Goran Jovanovic

Managing membrane stress: the phage shock protein (Psp) response, from molecular mechanisms to physiology.

The bacterial phage shock protein (Psp) response functions to help cells manage the impacts of agents impairing cell membrane function. The system has relevance to biotechnology and to medicine. Originally discovered in Escherichia coli, Psp proteins and homologues are found in Gram-positive and Gram-negative bacteria, in archaea and in plants. Study of the E. coli and Yersinia enterocolitica Psp systems provides insights into how membrane-associated sensory Psp proteins might perceive membrane stress, signal to the transcription apparatus and use an ATP-hydrolysing transcription activator to produce effector proteins to overcome the stress. Progress in understanding the mechanism of signal transduction by the membrane-bound Psp proteins, regulation of the psp gene-specific transcription activator and the cell biology of the system is presented and discussed. Many features of the action of the Psp system appear to be dominated by states of self-association of the master effector, PspA, and the transcription activator, PspF, alongside a signalling pathway that displays strong conditionality in its requirement.

Induction and Function of the Phage Shock Protein Extracytoplasmic Stress Response in Escherichia coli

The phage shock protein (Psp) F regulon response in Escherichia coli is thought to be induced by impaired inner membrane integrity and an associated decrease in proton motive force (pmf). Mechanisms by which the Psp system detects the stress signal and responds have so far remained undetermined. Here we demonstrate that PspA and PspG directly confront a variety of inducing stimuli by switching the cell to anaerobic respiration and fermentation and by down-regulating motility, thereby subtly adjusting and maintaining energy usage and pmf. Additionally, PspG controls iron usage. We show that the Psp-inducing protein IV secretin stress, in the absence of Psp proteins, decreases the pmf in an ArcB-dependent manner and that ArcB is required for amplifying and transducing the stress signal to the PspF regulon. The requirement of the ArcB signal transduction protein for induction of psp provides clear evidence for a direct link between the physiological redox state of the cell, the electron transport chain, and induction of the Psp response. Under normal growth conditions PspA and PspD control the level of activity of ArcB/ArcA system that senses the redox/metabolic state of the cell, whereas under stress conditions PspA, PspD, and PspG deliver their effector functions at least in part by activating ArcB/ArcA through positive feedback.

The PspA protein of Escherichia coli is a negative regulator of sigma(54)-dependent transcription.

In Eubacteria, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor sigma(54) is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the sigma(54)-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the sigma(54)-dependent pspA promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of Escherichia coli, which negatively regulates expression of the pspA promoter without binding DNA directly.

In vivo localizations of membrane stress controllers PspA and PspG in Escherichia coli

The phage shock protein (Psp) response in Gram‐negative bacteria counteracts membrane stress. Transcription of the PspF regulon (pspABCDE and pspG) in Escherichia coli is induced upon stresses that dissipate the proton motive force (pmf). Using GFP fusions we have visualized the subcellular localizations of PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp). It has previously been proposed that PspA evenly coates the inner membrane of the cell. We now demonstrate that instead of uniformly covering the entire cell, PspA (and PspG) is highly organized into what appear to be distinct functional classes (complexes at the cell pole and the lateral cell wall). Real‐time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain pmf under stress conditions. The two spatial subspecies therefore appear to be dynamically and functionally distinct with the polar clusters being associated with sensory function and the mobile complexes with maintenance of pmf.

A lower-order oligomer form of phage shock protein A (PspA) stably associates with the hexameric AAA(+) transcription activator protein PspF for negative regulation.

To survive and colonise their various environments, including those used during infection, bacteria have developed a variety of adaptive systems. Amongst these is phage shock protein (Psp) response, which can be induced in Escherichia coli upon filamentous phage infection (specifically phage secretin pIV) and by other membrane-damaging agents. The E. coli Psp system comprises seven proteins, of which PspA is the central component. PspA is a bifunctional protein that is directly involved in (i) the negative regulation of the psp-specific transcriptional activator PspF and (ii) the maintenance of membrane integrity in a mechanism proposed to involve the formation of a 36-mer ring complex. Here we established that the PspA negative regulation of PspF ATPase activity is the result of a cooperative inhibition. We present biochemical evidence showing that an inhibitory PspA–PspF regulatory complex, which has significantly reduced PspF ATPase activity, is composed of around six PspF subunits and six PspA subunits, suggesting that PspA exists in at least two different oligomeric assemblies. We now establish that all four putative helical domains of PspA are critical for the formation of the 36-mer. In contrast, not all four helical domains are required for the formation of the inhibitory PspA–PspF complex. Since a range of initial PspF oligomeric states permit formation of the apparent PspA–PspF dodecameric assembly, we conclude that PspA and PspF demonstrate a strong propensity to self-assemble into a single defined heteromeric regulatory complex.

The Evolution of the Phage Shock Protein Response System: Interplay between Protein Function, Genomic Organization, and System Function

Sensing the environment and responding appropriately to it are key capabilities for the survival of an organism. All extant organisms must have evolved suitable sensors, signaling systems, and response mechanisms allowing them to survive under the conditions they are likely to encounter. Here, we investigate in detail the evolutionary history of one such system: The phage shock protein (Psp) stress response system is an important part of the stress response machinery in many bacteria, including Escherichia coli K12. Here, we use a systematic analysis of the genes that make up and regulate the Psp system in E. coli in order to elucidate the evolutionary history of the system. We compare gene sharing, sequence evolution, and conservation of protein-coding as well as noncoding DNA sequences and link these to comparative analyses of genome/operon organization across 698 bacterial genomes. Finally, we evaluate experimentally the biological advantage/disadvantage of a simplified version of the Psp system under different oxygen-related environments. Our results suggest that the Psp system evolved around a core response mechanism by gradually co-opting genes into the system to provide more nuanced sensory, signaling, and effector functionalities. We find that recruitment of new genes into the response machinery is closely linked to incorporation of these genes into a psp operon as is seen in E. coli, which contains the bulk of genes involved in the response. The organization of this operon allows for surprising levels of additional transcriptional control and flexibility. The results discussed here suggest that the components of such signaling systems will only be evolutionarily conserved if the overall functionality of the system can be maintained.

Physical, functional and conditional interactions between ArcAB and phage shock proteins upon secretin-induced stress in Escherichia coli

The phage shock protein (Psp) system found in enterobacteria is induced in response to impaired inner membrane integrity (where the Psp response is thought to help maintain the proton motive force of the cell) and is implicated in the virulence of pathogens such as Yersinia and Salmonella. We provided evidence that the two‐component ArcAB system was involved in induction of the Psp response in Escherichia coli and now report that role of ArcAB is conditional. ArcAB, predominantly through the action of ArcA regulated genes, but also via a direct ArcB–Psp interaction, is required to propagate the protein IV (pIV)‐dependent psp‐inducing signal(s) during microaerobiosis, but not during aerobiosis or anaerobiosis. We show that ArcB directly interacts with the PspB, possibly by means of the PspB leucine zipper motif, thereby allowing cross‐communication between the two systems. In addition we demonstrate that the pIV‐dependent induction of psp expression in anaerobiosis is independent of PspBC, establishing that PspA and PspF can function as a minimal Psp system responsive to inner membrane stress.

Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in Escherichia coli

The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein–protein interactions, resulting in the release of the PspA–PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

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

ABSTRACT Phage 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 cells inner membrane experiences increased SCE stress. IMPORTANCE All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response.

Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells

Bacterial enhancer-dependent transcription systems support major adaptive responses and offer a singular paradigm in gene control analogous to complex eukaryotic systems. Here we report new mechanistic insights into the control of one-membrane stress-responsive bacterial enhancer-dependent system. Using millisecond single-molecule fluorescence microscopy of live cells we determine the localizations, two-dimensional diffusion dynamics and stoichiometries of complexes of the bacterial enhancer-binding ATPase PspF during its action at promoters as regulated by inner membrane interacting negative controller PspA. We establish that a stable repressive PspF–PspA complex is located in the nucleoid, transiently communicating with the inner membrane via PspA. The PspF as a hexamer stably binds only one of the two psp promoters at a time, suggesting that psp promoters will fire asynchronously and cooperative interactions of PspF with the basal transcription complex influence dynamics of the PspF hexamer–DNA complex and regulation of the psp promoters.