Behavioural phenotyping of C. elegans on UV-killed E. coli mutants v1

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour when killed by ultraviolet (UV) light

Antioxidant rescue of C. elegans behaviour on Keio E. coli mutants v1

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour in the presence of antioxidants.

Setting the Stage: Genes Controlling Mechanosensation and Ca2+ Signaling in Escherichia coli

ABSTRACT Although mechanistic understanding of calcium signaling in bacteria remains inchoate, current evidence clearly links Ca2+ signaling with membrane potential and mechanosensation. Adopting a radically new approach, Luder et al. scanned the Keio collection of Escherichia coli gene knockouts (R. Luder, G. N. Bruni, and J. M. Kralj, J Bacteriol 203:e00509-20, 2021, https://doi.org/10.1128/JB.00509-20) to identify mutations that cause changes in Ca2+ transients. They identify genes associating Ca2+ signaling with outer membrane biogenesis, proton motive force, and, surprisingly, long-term DNA damage. Their work has major implications for electrophysiological communication between bacteria and their environment.

Genetic analysis of tellurate reduction reveals the selenate/tellurate reductase genes ynfEF and the transcriptional regulation of moeA by NsrR in Escherichia coli

Several bacteria can reduce tellurate into the less toxic elemental tellurium, but the genes responsible for this process have not yet been identified. In this study, we screened the Keio collection of single-gene knockouts of Escherichia coli responsible for decreased tellurate reduction and found that deletions of 29 genes, including those for molybdenum cofactor (Moco) biosynthesis, iron-sulfur biosynthesis, and the twin-arginine translocation pathway resulted in decreased tellurate reduction. Among the gene knockouts, deletions of nsrR, moeA, yjbB, ynbA, ydaS, and yidH affected tellurate reduction more severely than those of other genes. Based on our findings, we determined that the ynfEF genes, which code for the components of the selenate reductase YnfEFGH, are responsible for tellurate reduction. Assays of several molybdoenzymes in the knockouts suggested that nsrR, yjbB, ynbA, ydaS, and yidH are essential for the activities of molybdoenzymes in E. coli. Furthermore, we found that the nitric oxide sensor NsrR positively regulated the transcription of the Moco biosynthesis gene moeA. These findings provided new insights into the complexity and regulation of Moco biosynthesis in E. coli.

Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.