dispatch for spatiotemporally separate analysis while offering much better amplification quality with a small increase in time and minimal increase in expendables/chemicals needed.

Protocols in Past Issues
0 Q&A 130 Views Apr 5, 2025

Laboratory-developed tests (LDTs) are optimal molecular diagnostic modalities in circumstances such as public health emergencies, rare disease diagnosis, limited budget, or where existing commercial alternatives are unavailable, limited in supply, or withdrawn, either temporarily or permanently. These tests reduce access barriers and enhance equitable clinical practice and healthcare delivery. Despite recommendations for the development of nucleic acid amplification tests, procedural details are often insufficient, inconsistent, and arbitrary. This protocol elucidates the methodology used in the development of a fully automated real-time polymerase chain reaction (qPCR)-based test, using the Panther Fusion® Open AccessTM functionality, for the detection of Streptococcus agalactiae in pregnant women, using selectively enriched rectovaginal swabs. In addition, guidelines are provided for oligonucleotide design (primers and TaqMan probes), in silico and in vitro evaluation of design effectiveness, optimization of the physicochemical conditions of the amplification reaction, and result analysis based on experimental designs and acceptance criteria. Furthermore, recommendations are provided for the analytical and clinical validation of the intended use. Our approach is cost-effective, particularly during the design and optimization phases. We primarily used open-source bioinformatics software and tools for in silico evaluations for the test design. Subsequently, the process was manually optimized using a CFX96 Dx analyzer, whose technical specifications and performance are homologous to that of the final platform (Panther Fusion®). Unlike Panther Fusion®, the CFX96 Dx does not require excess volumes of reagents, samples, and evaluation materials (dead volume) to accommodate potential robotic handling-associated imprecisions. The utilization of the CFX96 Dx analyzer represents a strategic approach to enhancing the efficiency of resources and the optimization of time during LDT optimization.

0 Q&A 243 Views Apr 5, 2025

Enteroviruses are abundant pathogens of humans and animals. Their replication is strictly dependent on the conserved, viral AAA+ ATPase 2C. 2C is an oligomerizing, peripheral membrane protein, and its low solubility as recombinant protein has hampered functional studies of the full-length, recombinant protein bound to a membrane. Here, we describe a modification of the classical, ultracentrifugation-based liposome flotation assay optimized to study the interaction of recombinant 2C with membranes and the functions of membrane-bound, full-length recombinant 2C. The assay takes advantage of the high solubility of recombinant 2C while fused to a maltose-binding protein. Removing this solubility-enhancing tag by specific protease cleavage in the presence of liposomes allows 2C to associate with membranes prior to aggregating. Fluorophore labeling of protein and liposomes allows rapid and precise quantitation of 2C’s association with membranes. This assay is adaptable to any peripheral membrane protein that can be fluorophore-labeled and expressed as a solubility-enhancing fusion protein.

0 Q&A 108 Views Apr 5, 2025

Protein palmitoylation is a lipid modification where a palmitoyl group is covalently attached via a thioester linkage to one or more cysteines on a substrate protein. This modification, catalyzed by a group of enzymes named DHHC enzymes after their conserved Asp-His-His-Cys motif, plays a significant role in regulating the localization, stability, and function of a wide range of cellular and viral proteins. By influencing how and where proteins interact within the cell, palmitoylation is essential for various cellular processes, including signaling pathways, membrane dynamics, and protein–protein interactions. Here, we describe the acyl-RAC assay, a biochemical technique designed to specifically enrich and analyze palmitoylated proteins from complex biological samples, such as cell lysates or tissue extracts. The assay begins by reducing and blocking free cysteine thiol groups on proteins, ensuring that only those thiols involved in thioester bonds with palmitates are accessible for downstream analysis. These thioester bonds are then cleaved to release the fatty acids from the cysteines, which are subsequently captured using thiopropyl Sepharose beads that bind to the newly exposed thiol groups. The captured proteins are eluted from the beads by breaking the bond between the thiol and the resin with reducing agents, and the proteins are then analyzed by SDS-PAGE followed by western blotting to identify and quantify them. The acyl-RAC assay's specificity for S-palmitoylated proteins makes it an invaluable tool for exploring this modification. It not only allows for the identification of previously unknown palmitoylated proteins, thereby deepening our understanding of palmitoylation in cellular processes and viral infections, but it also enables quantitative comparisons of protein palmitoylation under different experimental conditions or treatments.

0 Q&A 214 Views Mar 20, 2025

The early detection of meningitis pathogens—including Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella pneumoniae—through point-of-care (POC) systems is essential for mitigating the risk of neurological damage, enhancing patient outcomes, and facilitating prompt clinical decision-making. Nucleic acid amplification testing (NAAT) is a promising tool for improving the diagnosis process of bacterial pathogens associated with brain inflammation. This is due to its high sensitivity, rapidity, and compatibility with portable diagnostic platforms, making it particularly suitable for POC applications. This protocol introduces an innovative diagnostic approach designed to function effectively without the need for advanced laboratory equipment. By leveraging dual-priming isothermal amplification (DAMP), the assay uses custom internal primers to enhance specificity and minimize false results. Brilliant Green is used in this assay for fluorescence detection due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay demonstrated excellent specificity, absence of false positives, sensitivity comparable to loop-mediated isothermal amplification (LAMP), and a high S/B ratio.

0 Q&A 825 Views Mar 20, 2025

Traditional approaches for the detection and differentiation of Bacillus cereus group species often face challenges due to the complexity of genetic discrimination between species. In this protocol, we propose a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay incorporates a universal fluorescent reporter and four DNA binding fragments, three of which are responsible for “opening up” the folded rRNA while the fourth strand is responsible for detecting single nucleotide variation (SNV) with high selectivity. The binding of the DNM to 16S rRNA results in the formation of the 10-23 DNAzyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. The developed biplex assay enables the detection of B. thuringiensis 16S rRNA and B. mycoides at fluorescein and Cy5 channels, respectively. The protocol offers two detection options: one utilizing extracted total RNA and the other involving crude cell lysate. The latter enables a fast and straightforward detection after 1.5 h with a hands-on time of ~15 min. The new protocol may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis.

0 Q&A 1196 Views Mar 20, 2025

This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.

0 Q&A 850 Views Mar 20, 2025

Inflammatory bowel disease (IBD) is highly prevalent globally and, in the majority of cases, remains asymptomatic during its initial stages. The gastrointestinal microbiota secretes volatile organic compounds (VOCs), and their composition alters in IBD. The examination of VOCs could prove beneficial in complementing diagnostic techniques to facilitate the early identification of IBD risk. In this protocol, a model of sodium dextran sulfate (DSS)-induced colitis in rats was successfully implemented for the non-invasive metabolomic assessment of different stages of inflammation. Headspace–gas chromatography–mass spectrometry (HS–GC–MS) was used as a non-invasive method for inflammation assessment at early and remission stages. The disease activity index (DAI) and histological method were employed to assess intestinal inflammation. The HS–GC–MS method demonstrated high sensitivity to intestine inflammation, confirmed by DAI and histology assay, in the acute and remission stages, identifying changes in the relative content of VOCs in stools. HS–GC–MS may be a useful and non-invasive method for IBD diagnostics and therapy effectiveness control.

0 Q&A 417 Views Mar 20, 2025

Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.

0 Q&A 232 Views Mar 5, 2025

Microbial biofilms are structured communities of microorganisms embedded in a self-produced extracellular matrix, adhering to surfaces. These biofilms enhance bacterial resistance to antibiotics, immune responses, and environmental stress. Current microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are commonly used to visualize and differentiate biofilms. However, their high cost and complexity often render them impractical. In contrast, simpler methods like crystal violet and Congo red staining are limited in distinguishing bacterial cells from the biofilm matrix. This study introduces a cost-effective, dual-staining method using Maneval’s stain to visualize and differentiate microbial biofilms. It requires only basic equipment and minimal reagents, making it ideal for routine use in clinical diagnosis and microbial research.

0 Q&A 262 Views Mar 5, 2025

Capturing produced, consumed, or exchanged metabolites (metabolomics) and the result of gene expression (transcriptomics) require the extraction of metabolites and RNA. Multi-omics approaches and, notably, the combination of metabolomics and transcriptomic analyses are required for understanding the functional changes and adaptation of microorganisms to different physico-chemical and environmental conditions. A protocol was developed to extract total RNA and metabolites from less than 6 mg of a kind of phototrophic biofilm: oxygenic photogranules. These granules are aggregates of several hundred micrometers up to several millimeters. They harbor heterotrophic bacteria and phototrophs. After a common step for cell disruption by bead-beating, a part of the volume was recovered for RNA extraction, and the other half was used for the methanol- and dichloromethane-based extraction of metabolites. The solvents enabled the separation of two phases (aqueous and lipid) containing hydrophilic and lipophilic metabolites, respectively. The 1H nuclear magnetic resonance (NMR) analysis of these extracts produced spectra that contained over a hundred signals with a signal-to-noise ratio higher than 10. The quality of the spectra enabled the identification of dozens of metabolites per sample. Total RNA was purified using a commercially available kit, yielding sufficient concentration and quality for metatranscriptomic analysis. This novel method enables the co-extraction of RNA and metabolites from the same sample, as opposed to the parallel extraction from two samples. Using the same sample for both extractions is particularly advantageous when working with inherently heterogeneous complex biofilm. In heterogeneous systems, differences between samples may be substantial. The co-extraction will enable a holistic analysis of the metabolomics and metatranscriptomics data generated, minimizing experimental biases, including technical variations and, notably, biological variability. As a result, it will ensure more robust multi-omics analyses, particularly by improving the correlation between metabolic changes and transcript modifications.




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