The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim domains.

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

Regulatory interactions between daptomycin- and bacitracin-responsive pathways coordinate the cell envelope antibiotic resistance response of Enterococcus faecalis.

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.

The Wsp system of Pseudomonas aeruginosa links surface sensing and cell envelope stress.

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.

Membrane Lipids Augment Cell Envelope Stress Signaling via the MadRS System to Defend Against Antimicrobial Peptides and Antibiotics in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential ß-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.

RNA-seq reveals multifaceted gene expression response to Fab production in Escherichia coli fed-batch processes with particular focus on ribosome stalling.

BACKGROUND: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations. RESULTS: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising. CONCLUSIONS: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.

A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol.

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function.

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the ß-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

NlpE Is an OmpA-Associated Outer Membrane Sensor of the Cpx Envelope Stress Response.

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multilayered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors, such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates surface adhesion to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the major OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Mutation of OmpA peptidoglycan-binding residues abrogates signaling during OmpA overexpression, suggesting that NlpE signaling from the OM through the cell wall is coordinated via OmpA. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. IMPORTANCE The envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of OM ß-barrel protein and lipoprotein complexes to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

The implication of viability and pathogenicity by truncated lipopolysaccharide in Yersinia enterocolitica.

The fast envelope stress responses play a key role in the transmission and pathogenesis of Yersinia enterocolitica, one of the most common foodborne pathogens. Our previous study showed that deletion of the waaF gene, essential for the biosynthesis of lipopolysaccharide (LPS) core polysaccharides, led to the formation of a truncated LPS structure and induced cell envelope stress. This envelope stress may disturb the intracellular signal transduction, thereby affecting the physiological functions of Y. enterocolitica. In this study, truncated LPS caused by waaF deletion was used as a model of envelope stress in Y. enterocolitica. We investigated the mechanisms of envelope stress responses and the cellular functions affected by truncated LPS. Transcriptome analysis and phenotypic validation showed that LPS truncation reduced flagellar assembly, bacterial chemotaxis, and inositol phosphate metabolism, presenting lower pathogenicity and viability both in vivo and in vitro environments. Further 4D label-free phosphorylation analysis confirmed that truncated LPS perturbed multiple intracellular signal transduction pathways. Specifically, a comprehensive discussion was conducted on the mechanisms by which chemotactic signal transduction and Rcs system contribute to the inhibition of chemotaxis. Finally, the pathogenicity of Y. enterocolitica with truncated LPS was evaluated in vitro using IPEC-J2 cells as models, and it was found that truncated LPS exhibited reduced adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. Our research provides an understanding of LPS in the regulation of Y. enterocolitica viability and pathogenicity and, thus, opening new avenues to develop novel food safety strategies or drugs to prevent and control Y. enterocolitica infections. KEY POINTS: • Truncated LPS reduces flagellar assembly, chemotaxis, and inositol phosphate metabolism in Y. enterocolitica. • Truncated LPS reduces adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. • Truncated LPS regulates intracellular signal transduction of Y. enterocolitica.

The anti-sigma factor MucA is required for viability in Pseudomonas aeruginosa.

During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by the overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that the essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

Conformation control of the histidine kinase BceS of Bacillus subtilis by its cognate ABC-transporter facilitates need-based activation of antibiotic resistance.

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

Impact of Nuclear Envelope Stress on Physiological and Pathological Processes in Central Nervous System.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm and provides the molecular platforms for nucleocytoplasmic transport, higher-order chromatin organization, and physical links between the nucleus and cytoskeleton. Recent studies have shown that the NE is often damaged by various stresses termed "NE stress", leading to critical cellular dysfunction. Accumulating evidence has revealed the crucial roles of NE stress in the pathology of a broad spectrum of diseases. In the central nervous system (CNS), NE dysfunction impairs neural development and is associated with several neurological disorders, such as Alzheimer's disease and autosomal dominant leukodystrophy. In this review, the structure and functions of the NE are summarized, and the concepts of NE stress and NE stress responses are introduced. Additionally, the significant roles of the NE in the development of CNS and the mechanistic connections between NE stress and neurological disorders are described.

Development of a whole-cell biosensor for detection of antibiotics targeting bacterial cell envelope in Bacillus subtilis.

It is an urgent need to develop novel antibiotics to treat infections caused by multi-drug-resistant bacteria. One promising strategy could be the use of whole-cell biosensors, which have been extensively studied to monitor environmental pollutants and intracellular metabolites. Here, we used the σM-mediated regulatory system of Bacillus subtilis to construct a whole-cell biosensor for the detection of cell envelope-acting antibiotics. Using polymyxin B as the inducer for bacterial cell envelope stress and enhanced green fluorescent protein (EGFP) as the reporter, we found that the promoter of ypuA (PypuA) had the lowest background noise and the most significant changes in the fluorescence output. The whole-cell biosensor displayed dose-dependent and time-dependent responses in fluorescence signals. The detection range of this biosensor for polymyxin B was between 0.125 and 12 µg/mL. The response of the biosensor is specific to antibiotics that target the cell envelope. Besides determination in liquid cultures, the output signal of the biosensor can be easily determined on agar surfaces. Using this biosensor, we successfully detected polymyxins secreted by its producing strain and bacteria that produce cell envelope-acting antibiotics. KEY POINTS: • A whole-cell biosensor was constructed based on the σM-mediated regulatory system. • The response of the biosensor is specific to cell envelope-acting antibiotics. • The biosensor can be used to screen novel cell envelope-acting antibiotics.

Cell Envelope Stress Response in Pseudomonas aeruginosa.

Bacteria sense their environment via the cell envelope, which in Gram-negative bacteria comprises the outer membrane, the periplasmic space, and the inner membrane. Pseudomonas aeruginosa is an opportunistic pathogen which is exposed to different cell wall stresses imposed by exposure to antibiotics, osmotic pressure, and long-time colonization of host tissues such as the lung in cystic fibrosis patients. In response to these stresses, P. aeruginosa is able to respond by establishing a cell envelope stress response involving different regulatory pathways including the extra-cytoplasmic sigma factors AlgU, SigX, and SbrI and other two-component sensor/response regulators and effectors. This chapter aims to review the different factors leading to the activation of the cell envelope stress response in P. aeruginosa and the genetic determinants involved in this response, which is crucial for the survival of the bacterium upon exposure to different stressful conditions.

Crystal structure of the DNA-binding domain of Bacillus subtilis CssR.

Bacteria utilize two-component systems to regulate gene expression in response to changes in environmental stimuli. CssS/CssR, a two-component system in Bacillus subtilis, is responsible for overcoming envelope stresses caused by heat shock and secretion overload. During stress, the sensor component CssS is auto-phosphorylated and transfers the phosphoryl group to the response regulator CssR. Phosphorylated CssR then directly regulates the transcription of genes required to counteract the stress. Here, the crystal structure of the DNA-binding domain of CssR, determined at 1.07 Å resolution, is reported. The structure shows that the DNA-binding domain of CssR harbors a winged helix-turn-helix motif that is conserved in the OmpR/PhoB subfamily of response regulators. Based on the crystal structure, the dimeric architecture of the full-length CssR and its DNA-binding mode were suggested.

Revisiting long-chain fatty acid metabolism in Escherichia coli: integration with stress responses.

Long-chain fatty acids (LCFAs) are a tremendous source of metabolic energy, an essential component of membranes, and important effector molecules that regulate a myriad of cellular processes. As an energy-rich nutrient source, the role of LCFAs in promoting bacterial survival and infectivity is well appreciated. LCFA degradation generates a large number of reduced cofactors that may confer redox stress; therefore, it is imperative to understand how bacteria deal with this paradoxical situation. Although the LCFA utilization pathway has been studied in great detail, especially in Escherichia coli, where the earliest studies date back to the 1960s, the interconnection of LCFA degradation with bacterial stress responses remained largely unexplored. Recent work in E. coli shows that LCFA degradation induces oxidative stress and also impedes oxidative protein folding. Importantly, both issues arise due to the insufficiency of ubiquinone, a lipid-soluble electron carrier in the electron transport chain. However, to maintain redox homeostasis, bacteria induce sophisticated cellular responses. Here, we review these findings in light of our current knowledge of the LCFA metabolic pathway, metabolism-induced oxidative stress, the process of oxidative protein folding, and stress combat mechanisms. We discuss probable mechanisms for the activation of defense players during LCFA metabolism and the likely feedback imparted by them. We suggest that besides defending against intrinsic stresses, LCFA-mediated upregulation of stress response pathways primes bacteria to adapt to harsh external environments. Collectively, the interplay between LCFA metabolism and stress responses is likely an important factor that underlies the success of LCFA-utilizing bacteria in the host.

Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins.

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.