Psp System Research Articles

Are envelope stress responses essential for persistence to β-lactams in Escherichia coli?

Bacterial persistence to antibiotics defines the ability of small sub-populations of sensitive cells within an isogenic population to survive high doses of bactericidal antibiotics. Here, we investigated the importance of the five main envelope stress responses (ESRs) of Escherichia coli in persistence to five bactericidal β-lactam antibiotics by combining classical time-kill curve experiments and single-cell analysis using time-lapse microscopy. We showed that the survival frequency of mutants for the Bae, Cpx, Psp, and Rcs systems treated with different β-lactams is comparable to that of the wild-type strain, indicating that these ESRs do not play a direct role in persistence to β-lactams. Since the σE-encoding gene is essential, we could not directly test its role. Using fluorescent reporters to monitor the activation of ESRs, we observed that σE is induced by high doses of meropenem. However, the dynamics of σE activation during meropenem treatment did not reveal any difference in persister cells compared to the bulk of the population, indicating that σE activation is not a hallmark of persistence. The Bae, Cpx, Psp, and Rcs responses were neither induced by ampicillin nor by meropenem. However, pre-induction of the Rcs system by polymyxin B increased survival to meropenem in an Rcs-dependent manner, suggesting that this ESR might confer some yet uncharacterized advantages during meropenem treatment or at the post-antibiotic recovery step. Altogether, our data suggest that ESRs are not key actors in E. coli persistence to β-lactams in the conditions we tested.

Comparison of the Effect of the Time Under the Three Primary Color Lighting of Led Production Before Scanning of Phosphorus Plates

Purpose
 This study aims to compare the effect of light exposure in red, green and blue (RGB) colors prior to scanning of the PSP plates.
 Materials & Methods
 An Arduino-based system is produced for standardized light exposure to the irradiated PSP plates. The system consisted of an Arduino Mega 2560 developer board, 2 RGB LED light sources, a TSL2591 digital light sensor and a DHT11 temperature and humidity sensor. A light-tight platform is produced with additive manufacturing and electronical units are integrated into this platform. A two-step alloy was used to create contrast. PSP system (VistaScan, Dürr Dental, Germany) is irradiated with fixed parameters of 70 kV, 8 mA and 0.5 seconds. Scanning of the PSPs were delayed for 1-,3-,5-, and 10-minutes, and half of the active surfaces are exposed to RGB lights independently in full brightness (PWM) and calibrated with lux while the rest is protected. MGVs are measured in 6 regions per image. The MGV differences in regions between conditions were examined by Kruskal-Wallis test. A p-value

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into μm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Comparison of two digital intraoral radiography imaging systems as a function of contrast resolution and exposure time.

To compare the image quality of two different digital imaging systems; one photostimulable phosphor plate system (PSP) and a direct digital radiography system with CMOS imaging sensor; via evaluating contrast resolution among four different exposure times. Endodontically treated incisor teeth embedded in paraffin blocks are aligned next to a 99.5% Al wedge and exposed for 0.8, 0.1,0.125 and 0.16 seconds using both the CMOS and PSP systems. Using ImageJ software, 5 isometric and isogridded ROI from each root filling area and isometric ROI from the Al stepwedge were calculated. Evaluation of the total of 120 images displayed that PSP system produced significantly higher contrast resolution (P<0.05) in regard to pixel values than the CMOS. The CMOS system was non- responsive to increasing dose (P=0.000). Regarding the EqAl values, no significant difference was determined between groups (P>0.05). The contrast resolution was higher using the PSP system. It can be estimated that, filling material will be more obvious under lower doses using PSP.

Radiographic Diagnosis of Simulated External Root Resorption in Multi-Rooted Teeth: The Influence of Spatial Resolution.

The aim of this study was to evaluate the influence of spatial resolution (line pairs per millimetre - lp/mm) on the diagnosis of simulated external root resorption (ERR) in multi-rooted teeth by using digital periapical radiography. Forty human mandibular molars (80 roots) were used. The roots were divided into the following groups (n = 10): control without root filling (WORF), control with root filling (WRF), small ERRWORF, small ERR-WRF, moderate ERR-WORF, moderate ERR-WRF, extensive ERR-WORF and extensive ERR-WRF. Four digital radiographs (phosphor storage plates - PSP system) were taken of each tooth in three angulations. The PSPs were scanned with 10, 20, 25 and 40 lp/mm. All images were assessed by three endodontists who used a five-point scale for presence and absence of ERR and classified its location (cervical, middle or apical third). ROC curves and one-way ANOVA were performed (p &lt; 0.01). Diagnosis of ERR in nonroot-filled teeth showed higher values of sensitivity for 20 lp/mm and higher values of both specificity and accuracy for 40 lp/mm. In root-filled teeth, sensitivity and accuracy were higher for 25 lp/mm and spatial resolution had no influence on specificity. The best resolution for diagnosis of small and extensive ERR was 25 lp/mm, whereas for moderate ERR, it was 40 lp/mm. Cervical ERR was the most difficult to diagnose, regardless of the spatial resolution. Higher spatial resolutions have improved the radiographic diagnosis of simulated ERR in multi-rooted teeth and this should be considered when performing digital radiographs.

Evaluation of two dental digital imaging systems based on quality scorings, burn-out effects and cervical width determination

Background/Aim: The aim of the present study is to evaluate diagnostic accuracy of two generic image receptors with CMOS and PSP sensors for image quality scoring (IQS), burnout incidences (BI) and cervical widths (CW) with regard to four different exposure times. Material and Methods: 43 incisor teeth within 15 paraffin block models were exposed at 4 different exposure times both for the CMOS and PSP groups, and a total number of 120 images were obtained. All images were evaluated by 3 dentomaxillofacial radiologists via 3 different criteria; IQS, BI, CW. Results: Diagnostic quality scorings between groups displayed statistically significant difference for 0,1; 0,125 and 0,16 SEC exposure times. PSP group revealed higher IQS. For 0,125 and 0,16 seconds exposure times, PSP group showed higher percentages of BI. Average CW were lower in PSP group. Although no statistically significant difference was found between average CW vs. exposure times in the CMOS group; PSP group revealed significant differences among exposure times. We can state that, the PSP system displayed higher image quality so exposure times can be reduced, alas, the same conclusion is not possible with CMOS system used. Conclusions: Image quality perception is higher in PSP system we used, compared with CMOS system. PSP system display more burnout effects with increasing exposure times, while CMOS system is constant.

Characterization of the Vibrio cholerae Phage Shock Protein Response.

The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Evidence for a second regulatory binding site on PspF that is occupied by the C-terminal domain of PspA.

PspA is a key component of the bacterial Psp membrane-stress response system. The biochemical and functional characterization of PspA is impeded by its oligomerization and aggregation properties. It was recently possible to solve the coiled coil structure of a completely soluble PspA fragment, PspA(1–144), that associates with the σ54 enhancer binding protein PspF at its W56-loop and thereby down-regulates the Psp response. We now found that the C-terminal part of PspA, PspA(145–222), also interacts with PspF and inhibits its activity in the absence of full-length PspA. Surprisingly, PspA(145–222) effects changed completely in the presence of full-length PspA, as promoter activity was triggered instead of being inhibited under this condition. PspA(145–222) thus interfered with the inhibitory effect of full-length PspA on PspF, most likely by interacting with full-length PspA that remained bound to PspF. In support of this view, a comprehensive bacterial-2-hybrid screen as well as co-purification analyses indicated a self-interaction of PspA(145–222) and an interaction with full-length PspA. This is the first direct demonstration of PspA/PspA and PspA/PspF interactions in vivo that are mediated by the C-terminus of PspA. The data indicate that regulatory binding sites on PspF do not only exist for the N-terminal coiled coil domain but also for the C-terminal domain of PspA. The inhibition of PspF by PspA-(145–222) was reduced upon membrane stress, whereas the inhibition of PspF by PspA(1–144) did not respond to membrane stress. We therefore propose that the C-terminal domain of PspA is crucial for the regulation of PspF in response to Psp system stimuli.

Variations on a theme: evolution of the phage-shock-protein system in Actinobacteria.

The phage shock protein (Psp) stress-response system protects bacteria from envelope stress through a cascade of interactions with other proteins and membrane lipids to stabilize the cell membrane. A key component of this multi-gene system is PspA, an effector protein that is found in diverse bacterial phyla, archaea, cyanobacteria, and chloroplasts. Other members of the Psp system include the cognate partners of PspA that are part of known operons: pspF||pspABC in Proteobacteria, liaIHGFSR in Firmicutes, and clgRpspAMN in Actinobacteria. Despite the functional significance of the Psp system, the conservation of PspA and other Psp functions, as well as the various genomic contexts of PspA, remain poorly characterized in Actinobacteria. Here we utilize a computational evolutionary approach to systematically identify the variations of the Psp system in~450 completed actinobacterial genomes. We first determined the homologs of PspA and its cognate partners (as reported in Escherichia coli, Bacillus subtilis, and Mycobacterium tuberculosis) across Actinobacteria. This survey revealed that PspA and most of its functional partners are prevalent in Actinobacteria. We then found that PspA occurs in four predominant genomic contexts within Actinobacteria, the primary context being the clgRpspAM system previously identified in Mycobacteria. We also constructed a phylogenetic tree of PspA homologs (including paralogs) to trace the conservation and evolution of PspA across Actinobacteria. The genomic context revealed that PspA shows changes in its gene-neighborhood. The presence of multiple PspA contexts or of other known Psp members in genomic neighborhoods that do not carry pspA suggests yet undiscovered functional implications in envelope stress response mechanisms.

Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

ABSTRACTThe bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica. It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers.

The Rcs-Regulated Colanic Acid Capsule Maintains Membrane Potential in Salmonella enterica serovar Typhimurium.

ABSTRACTThe Rcs phosphorelay and Psp (phage shock protein) systems are envelope stress responses that are highly conserved in gammaproteobacteria. The Rcs regulon was found to be strongly induced during metal deprivation of Salmonella enterica serovar Typhimurium lacking the Psp response. Nineteen genes activated by the RcsA-RcsB response regulator make up an operon responsible for the production of colanic acid capsular polysaccharide, which promotes biofilm development. Despite more than half a century of research, the physiological function of colanic acid has remained elusive. Here we show that Rcs-dependent colanic acid production maintains the transmembrane electrical potential and proton motive force in cooperation with the Psp response. Production of negatively charged exopolysaccharide covalently bound to the outer membrane may enhance the surface potential by increasing the local proton concentration. This provides a unifying mechanism to account for diverse Rcs/colanic acid-related phenotypes, including susceptibility to membrane-damaging agents and biofilm formation.

The Phage Shock Protein Response.

The phage shock protein (Psp) system was identified as a response to phage infection in Escherichia coli, but rather than being a specific response to a phage, it detects and mitigates various problems that could increase inner-membrane (IM) permeability. Interest in the Psp system has increased significantly in recent years due to appreciation that Psp-like proteins are found in all three domains of life and because the bacterial Psp response has been linked to virulence and other important phenotypes. In this article, we summarize our current understanding of what the Psp system detects and how it detects it, how four core Psp proteins form a signal transduction cascade between the IM and the cytoplasm, and current ideas that explain how the Psp response keeps bacterial cells alive. Although recent studies have significantly improved our understanding of this system, it is an understanding that is still far from complete.

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism.

PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

Phage 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 coiled-coil 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.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

ABSTRACTPhage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress.

Evaluation of the Radiopacities of Bulk-fill Restoratives Using Two Digital Radiography Systems.

This study investigated the radiopacity values of bulk-fill restoratives by using two digital radiography systems. Nine bulk-fill restoratives and a conventional composite were used in the study. Six disc-shaped specimens were prepared from each of these materials, three each at thicknesses of 1 mm and 2 mm, and tooth slices with these same thicknesses were obtained. As a control, an aluminum step wedge varying in thickness from 0.5 to 10 mm in was used. Three specimens of each of the materials, together with the tooth slice and the aluminum step wedge, were placed over a complementary metal oxide semiconductor (CMOS) sensor and a storage photostimulable phosphor (PPS) plate system and exposed using a dental x-ray unit. The images were analyzed using a software program to measure the mean gray values (MGVs). Five measurements were obtained from each of the restorative materials, the enamel, the dentin, and the stepwedge. The MGVs were converted to the equivalent aluminum thicknesses. Three-way analysis of variance (ANOVA) was used to determine the significance of the differences among the groups. A Tukey test was applied for pairwise comparisons (p<0.05). All composite-based restoratives were found to have greater radiopacities than enamel or dentin. Equia Fil had the lowest radiopacity value. Radiopacity increased as the thicknesses of the restorative material increased. The CMOS system showed significantly higher radiopacity values than the PSP system. In conclusion, all investigated bulk-fill restoratives passed the International Organization for Standardization and American National Standard Institute/American Dental Association requirements for radiopacity values when evaluated with the two digital radiography systems.

Efficacy of lead foil for reducing doses in the head and neck: a simulation study using digital intraoral systems.

To assess the efficacy of lead foils in reducing the radiation dose received by different anatomical sites of the head and neck during periapical intraoral examinations performed with digital systems. Images were acquired through four different manners: phosphor plate (PSP; VistaScan(®) system; Dürr Dental GmbH, Bissingen, Germany) alone, PSP plus lead foil, complementary metal oxide semiconductor (CMOS; DIGORA(®) Toto, Soredex(®), Tuusula, Finland) alone and CMOS plus lead foil. Radiation dose was measured after a full-mouth periapical series (14 radiographs) using the long-cone paralleling technique. Lithium fluoride (LiF 100) thermoluminescent dosemeters were placed in an anthropomorphic phantom at points corresponding to the tongue, thyroid, crystalline lenses, parotid glands and maxillary sinuses. Dosemeter readings demonstrated the efficacy of the addition of lead foil in the intraoral digital X-ray systems provided in reducing organ doses in the selected structures, approximately 32% in the PSP system and 59% in the CMOS system. The use of lead foils associated with digital X-ray sensors is an effective alternative for the protection of different anatomical sites of the head and neck during full-mouth periapical series acquisition.

The Psp system of Mycobacterium tuberculosis integrates envelope stress-sensing and envelope-preserving functions.

The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system since it encodes only pspA and no other psp ortholog. Intriguingly, pspA maps downstream from clgR, which encodes a transcription factor regulated by the MprAB-σ(E) envelope-stress-signaling system. clgR inactivation lowered ATP concentration during stress and protonophore treatment-induced clgR-pspA expression, suggesting that these genes express Psp-like functions. We identified a four-gene set - clgR, pspA (rv2744c), rv2743c, rv2742c - that is regulated by clgR and in turn regulates ClgR activity. Regulatory and protein-protein interactions within the set and a requirement of the four genes for functions associated with envelope integrity and surface-stress tolerance indicate that a Psp-like system has evolved in mycobacteria. Among Actinobacteria, the four-gene module occurred only in tuberculous mycobacteria and was required for intramacrophage growth, suggesting links between its function and mycobacterial virulence. Additionally, the four-gene module was required for MprAB-σ(E) stress-signaling activity. The positive feedback between envelope-stress-sensing and envelope-preserving functions allows sustained responses to multiple, envelope-perturbing signals during chronic infection, making the system uniquely suited to tuberculosis pathogenesis.

Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners

The bacterial phage shock protein (Psp) system is a highly conserved cell envelope stress response required for virulence in Yersinia enterocolitica and Salmonella enterica. In non-inducing conditions the transcription factor PspF is inhibited by an interaction with PspA. In contrast, PspA associates with the cytoplasmic membrane proteins PspBC during inducing conditions. This has led to the proposal that PspBC exists in an OFF state, which cannot recruit PspA, or an ON state, which can. However, nothing was known about the difference between these two states. Here, we provide evidence that it is the C-terminal domain of Y. enterocolitica PspC (PspC(CT)) that interacts directly with PspA, both in vivo and in vitro. Site-specific photocross-linking revealed that this interaction occurred only during Psp-inducing conditions in vivo. Importantly, we have also discovered that PspC(CT) can interact with the C-terminal domain of PspB (PspC(CT)·PspB(CT)). However, the PspC(CT)·PspB(CT) and PspC(CT)·PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspC(CT) contacted PspB(CT) in the OFF state, whereas it contacted PspA in the ON state. These findings provide the first description of the previously proposed PspBC OFF and ON states and reveal that the regulatory switch is centered on a PspC(CT) partner-switching mechanism.

Is the cellular and molecular machinery docile in the stationary phase of Escherichia coli?

The bacterial cell envelope retains a highly dense cytoplasm. The properties of the cytoplasm change with the metabolic state of the cell, the logarithmic phase (log) being highly active and the stationary phase metabolically much slower. Under the differing growth phases, many different types of stress mechanisms are activated in order to maintain cellular integrity. One such response in enterobacteria is the phage shock protein (Psp) response that enables adaptation to the inner membrane (IM) stress. The Psp system consists of a transcriptional activator PspF, negative regulator PspA, signal sensors PspBC, with PspA and PspG acting as effectors. The single molecule imaging of the PspF showed the existence of dynamic communication between the nucleoid-bound states of PspF and membrane via negative regulator PspA and PspBC sensors. The movement of proteins in the cytoplasm of bacterial cells is often by passive diffusion. It is plausible that the dynamics of the biomolecules differs with the state of the cytoplasm depending on the growth phase. Therefore, the Psp response proteins might encounter the densely packed glass-like properties of the cytoplasm in the stationary phase, which can influence their cellular dynamics and function. By comparing the properties of the log and stationary phases, we find that the dynamics of PspF are influenced by the growth phase and may be controlled by the changes in the cytoplasmic fluidity.