Microbiology

The fission yeast Schizosaccharomyces pombe is an excellent genetically tractable model organism used in the study of conserved eukaryotic cellular biology. One genetic tool in the assessment of gene function is the in vivo overexpression of proteins. Existing overexpression tools have limitations of induction kinetics, dynamic range, and/or system-wide changes due to the induction conditions or inducer. Here, I describe the methodology for the use of a plasmid-based long non-coding RNA (lncRNA)-regulated overexpression system that is induced by the addition of thiamine. This system, termed the pTIN-system (thiamine inducible), utilizes the fast repression kinetics of the thiamine-regulated nmt1⁺ promoter integrated with the lncRNA regulated tgp1⁺ promoter. The advantages of the pTIN-system are rapid induction kinetics of gene expression, broad dynamic range, and tunable expression.

Phototrophic microorganisms are frequently engineered to regulate the expression and the activity of targeted enzymes of interest for specific biotechnological and agricultural applications. This protocol describes a method to evaluate the expression of RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) in the model cyanobacterium Synechococcus elongatus PCC 7942, at both the transcript and protein levels by quantitative PCR and Western blot, respectively. We further describe an experimental method to determine photosynthetic activity using an oxygen electrode that measures the rate of molecular oxygen production by cyanobacterial cultures. Our protocol can be utilized to assess the effects of RuBisCO engineering at the metabolic and physiological levels.

Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities. Current methodologies commonly involve static biofilm setups (such as biofilm assays in microplates, bead biofilms, or biofilms on glass-slides) and fluidic flow biofilm systems (such as drip-flow biofilm reactors, 3-channel biofilm reactors, or tubing biofilm reactors). Continuous flow systems take into consideration the contribution of hydrodynamic shear forces, nutrient supply, and physical transport of dispersed cells, which define the habitat for biofilm development in most natural and engineered systems. This protocol describes the assembly of 3 flow-system setups to cultivate Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 model biofilms, including the respective quantitation and observation approaches. The standardized flow systems promise productive and reproducible biofilm experimental results, which can be further modified according to specific research projects.

Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans.

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.

Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis.

Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of ribosome profiling in bacteria has been problematic, we report here a systematically optimized protocol for E. coli that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high magnesium concentration without the use of antibiotics. These improvements eliminate artifacts induced by harvesting cells by centrifugation or filtration and by use of chloramphenicol to arrest translation. These improvements are especially appropriate for studies where the exact position of the ribosome is critical, and not merely the number of ribosomes per message, such as studies aimed at monitoring differences in local elongation rates.

Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast represents a major challenge for single-cell transcriptomics, due to their small size, rigid cell wall, and low RNA content per cell. Because of these technical challenges, yeast-specific scRNA-seq methodologies have recently started to appear, each one of them relying on different cell-isolation and library-preparation methods. Consequently, each approach harbors unique strengths and weaknesses that need to be considered. We have recently developed a yeast single-cell RNA-seq protocol (yscRNA-seq), which is inexpensive, high-throughput and easy-to-implement, tailored to the unique needs of yeast. yscRNA-seq provides a unique platform that combines single-cell phenotyping via index sorting with the incorporation of unique molecule identifiers on transcripts that allows to digitally count the number of molecules in a strand- and isoform-specific manner. Here, we provide a detailed, step-by-step description of the experimental and computational steps of yscRNA-seq protocol. This protocol will ease the implementation of yscRNA-seq in other laboratories and provide guidelines for the development of novel technologies.

Genomics, transcriptomics and metabolomics are powerful technologies for studying microbial interactions. The main drawback of these methods is the requirement for destructive sampling. We have established an alternative but complementary technique based on a microplate system combined with promoter fusions for visualizing gene expression in space and time. Here we provide a protocol for measuring spatial and temporal gene expression of a bacterial reporter strain interacting with a fungus on a solid surface.