Summary The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.

SUMMARY As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, β-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotrantslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors.

In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σE stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σE transcription factor, thereby modulating expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.

Proteolytic cascades are widely implicated in signaling between cellular compartments. In Escherichia coli, accumulation of unassembled outer membrane porins (OMPs) in the envelope leads to expression of E -dependent genes in the cytoplasmic cellular compartment. A proteolytic cascade conveys the OMP signal by regulated proteolysis of RseA, a membrane-spanning anti-sigma factor whose cytoplasmic domain inhibits E -dependent transcription. Upon activation by OMP C termini, the membrane localized DegS protease cleaves RseA in its periplasmic domain, the membrane-embedded protease RseP (YaeL) cleaves RseA near the inner membrane, and the released cytoplasmic RseA fragment is further degraded. Initiation of RseA degradation by activated DegS makes the system sensitive to a wide range of OMP concentrations and unresponsive to variations in the levels of DegS and RseP proteases. These features rely on the inability of RseP to cleave intact RseA. In the present report, we demonstrate that RseB, which binds to the periplasmic face of RseA, and DegS each independently inhibits RseP cleavage of intact RseA. Thus, the function of RseB, widely conserved among bacteria using the E pathway, and the second role of DegS (in addition to RseA proteolysis initiation) is to improve the performance characteristics of this signal transduction system.

Proteolytic cascades often transduce signals between cellular compartments, but the features of these cascades that permit efficient conversion of a biological signal into a transcriptional output are not well elucidated. E mediates an envelope stress response in Escherichia coli, and its activity is controlled by regulated degradation of RseA, a membrane-spanning anti-factor. Examination of the individual steps in this protease cascade reveals that the initial, signal-sensing cleavage step is rate-limiting; that multiple ATP-dependent proteases degrade the cytoplasmic fragment of RseA and that dissociation of E from RseA is so slow that most free E must be generated by the active degradation of RseA. As a consequence, the degradation rate of RseA is set by the amount of inducing signal, and insulated from the "load" on and activity of the cytoplasmic proteases. Additionally, changes in RseA degradation rate are rapidly reflected in altered E activity. These design features are attractive as general components of signal transduction pathways governed by unstable negative regulators. Proteolytic cascades are widely used to transduce signals across membranes to enable cells to respond to environmental stress and coordinate processes in different cellular compartments. However, the molecular properties of these cascades that facilitate the desired outputs have rarely been examined. In this work, we examine the individual steps in the protease cascade governing the activity of the E -mediated envelope stress response in Escherichia coli to determine the construction features of this cascade that facilitate faithful transmission of signal and a rapid output.E directs RNA polymerase to transcribe genes encoding proteins that ensure the synthesis, assembly, and homeostasis of outer membrane porins and lipopolysaccharide, the two major components of the unique outer membrane of Gram-negative bacteria (Dartigalongue et al. 2001;Rezuchova et al. 2003;Rhodius et al. 2006). Envelope integrity is required under all growth conditions, and E is an essential transcription factor (De Las Penas et al. 1997a). Perturbations in the integrity and protein-folding state of the envelope caused by temperature upshift, chaperone depletion, or accumulation of unassembled porins increase E activity; conversely, temperature downshift and/or depletion of porins decrease E activity (Mecsas et al. 1993;Hiratsu et al. 1995;Raina et al. 1995;Rouviere et al. 1995;Missiakas et al. 1996;Rouviere and Gross 1996;Ades et al. 2003).The components of the signal transduction system that control E activity are shown in Figure 1. RseA, a membrane-spanning anti-factor, inhibits E activity. The cytoplasmic domain of RseA (RseA 1-108 ) binds to E and its periplasmic domain binds to RseB (De Las Penas et al. 1997b;Missiakas et al. 1997