Microbiology

The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example.

Sheath blight, caused by Rhizoctonia solani, is a major fungal disease of rice that leads to significant yield losses globally. Conventional inoculation methods often fail to achieve consistent and uniform infection, limiting their applicability in antifungal screening studies. This protocol describes a reliable in planta inoculation method for R. solani using mature sclerotia placed at the internodal region of tillering-stage rice seedlings. The procedure includes step-by-step instructions for seed germination, seedling preparation, pathogen culture, artificial inoculation, and post-infection application of antifungal treatments, including botanical compounds such as Ocimum gratissimum essential oil and thymol. Lesion development is monitored and quantified over time, and data are analyzed statistically to evaluate treatment efficacy. The protocol is optimized for reproducibility, scalability, and compatibility with sustainable disease management approaches. It provides a robust platform for evaluating antifungal agents in a biologically relevant and controlled environment.

The rhizosphere, a 2–10 mm region surrounding the root surface, is colonized by numerous microorganisms, known as the rhizosphere microbiome. These microorganisms interact with each other, leading to emergent properties that affect plant fitness. Mapping these interactions is crucial to understanding microbial ecology in the rhizosphere and predicting and manipulating plant health. However, current methods do not capture the chemistry of the rhizosphere environment, and common plant–microbe interaction study setups do not map bacterial interactions in this niche. Additionally, studying bacterial interactions may require the creation of transgenic bacterial lines with markers for antibiotic resistance/fluorescent probes and even isotope labeling. Here, we describe a protocol for both in silico prediction and in vitro validation of bacterial interactions that closely recapitulate the major chemical constituents of the rhizosphere environment using a widely used Murashige & Skoog (MS)-based gnotobiotic plant growth system. We use the auto-fluorescent Pseudomonas, abundantly found in the rhizosphere, to estimate their interactions with other strains, thereby avoiding the need for the creation of transgenic bacterial strains. By combining artificial root exudate medium, plant cultivation medium, and a synthetic bacterial community (SynCom), we first simulate their interactions using genome-scale metabolic models (GSMMs) and then validate these interactions in vitro, using growth assays. We show that the GSMM-predicted interaction scores correlate moderately, yet significantly, with their in vitro validation. Given the complexity of interactions among rhizosphere microbiome members, this reproducible and efficient protocol will allow confident mapping of interactions of fluorescent Pseudomonas with other bacterial strains within the rhizosphere microbiome.

Xenobiotics, including environmental pollutants such as bisphenols, phthalates, and parabens, are widely present in food, cosmetics, packaging, and water. These compounds can reach the gastrointestinal tract and interact with the gut microbiota (GM), a complex microbial community that plays a key role in host immunity, metabolism, and barrier function. The GM engages in bidirectional communication with the host via the production of bioactive metabolites, including short-chain fatty acids, neurotransmitter precursors, and bile acid derivatives. Dysbiosis induced by xenobiotics can disrupt microbial metabolite production, impair gut barrier integrity, and contribute to the development of systemic disorders affecting distant organs such as the liver or brain. On the other hand, the GM can biotransform xenobiotics into metabolites with altered bioactivity or toxicity. In vitro models of the human GM offer a valuable tool to complement population-based and in vivo studies, enabling controlled investigation of causative effects and underlying mechanisms. Here, we present an optimized protocol for the collection, cryopreservation, and cultivation of human GM under strictly anaerobic conditions for toxicomicrobiomics applications. The method allows the assessment of xenobiotic–GM interactions in a cost-effective and ethically sustainable way. It is compatible with a wide range of downstream applications, including 16S rDNA sequencing, metabolomics, and endocrine activity assays. The protocol has been optimized to minimize oxygen exposure to less than 2 min, ensuring the viability of obligate anaerobes that dominate the gut ecosystem. This approach facilitates reproducible, mechanistic studies on the impact of environmental xenobiotics on human GM.

Salt-tolerant bacteria can enhance plant growth through various mechanisms, including the production of auxin, siderophores, hydrogen cyanide, and the solubilization of insoluble phosphate. This study investigated the production of these growth-stimulating factors by salt- and drought-tolerant bacteria isolated from the arid and saline farmlands of Jiroft. Initially, we screened for bacterial strains that exhibited the highest levels of these factors. We then evaluated their effects on improving the growth indices of cucumber seedlings. Additionally, we optimized the protocols for isolating auxin, siderophores, hydrogen cyanide, and phosphate solubilization, which can also be applied to other host rhizobacteria to assess their growth-promoting compounds.

Fusarium crown rot (FCR), mainly caused by Fusarium pseudograminearum, is a devastating soil-borne disease of wheat that results in severe yield and quality reduction. FCR is characterized by stem base necrosis and whitehead development. In recent years, FCR has escalated in both incidence and severity, emerging as a critical threat to global wheat production, particularly within key cultivation zones such as China's Huang-Huai-Hai Plain. The development of resistant cultivars is an effective and environmentally sustainable strategy for FCR disease control. However, the lack of standardized and reproducible inoculation protocols has hindered the accurate assessment and screening of disease-resistant wheat germplasms. To address this limitation, we established a robust FCR inoculation system utilizing F. pseudograminearum propagated on a millet grain substrate, facilitating rapid and reliable evaluation of both host resistance and fungal pathogenicity. Laboratory validation demonstrated high infection efficiency and strong reproducibility of this method.

Hydrogen cyanide (HCN) is a volatile, nitrogen-containing secondary metabolite produced by various bacterial species, primarily during the idiophase of growth under nutrient-limiting or competitive conditions. It plays a significant ecological role as a biocontrol agent by inhibiting the respiratory enzymes of plant pathogens and modulating microbial competition in the rhizosphere. Although protocols for detecting HCN production have existed for over a century, they have largely remained qualitative and are rarely optimized for quantitative assessment. This is mainly due to the volatile nature of HCN, unidentified stable reference standards, and the absence of a robust, universally accepted protocol that ensures consistency across diverse microbial types. In this study, we present a simplified and efficient colorimetric method to quantify HCN production in both Gram-positive and Gram-negative bacteria. Qualitatively, HCN production was observed by a color change due to the isopurpurate complex. This compound was then eluted and quantified by measuring absorbance at 625 nm. The method uses potassium ferrocyanide as a standard, whose slow dissociation constant enables a stable and controlled release of cyanide ions for calibration, unlike highly dissociative salts like KCN that introduce early volatilization errors. This protocol demonstrated high sensitivity, capable of detecting HCN at concentrations as low as ppm levels, with strong correlation to the standard curve (R² > 0.99). Achieving such sensitivity with other conventional methods, such as gas detection tubes or electrochemical sensors, often requires more sophisticated instrumentation and strict handling conditions. In contrast, this approach offers a cost-effective, reproducible, and user-friendly alternative. While a universally adopted method is still lacking due to standardization challenges and HCN volatility, the proposed protocol marks a significant advancement toward accurate and accessible quantitative assessment in microbiological and agricultural applications.

No specific ecological niche has been identified for Serratia proteamaculans. Different strains of the bacterium have been described as opportunistic pathogens of plants, animals, and humans, as plant symbionts, and as free-living bacteria. This makes S. proteamaculans and its particular strains promising models for research, particularly aimed at studying the role of various genes in interspecific interactions. Genome editing is one of the most significant approaches used to study gene function. However, as each bacterial species has its own characteristics, editing methods often need to be adapted. In this study, we adapted a conventional approach based on homologous recombination—the allelic exchange method—to edit the genome of S. proteamaculans, with the aim of examining the biological role of protealysin. Plasmids for recombination were created using the suicidal vector pRE118, and then an auxotrophic Escherichia coli ST18 strain was used to deliver these plasmids to S. proteamaculans through conjugation. This method is valid and can potentially be used to create knockouts, knockins, and point mutations in the S. proteamaculans genome, without the need to insert a selective marker into the genome.

Oomycetes are a predominantly plant-pathogenic group of organisms often considered and managed as fungi; however, due to their evolutionary divergence from true fungi, many conventional fungicides are ineffective against them. Their unique physiological characteristics make them challenging to work with, highlighting the need for a standardized and reproducible procedure for anti-oomycete assays. Previous studies describe methods to obtain sporulation forms in the laboratory, but there remains a disconnect between spore production and the subsequent screening process for potential biological pesticides based on microbial organic extracts. This protocol bridges that gap by providing a complete and reliable workflow from spore production to screening. In this study, we present an efficient in vitro protocol to identify microbial extracts with activity against Phytophthora capsici and Pythium ultimum. The protocol includes a method for obtaining zoospores of P. capsici and oospores of P. ultimum, followed by a simple and rapid screening assay to detect microbial extracts that inhibit the growth of these pathogens. The extracts are dispensed onto plates in two concentrations and allowed to dry. This facilitates pauses in the protocol and allows for storage of the plates until the biological material is ready for the assay. The protocol’s effectiveness has been validated with these two oomycetes, resulting in the identification of active extracts in both cases. Moreover, it can be adapted to other pathogens.

Candida albicans is the pathogenic fungus that most frequently causes infections in humans. It is part of the microbiota commonly found in the skin, gastrointestinal tract, and vaginal mucosa. However, certain conditions, including immunosuppression, excessive use of antibiotics, hormonal changes, the use of medical devices in patients, and individual nutritional status, promote the development of opportunistic infections caused by this fungus. One of the main fungal structures interacting with the host is the cell wall, which is principally composed of chitin, glucan, and proteins. The cell wall plays key functions for the cell, such as osmotic protection; it is also responsible for cellular shape and acts as a signaling hub in response to environmental changes. Cell wall proteins participate in diverse cellular functions, such as attachment to surfaces and cell wall structure; some possess catalytic or transport activities. In this protocol, we show the methodology for isolating cell wall proteins covalently linked or not to cell wall components that can be previously labeled with [¹⁴C]-L-lysine by the action of the fungal transglutaminase localized in the cell wall. We use an extraction method by mechanical cell disruption and washing with 2 M NaCl, whose ionic strength eliminates contaminating proteins from other organelles, through subsequent serial treatments with SDS, chitinase, and zymolyase.