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Highlights d A universal bacterial system for titrating CRISPRi using partially mismatched sgRNAs d Determined expression-fitness relationships of E. coli and B. subtilis essentialome d Expression-fitness relationships are shared within pathways and between homologs d Shared homeostatic constraints underlie the optimization of essential gene expression

The phage shock protein A (PspA) of Escherichia coli stabilizes the cytoplasmic membrane under stress conditions. Here we demonstrate that PspA can form hollow spherical or prolate spheroidal particles of about 30-40 nm diameter with a scaffold-like arrangement of protein subunits at the surface. The ÔPspA-scaffoldÕ is the basic structure that is common to all particles. The PspA-scaffold may be of fundamental importance, as it could allow PspA to stabilize the integrity of membranes through numerous contact points over a large surface area.

SummaryPhage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ 54 enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ 54 -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiledcoil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains.

Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them.

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High-throughput functional genomic technologies are accelerating progress in understanding the diversity of bacterial life and in developing a systems-level understanding of model bacterial organisms. Here we highlight progress in deep-sequencing-based functional genomics, show how phenotyping based on whole genome sequencing is enabling phenotyping in organisms recalcitrant to genetic approaches, and recount the rapid proliferation of functional genomic approaches to non-growth phenotypes, and discuss how advances are enabling genome-scale resource libraries for many different bacteria.

Background:The Tat translocon transports folded proteins across energized prokaryotic cytoplasmic membranes. Results: The N-terminal transmembrane domain of TatA interacts with the membrane-stabilizing Psp machinery. Conclusion: Membrane-stabilization takes place where folded proteins are Tat-dependently translocated. Significance: Membrane stress is directly related to Tat transport.