微生物学

The study of carbohydrate–protein interactions is crucial for clarifying biological processes and identifying potential drug candidates. However, due to the complex structure of carbohydrates, such as high molecular weight, dynamic flexibility, and high solution viscosity, it is challenging to study their interactions with diverse proteins. Conventional analytical techniques like isothermal titration calorimetry (ITC), X-ray crystallography, molecular dynamics (MD) simulations, and nuclear magnetic resonance (NMR) spectroscopy have limitations in revealing these molecular interactions. Surface plasmon resonance (SPR), an advanced optical biosensor technique, overcomes these limitations. It enables real-time, label-free monitoring of the interaction dynamics between carbohydrates and proteins through a continuous flow over a chip surface. In this study, we utilized SPR-based techniques to explore the interaction of capsular polysaccharides (CPS) of Klebsiella pneumoniae and the enzyme KpACE (K. pneumoniae acetylated capsule esterase). Our SPR-based analytical platform has several advantages, including shorter experimental time, a simulated physiological state, and minimal sample requirements for investigating carbohydrate–protein interactions. This approach expands the applicability scope of SPR technology and provides a valuable tool for a wide range of research. By using SPR, we successfully verified that KpACE acts on the acetyl groups of CPS, demonstrating its enzymatic activity, which is crucial for understanding the pathogenic mechanism of K. pneumoniae and developing potential antibacterial drugs.

The target of rapamycin complex 1 (TORC1) is a highly conserved protein complex whose primary function is to link nutrient availability to cell growth in eukaryotes, particularly nitrogen sources. It was originally identified during the screening of Saccharomyces cerevisiae strains resistant to rapamycin treatment. For its part, S. cerevisiae is well known for being a key model organism in biological research and an essential microorganism for the fermentation of food and beverages. This yeast is widely distributed in nature, with domesticated and wild strains existing. However, little is known about what effects domestication has had on its different phenotypes; for example, how nitrogen sources are sensed for TORC1 activation and what impact domestication has had on TORC1 activation are questions that still have no complete answer. To study the genetic basis of TORC1 activation associated with domestication through approaches such as quantitative trait loci (QTL) mapping or genome-wide association studies (GWAS), and more generally for any study requiring TORC1 activity as a readout for a large number of individuals, it is necessary to have a high-throughput methodology that allows monitoring the activation of this pathway in numerous yeast strains. In this context, the present protocol was designed to assess phenotypical differences in TORC1 activation using a new reporter plasmid, the pTOMAN-G plasmid, specifically designed to monitor TORC1 activation. As a proof of concept, this methodology allowed phenotyping a large population of yeast strains derived from the 1002 Yeast Genomes Project, the most complete catalog of genetic variation in yeasts. This protocol proved to be an efficient alternative to assess TORC1 pathway activation compared to techniques based on immunoblot detection, which, although effective, are considerably more laborious. Briefly, the protocol involves the design and construction of the pTOMAN-G plasmid, which carries a construct containing the firefly luciferase gene (Luc) under the control of the TORC1-regulated RPL26A gene promoter (P_RPL26A). The protocol then details the process for selecting subgroups of yeasts based on their ability to grow under nutrient-limited conditions, using proline as the sole nitrogen source. These yeasts are then transformed with the TOMAN-G plasmid, using two alternative transformation methods. Finally, those yeasts that emit luminescence are selected, whose phenotype for TORC1 activation is measured by a nitrogen-upshift experiment in microculture. This approach, using the pTOMAN-G plasmid, offers a rapid and consistent method for assessing TORC1 signaling pathway activation in a large number of yeast strains, highlighting its usefulness to study the activation of the TORC1 pathway and the domestication process associated with it. In the future, a redesign of the plasmid could extend its use as a reporter tool to monitor the activation of the TORC1 pathway, or other pathways, in other yeast species.

Human intestinal barrier function is crucial for health. Beneficial microbes, such as commensal gut bacteria and probiotics, are known to contribute to the regulation of this barrier function. Interactions between bacteria and human intestinal cells can be analyzed by co-culturing bacteria with mammalian cells in vitro. Here, we describe a method to assess the effect of individual bacterial strains on intestinal barrier function using automated transepithelial electrical resistance (TEER) measurements. Caco-2 cells are used as a model of the intestinal epithelium, as these cells spontaneously differentiate into small intestinal epithelial-like cells characterized by tight junctions between adjacent cells. These cells are seeded on polyester filter inserts and cultured for 17 days to form a differentiated monolayer prior to the co-culture experiment. Bacteria are grown on agar, and a single colony is used to prepare a liquid culture in bacterial broth appropriate for the bacteria of interest. On the day of the co-culture experiment, the bacterial culture is resuspended in cell culture medium at the desired concentration. Inserts are transferred to cellZscope cell modules to enable automated TEER measurements, and the medium in the insert is replaced with cell culture medium containing the bacteria of interest. This method allows for intestinal tight junction barrier function to be assessed non-invasively and in real-time in response to probiotics. The use of the automated cellZscope system eliminates the need for labor-intensive manual TEER measurements, which reduces the variability in data that results from human handling and temperature changes that occur when cells are removed from the incubator.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi’s sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms.

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.

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.

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.

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.

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.