Summary Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (β′-yGFP) in live E. coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, Nucleoid-ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55,000 ribosomes. Only 10–15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome-rich endcaps. The predominant observed diffusion coefficient of ribosomes is Dribo = 0.04 μm2/s, attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of sub-diffusion, as would arise from tethering of ribosomes. The degree of DNA-ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome-rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.

Quorum sensing is a chemical communication process that bacteria use to regulate collective behaviors. Disabling quorum-sensing circuits with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. The human pathogen Pseudomonas aeruginosa uses quorum sensing to control virulence and biofilm formation. Here, we analyze synthetic molecules for inhibition of the two P. aeruginosa quorum-sensing receptors, LasR and RhlR. Our most effective compound, meta-bromo-thiolactone (mBTL), inhibits both the production of the virulence factor pyocyanin and biofilm formation. mBTL also protects Caenorhabditis elegans and human lung epithelial cells from killing by P. aeruginosa. Both LasR and RhlR are partially inhibited by mBTL in vivo and in vitro; however, RhlR, not LasR, is the relevant in vivo target. More potent antagonists do not exhibit superior function in impeding virulence. Because LasR and RhlR reciprocally control crucial virulence factors, appropriately tuning rather than completely inhibiting their activities appears to hold the key to blocking pathogenesis in vivo.

Summary In the wild, bacteria are predominantly associated with surfaces as opposed to existing as free-swimming, isolated organisms. They are thus subject to surface-specific mechanics including hydrodynamic forces, adhesive forces, the rheology of their surroundings and transport rules that define their encounters with nutrients and signaling molecules. Here, we highlight the effects of mechanics on bacterial behaviors on surfaces at multiple length scales, from single bacteria to the development of multicellular bacterial communities such as biofilms.

Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non-surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface-two host-nonspecific cues-explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity.T he bacterium Pseudomonas aeruginosa is a metabolically versatile pathogen that inhabits diverse environments and infects a remarkable range of hosts, including mammals, insects, worms, amoeba, fungi, and other bacteria. P. aeruginosa produces a large number of secreted and cell-associated virulence factors that are redundant and multifactorial (1, 2). Many of P. aeruginosa's virulence factors-including pyocyanin, elastase, and hydrogen cyanide-are host-nonspecific (3-5), bolstering the ability of P. aeruginosa to attack a large range of hosts. Although many of the virulence factors in P. aeruginosa have been identified, the cues that regulate their activity are less understood. Because many of the virulence factors are host-nonspecific, we explored whether virulence in P. aeruginosa is regulated by host-nonspecific cues.Host cell membranes and cell surfaces are the first line of defense against bacterial toxins and invasion. P. aeruginosa attaches to host cell surfaces early during the infection process. The presence of a surface could thus act as a cue for P. aeruginosa, signaling the presence of a host. Surface attachment is also a critical initial step that enables the establishment of biofilms (6-8). Although biofilms are clearly important for pathogenesis, it remains unclear whether they directly promote host cell killing or mediate other important processes such as long-term colonization.One host-nonspecific cue that could regulate virulence is the mechanical force that bacteria experience upon surface attachment. P. aeruginosa performs surface-associated behaviors (7, 8) such as swarming and twitching (9, 10), but it remains unclear whether P. aeruginosa senses the chemical or mechanical properties of surfaces. There is precedence for mechanotransduction in eukaryotes, in which surface substrate recognition is an important regulat...