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Abstract 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.

Phage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in Proteobacteria and Firmicutes. We now show that a minimal PSP system was present in the last universal common ancestor that evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate the use of domain architecture and genetic context for discovery in comparative genomics.

Eukaryotic members of the endosome sorting complex required for transport-III (ESCRT-III) family have been shown to form diverse higher-order assemblies. The bacterial phage shock protein A (PspA) has been identified as a member of the ESCRT-III superfamily, and PspA homo-oligomerizes to form rod-shaped assemblies. As observed for eukaryotic ESCRT-III, PspA forms tubular assemblies of varying diameters. Using electron cryo-electron microscopy, we determined 61 Synechocystis PspA structures and observed in molecular detail how the structural plasticity of PspA rods is mediated by conformational changes at three hinge regions in the monomer and by the fixed and changing molecular contacts between protomers. Moreover, we reduced and increased the structural plasticity of PspA rods by removing the loop connecting helices α3/α4 and the addition of nucleotides, respectively. Based on our analysis of PspA-mediated membrane remodeling, we suggest that the observed mode of structural plasticity is a prerequisite for the biological function of ESCRT-III members. Using cryo-EM, the authors revealed structures of ESCRT-III superfamily member PspA and the molecular basis of structural plasticity that enables assembly modulations by the addition of nucleotides and targeted mutations.

Macrophages are professional phagocytes that play a major role in engulfing and eliminating invading pathogens. Some intracellular pathogens, such as Salmonella enterica serovar Typhimurium, exploit macrophages as niches for their replication, which requires precise and dynamic modulation of bacterial gene expression in order to resist the hostile intracellular environment. Here, we present a comprehensive analysis of the global transcriptome of S. Typhimurium across four stages of infection of primary macrophages. Our results revealed a profound change in early-stage gene expression dominated by pathways linked to metabolic processes required for Salmonella adaptation to the proinflammatory conditions of the macrophage. We identified the phage shock protein (Psp) system to be highly expressed in intracellular S. Typhimurium, with sustained high expression over the course of infection. We determined that the Psp system is regulated by the virulence-associated two-component system SsrA-SsrB, which coordinates its expression with critical bacterial functions required for immune evasion and intracellular survival. Functional assays demonstrated that the Psp system mediates resistance to host antimicrobial peptides, including cathelicidin-related antimicrobial peptide (CRAMP), which we demonstrate supports bacterial persistence in host tissues and survival within macrophages. Our findings establish the Psp system as a new and critical adaptive mechanism for evading host immune defenses and highlight the utility of temporal transcriptomics in unraveling the genetic strategies employed by S. Typhimurium during macrophage infection.

Food waste due to perishable and unsafe food products is a major issue worldwide. For some high-quality perishable food products, such as fresh fish and cold-smoked salmon, traditional food preservation techniques are unsuitable as they can compromise sensory qualities such as flavor, texture, and freshness. These products often support the growth of the human pathogen Listeria monocytogenes , which can be present if thermal treatment is not applied. Thus, antilisterial bacteriocins, such as garvicin KS (GarKS), in combination with other technologies like high-pressure processing, are being investigated as hurdle strategies to increase the shelf life and food safety of fish products. In this study, we aimed to identify potential resistance development and genetic factors affecting the susceptibility of L. monocytogenes towards GarKS. We show that L. monocytogenes strains associated with fish products and fish processing plants are susceptible to GarKS with MIC values ranging from 20 to 275 nM. By RNA sequencing, we showed that exposure to GarKS resulted in an upregulation of genes involved in the phage shock protein ( psp ) response. Furthermore, isolation of resistant mutants indicated a low frequency of resistance to GarKS (10⁻ 9 to 10⁻ 11 ). The GarKS-tolerant mutants isolated (2-fold increased MIC values) were shown to harbor disruption mutations in lmo2468 , encoding a PspC-domain-containing protein. Overexpression of this gene increased susceptibility to GarKS two-fold and restored wild-type susceptibility in a disruption mutant. This study thus demonstrates that resistance development to GarKS is rare and identifies the phage shock protein response as a key player involved in susceptibility to GarKS.

Although phages shape bacterial evolution and physiology, the specificity of host transcriptomic responses to phage infection remains incompletely understood. Here, we performed global transcriptomic profiling of Escherichia coli exposed to two lytic phages, ΦX174 and T4, and the temperate phage λ, to explore both conserved and phage-specific host responses. All infections induced stress-related genes, including SOS and general stress pathways, along with repression of anabolic processes such as purine and amino acid biosynthesis, suggesting a metabolic shift to conserve resources. Notably, ΦX174 strongly activated the phage shock protein operon, while both ΦX174 and λ selectively induced soxS , a regulator of oxidative stress. Despite infecting the same host, each phage triggered distinct transcriptional signatures. These findings highlight the complexity of bacterial responses and the value of transcriptomics in decoding host-phage interactions, offering insights into resistance, survival, and co-evolution.

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 cell's 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.

Recently, we developed a spatial phage-assisted continuous evolution (SPACE) system. This system utilizes chemotaxis coupled with the growth of motile bacteria during their spatial range expansion in soft agar to provide fresh host cells for iterative phage infection and selection pressure for preserving evolved genes of interest carried by phage mutants. Controllable mutagenesis activated only in a subpopulation of the migrating cells is essential in this system to efficiently generate mutated progeny phages from which desired individuals are selected during the directed evolution process. But, the widely adopted small molecule-dependent inducible system could hardly fulfill this purpose because it always affects all cells homogeneously. In this study, we developed a phage infection-induced gene expression system using modified Escherichia coli (E. coli) phage shock protein operon or sigma factors from Bacillus subtilis. Results showed that this system enabled efficient control of gene expression upon phage infection with dynamic output ranges from small to large using combinations of different engineered phages and corresponding promoters. This system was incorporated into SPACE to function as a phage infection-induced mutagenesis module and successfully facilitated the evolution of T7 RNA polymerase, which generated diverse mutants with altered promoter recognition specificity. We expect that phage infection-induced gene expression system could be further extended to more applications involving partial induction in a portion of a population and targeted induction in specific strains among a mixed bacterial community, which provides an important complement to small molecule-dependent inducible systems.

Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections.The proton motive force (PMF) is a critical electrochemical gradient involved in bacterial energy metabolism. Here, Miaomiao Wang, Edward Wai Chi Chan et al. demonstrate that the PMF plays a key role in starvation-induced antibiotic tolerance in E. coli, suggesting that PMF disruption could be a future strategy to treat bacterial infection.

The 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. IMPORTANCE Colanic acid is a negatively charged polysaccharide capsule produced by Escherichia coli , Salmonella , and other gammaproteobacteria. Research conducted over the 50 years since the discovery of colanic acid suggests that this exopolysaccharide plays an important role for bacteria living in biofilms. However, a precise physiological role for colanic acid has not been defined. In this study, we provide evidence that colanic acid maintains the transmembrane potential and proton motive force during envelope stress. This work provides a new and fundamental insight into bacterial physiology. Colanic acid is a negatively charged polysaccharide capsule produced by Escherichia coli , Salmonella , and other gammaproteobacteria. Research conducted over the 50 years since the discovery of colanic acid suggests that this exopolysaccharide plays an important role for bacteria living in biofilms. However, a precise physiological role for colanic acid has not been defined. In this study, we provide evidence that colanic acid maintains the transmembrane potential and proton motive force during envelope stress. This work provides a new and fundamental insight into bacterial physiology.