Mark Goulian

Rewiring the Specificity of Two-Component Signal Transduction Systems

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.

Energetics of Inclusion-Induced Bilayer Deformations

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the proteins hydrophobic exterior surface and the average thickness of the bilayers hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.

Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells

Quantitative spatial distributions of ribosomes (S2‐YFP) and RNA polymerase (RNAP; β′‐yGFP) in live Escherichia 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−1, attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. 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.

Long-Range Forces in Heterogeneous Fluid Membranes

We find a new long-range interaction between foreign inclusions (e.g. mobile proteins) in a fluid membrane which is mediated by the membrane itself. The interaction falls off as 1/R4, can be attractive or repulsive depending on the temperature and the elastic properties of the inclusion and the membrane, and for large distances is large compared with electrostatic, van der Waals, and other lipid-mediated forces.

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

The EnvZ/OmpR system in Escherichia coli, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling. Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR. We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation. The model predicts that when EnvZ is much less abundant than OmpR, as is the case in E. coli, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ. The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high. To test these predictions, we have perturbed the porin regulatory circuit in E. coli by varying the expression levels of EnvZ and OmpR. We have constructed two-color fluorescent reporter strains in which ompF and ompC transcription can be easily measured in the same culture. Using these strains we have shown that, consistent with the predictions of our model, the transcription of ompC and ompF is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.

Tracking Single Proteins within Cells

We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.

Staphylococcus aureus RN6390 Replicates and Induces Apoptosis in a Pulmonary Epithelial Cell Line

ABSTRACT Staphylococcus aureus frequently colonizes the airways of patients with compromised airway defenses (e.g., cystic fibrosis [CF] patients) for extended periods. Persistent and relapsing infections may be related to live S. aureus bacteria actively residing inside epithelial cells. In this study, we infected a respiratory epithelial cell line, which was derived from a CF patient, with S. aureus RN6390. Internalization of S. aureus was found to be time and dose dependent and could be blocked by cytochalasin D. Transmission electron microscopy revealed that internalized bacteria resided within endocytic vacuoles without any evidence of lysosomal fusion in a 24-h period. The results of internalization experiments and time-lapse fluorescence microscopy of epithelial cells infected with green fluorescent S. aureusindicate that, after an initial lag period of 7 to 9 h, intracellular bacteria began to replicate, with three to five divisions in a 24-h period, leading to apoptosis of infected cells. Induction of apoptosis required bacterial internalization and is associated with intracellular replication. The slow and gradual replication of S. aureus inside epithelial cells hints at the role of host factors or signals in bacterial growth and further suggests possible cross talk between host cells and S. aureus.

The Escherichia coli CpxA-CpxR Envelope Stress Response System Regulates Expression of the Porins OmpF and OmpC

We performed transposon mutagenesis of a two-color fluorescent reporter strain to identify new regulators of the porin genes ompF and ompC in Escherichia coli. Screening of colonies by fluorescence microscopy revealed numerous mutants that exhibited interesting patterns of porin expression. One mutant harbored an insertion in the gene encoding the histidine kinase CpxA, the sensor for a two-component signaling system that responds to envelope stress. The cpxA mutant exhibited increased transcription of ompC and a very strong decrease in transcription of ompF under conditions in which acetyl phosphate levels were high. Subsequent genetic analysis revealed that this phenotype is dependent on phosphorylation of the response regulator CpxR and that activation of CpxA in wild-type cells results in similar regulation of porin expression. Using DNase I footprinting, we demonstrated that CpxR binds upstream of both the ompF and ompC promoters. It thus appears that two distinct two-component systems, CpxA-CpxR and EnvZ-OmpR, converge at the porin promoters. Within the context of envelope stress, outer membrane beta-barrel proteins have generally been associated with the sigma E pathway. However, at least for the classical porins OmpF and OmpC, our results show that the Cpx envelope stress response system plays a role in regulating their expression.

Gramicidin Channel Kinetics under Tension

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Mucosal penetration primes Vibrio cholerae for host colonization by repressing quorum sensing

To successfully infect a host and cause the diarrheal disease cholera, Vibrio cholerae must penetrate the intestinal mucosal layer and express virulence genes. Previous studies have demonstrated that the transcriptional regulator HapR, which is part of the quorum sensing network in V. cholerae, represses the expression of virulence genes. Here, we show that hapR expression is also modulated by the regulatory network that governs flagellar assembly. Specifically, FliA, which is the alternative σ-factor (σ28) that activates late-class flagellin genes in V. cholerae, represses hapR expression. In addition, we show that mucin penetration by V. cholerae is sufficient to break flagella and so cause the secretion of FlgM, the anti-σ factor that inhibits FliA activity. During initial colonization of host intestinal tissue, hapR expression is repressed because of low cell density. However, full repression of hapR expression does not occur in fliA mutants, which results in attenuated colonization. Our results suggest that V. cholerae uses flagellar machinery to sense particular intestinal signals before colonization and enhance the expression of virulence genes by modulating the output of quorum sensing signaling.