A Comprehensive Genetic Characterization of Bacterial Motility

Abstract

We have developed a powerful experimental framework that combines competitive selection and microarray-based genetic footprinting to comprehensively reveal the genetic basis of bacterial behaviors. Application of this method to Escherichia coli motility identifies 95% of the known flagellar and chemotaxis genes, and reveals three dozen novel loci that, to varying degrees and through diverse mechanisms, affect motility. To probe the network context in which these genes function, we developed a method that uncovers genome-wide epistatic interactions through comprehensive analyses of double-mutant phenotypes. This allows us to place the novel genes within the context of signaling and regulatory networks, including the Rcs phosphorelay pathway and the cyclic di-GMP second-messenger system. This unifying framework enables sensitive and comprehensive genetic characterization of complex behaviors across the microbial biosphere.

Large mutational target size for rapid emergence of bacterial persistence

“…1B, the fraction of persister cells in the mutant library substantially increases with each cycle of enrichment. To quantify the contribution of every genetic locus to increased persistence, we applied our microarray-based genetic footprinting (10) to the entire population of enriched cells. The hybridization value for each ORF on the array is compared with five hybridizations of the unselected library, yielding a positive z score for favorable mutations and a negative z score for deleterious insertions (10).…”

“…Before use, penicillinase was diluted to 50 U/mL in LB and filter sterilized. Genetic footprinting and subsequent hybridization to DNA spotted arrays were performed as described previously (10).…”

“…We used this experimental framework to identify genes that interact with hipA7 and metG::Tn, each of which confers a persistence frequency 10,000-fold higher than the wild type. The first step in this process involved creating double-mutant libraries in each of these genetic backgrounds (10). However, because metG::Tn already contained a transposon with the same selectable marker used to create libraries, we created a mutant by site-directed recombination to replace 21 nucleotides at the 3′ end of metG with a selectable marker (30) that was removed using Flippase recombination enzyme (31), resulting in a gene product missing seven amino acids at the C-A B C D Fig.…”

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…Although c-di-GMP appears to be a conserved signal, the outputs it regulates are diverse and vary among different systems. These outputs include regulation of extracellular polysaccharide production (5,6), adhesin secretion and localization (7), flagellar function (6,8), and transcription of genes that direct attachment (9). Although c-di-GMP signaling has been established as a conserved modality in biofilm regulation, the specific mechanisms by which the signal is received and acted upon are largely unknown.…”

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

“…E. acetylicum was mutagenized with 2.5-20 mg/mL EMS or N-ethyl-N-nitrosourea (ENU) (Sigma-Aldrich) in PBS for 1 h. Mutant bacteria were inoculated in 0.3% BHI agar plates. After overnight incubation, bacteria at the initial site of inoculation were collected and reinoculated in the center of another 0.3% agar plate to enrich for nonswarming mutants (45). After three rounds of enrichment, individual bacterial colonies were tested in a 96-well plating assay (46) to identify mutants that failed to swarm in lowpercentage agar.…”