Microbiology

Light is a double-edged sword: it is essential for life on the planet but also causes cellular damage and death. Consequently, organisms have evolved systems not only for harvesting and converting light energy into chemical energy but also for countering its toxic effects. Despite the omnipresence and importance of such light-dependent effects, there are very few unbiased genetic screens, if any, investigating the mechanistic consequences that visible light has on cells. Baker’s yeast, Saccharomyces cerevisiae, is one of the best annotated organisms thanks to several easily available mutant collections and its amenability to high-throughput genetic screening. However, until recently this yeast was thought to lack receptors for visible light, therefore its response to visible light was poorly understood. Nevertheless, a couple of years ago it was discovered that yeast senses light via a novel and unconventional pathway involving a peroxisomal oxidase, hydrogen peroxide, and a particular type of antioxidant protein, called peroxiredoxin. Here, we describe in detail a protocol for scoring yeast genes involved in the resistance to visible light (400-700 nm) on a genome-wide scale. Because cells in dense cultures shield each other from light exposure, resulting in apparent light resistance, our method involves adaptations to reduce inoculum size under conditions amenable to high-throughput screens, to properly be able to identify light-sensitive mutants. We also describe how to measure growth in the presence of light, including two follow-up validation tests. In this way, this method makes it possible to score light-sensitivity on a genome-wide scale with high confidence.

Graphic abstract:

Overview of strategy for high-throughput determination of yeast growth upon visible light stress.

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.

Bacterial chemotaxis is a motility-based response that biases cell movement toward beneficial molecules, called attractants, and away from harmful molecules, also known as repellents. Since the species of the genus Pseudomonas are characterized by a metabolic versatility, these bacteria have developed chemotactic behaviors towards a wide range of different compounds. The specificity of a chemotactic response is determined by the chemoreceptor, which is at the beginning of the signaling cascade and which receives the signal input. The basic elements of a typical chemoreceptor are the periplasmic ligand binding domain (LBD), responsible for sensing environmental stimuli, and the cytosolic methyl-accepting (MA) domain, that interacts with other components of the cellular signaling cascade. Escherichia coli (E. coli), the traditional model in chemotaxis research, has 5 well-characterized chemoreceptors. However, genome sequence analyses have revealed that many other bacteria possess many more chemoreceptors, some of which with partially overlapping signal profiles. This high number of chemoreceptors complicates their study by the analysis of single chemoreceptor mutants. We have pursued an alternative strategy for chemoreceptor characterization which corresponds to the generation of chimeric receptors composed of the LBD of the chemoreceptor under investigation and the MA domain of an E. coli receptor (Tar). The chimer is then introduced into a chemoreceptor free mutant of E. coli and the chemotaxis of the resulting strain is entirely due to the action of this chimeric receptor. In this publication we describe the use of quantitative capillary and gradient plate assays to study Pseudomonas chemotaxis as well as E. coli strains harboring chimeric receptors.