This protocol describes a step by step method for heterologous expression of SARS-CoV2 Nucleocapsid (N) protein in Escherichia coli. Moreover, this protocol includes steps to purify the N protein to high purity and homogeneity. Thus, purified protein can be used for ligand binding assays and other biochemical experiments.

Microbiome studies are quickly gaining momentum. Since most of the resident microbes (consisting of bacteria, fungi, and viruses) are difficult to culture, sequencing the microbial genome is the method of choice to characterize them. It is therefore important to have efficient methodology for gDNA isolation of gut microbes. Mouse models are widely used to understand human disease etiology while avoiding human ethics-related complications. However, the widely used kit-based methods are costly, and sometimes yields (in terms of quality and quantity) are sub-optimal. To overcome this problem, we developed a straightforward, standardized DNA isolation procedure from mouse cecal content for further microbiome-related studies. The reagents we used to standardize the procedure are readily available even in a not-so-well-equipped laboratory, and the reagents are not expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods.

Additionally, we modified the kit-based method of RNA isolation from the colon tissue sample of the mouse for better yield. Churning the tissue with liquid nitrogen at the beginning of the procedure improves RNA quality and quantity.

Graphical abstract:

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution.

Graphic abstract:

Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion (Steager et al., 2008). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales-Soto et al., 2015); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a “homemade,” environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.

Development of biofilm associated candidemia for patients with implanted biomaterials causes an urgency to develop antimicrobial and biofilm inhibitive coatings in the management of recalcitrant Candida infections. Recently, there is an increase in the number of patients with biofilm formation and resistance to antifungal therapy. Therefore, there is a growing interest to use essential oils as coating agents in order to prevent biomaterial-associated Candida infections. Often high costs, complicated and laborious technologies are used for both applying the coating and determination of the antibiofilm effects hampering a rapid screening of essential oils. In order to determine biofilm formation of Candida on essential oil coated surfaces easier, cheaper and faster, we developed an essential oil (lemongrass oil) coated surface (silicone-rubber) by using a hypromellose ointment/essential oil mixture. Furthermore, we modified the “crystal violet binding assay” to quantify the biofilm mass of Candida biofilm formed on the lemongrass oil coated silicone rubber surface. The essential oil coating and the biomass determination of biofilms on silicone rubber can be easily applied with simple and accessible equipment, and will therefore provide rapid information about whether or not a particular essential oil is antiseptic, also when it is used as a coating agent.

Spherical zoospores of a rare actinomycete, Actinoplanes missouriensis, adhere to various hydrophobic solid surfaces by means of type IV pili. The purpose of this protocol is to provide detailed descriptions of the preparation of A. missouriensis zoospores and an assay for the adhesion of the zoospores to solid surfaces. This adhesion assay, which measures numbers of zoospores that adhered to the dish surface and swimming zoospores in a tunnel chamber by using a phase-contrast microscope, can also be used for swimming cells of other microorganisms.

Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although many ssDNA detection technologies exist, almost all methods have been developed for eukaryotic genomes. To apply these to bacterial genomes, which harbor a smaller amount of DNA than those of eukaryotes, more efficient, new methods are required. Therefore, we developed a novel strand-specific ssDNA sequencing method, called 4S-seq, for the bacterial genome. The 4S-seq method enriches ssDNA in the extracted genomic DNA by a dsDNA-specific nuclease and implements a strand-specific library using a biotin label with a customized tag. As a result, the 4S-seq is able to calculate the ssDNA content in each strand (Watson/Crick) at each position of the genome efficiently.

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. To be able to eliminate such biofilms, it is important to understand the key bacterial factors that contribute to their formation. A common practice in the lab setting is to study biofilms grown in laboratory media. However, these media do not fully reflect the host environment conditions, potentially masking relevant biological determinants. This is the case during urinary catheterization, where a key element for Enterococcus faecalis and Staphylococcus aureus colonization and biofilm formation is the release of fibrinogen (Fg) into the bladder and its deposition on the urinary catheter. To recapitulate bladder conditions during catheter-associated urinary tract infection (CAUTI), we have developed a fibrinogen-coated catheter and 96-well plate biofilm assay in urine. Notably, enterococcal biofilm factors identified in these in vitro assays proved to be important for biofilm formation in vivo in a mouse model of CAUTI. Thus, the method described herein can be used to uncover biofilm-promoting factors that are uniquely relevant in the host environment, and that can be exploited to develop new antibacterial therapies.

Biogenic volatile compounds (VCs) mediate various types of crucial intra- and inter-species interactions in plants, animals, and microorganisms owing to their ability to travel through air, liquid, and porous soils. To study how VCs produced by Verticillium dahliae, a soilborne fungal pathogen, affect plant growth and development, we slightly modified a method previously used to study the effect of bacterial VCs on plant growth. The method involves culturing microbial cells and plants in I plate to allow only VC-mediated interaction. The modified protocol is simple to set up and produces reproducible results, facilitating studies on this poorly explored form of plant-fungal interactions. We also optimized conditions for extracting and identifying fungal VCs using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS).

Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide gel electrophoresis (PAGE) method for both WTA and LTA. To extract crude WTA, the peptidoglycan sacculus is first isolated and WTA is then liberated by hydrolysis. LTA is extracted by 1-butanol and pre-treated with lipase to prevent aggregation and improve single-band resolution by PAGE. Crude extracts of both TAs are then subjected to PAGE followed by Alcian blue and silver staining. These protocols are easily adoptable by laboratories interested in rapidly analyzing TAs and can be used determine the relative abundance, relative polymer length and whether TAs are glycosylated. More detailed TA structural and compositional information can be obtained using the described purification protocols by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) analysis.