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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 β-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.

Escherichia coli is both a harmless commensal in the intestines of many mammals, as well as a dangerous pathogen. The evolutionary paths taken by strains of this species in the commensal-to-pathogen transition are complex and can involve changes both in the core genome, as well in the pan-genome. One way to understand the likely paths that a commensal strain of E . coli takes when evolving pathogenicity is through experimentally evolving the strain under the selective pressures that it will have to withstand as a pathogen. Here, we report that a commensal strain, under continuous pressure from macrophages, recurrently acquired a transposable element insertion, which resulted in two key phenotypic changes: increased intracellular survival, through the delay of phagosome maturation and increased ability to escape macrophages. We further show that the acquisition of the pathoadaptive traits was accompanied by small but significant changes in the transcriptome of macrophages upon infection. These results show that under constant pressures from a key component of the host immune system, namely macrophage phagocytosis, commensal E . coli rapidly acquires pathoadaptive mutations that cause transcriptome changes associated to the host-microbe duet.

Multiple genetic changes in the enteric pathogen Yersinia pseudotuberculosis have driven the emergence of Yesinia pestis , the arthropod-borne, etiological agent of plague. These include developing the capacity for biofilm-dependent blockage of the flea foregut to enable transmission by flea bite. Previously, we showed that pseudogenization of rcsA , encoding a component of the Rcs signalling pathway, is an important evolutionary step facilitating Y. pestis flea-borne transmission. Additionally, rcsD, another important gene in the Rcs system, harbours a frameshift mutation. Here, we demonstrated that this rcsD mutation resulted in production of a small protein composing the C-terminal RcsD histidine-phosphotransferase domain (designated RcsD-Hpt) and full-length RcsD. Genetic analysis revealed that the rcsD frameshift mutation followed the emergence of rcsA pseudogenization. It further altered the canonical Rcs phosphorylation signal cascade, fine-tuning biofilm production to be conducive with retention of the pgm locus in modern lineages of Y. pestis . Taken together, our findings suggest that a frameshift mutation in rcsD is an important evolutionary step that fine-tuned biofilm production to ensure perpetuation of flea-mammal plague transmission cycles. Yersinia pestis , the agent responsible for the plague, emerged 6,000 to 7,000 years ago from Yersinia pseudotuberculosis , another type of bacteria which still exists today. Although they are highly similar genetically, these two species are strikingly different. While Y. pseudotuberculosis spreads via food and water and causes mild stomach distress, Y. pestis uses fleas to infect new hosts and has killed millions. A small set of genetic changes has contributed to the emergence of Y. pestis by allowing it to thrive inside a flea and maximise its transmission. In particular, some of these mutations have led to the bacteria being able to come together to form a sticky layer that adheres to the gut of the insect, with this ‘biofilm’ stopping the flea from feeding on blood. The starving flea keeps trying to feed, and with each bite comes another opportunity for Y. pestis to jump host. However, it remains unclear exactly how the mutations have influenced biofilm formation to allow for this new transmission mechanism to take place. To examine this phenomenon, Guo et al. focused on rcsD , a gene that codes for a component of the signalling system that controls biofilm creation. In Y. pestis this sequence has been mutated to become a ‘pseudogene’, a type of sequence which is often thought to be non-functional. However, the experiments showed that, in Y. pestis, rcsD could produce small amounts of a full-length RcsD protein similar to the one found in Y. pseudotuberculosis . However, the gene mostly produces a short ‘RcsD-Hpt’ protein that can, in turn, alter the expression of many genes, including those that decrease biofilm formation. This may prove to be beneficial for Y. pestis , for example when the bacteria switches from living in fleas to living in humans, where it does not require a biofilm. Guo et al. further investigated the impact of rcsD becoming a pseudogene in Y. pestis , showing that if normal amounts of the full-length RcsD protein are produced, the bacteria quickly lose the gene that allows them to form biofilm in fleas, and cause disease in humans. In fact, additional analyses revealed that all sequenced strains of ancient and modern Y. pestis bacteria can produce RcsD-Hpt, even if they do not carry the same exact rcsD mutation. Overall, these results indicate that rcsD turning into a pseudogene marked an important step in the emergence of Y. pestis strains that can cause lasting plague outbreaks. They also point towards pseudogenes having more important roles in evolution than previously thought.

Bacteria must coordinate their growth rate, shape, and division to survive and flourish, yet how these cellular properties are maintained in the face of environmental stresses is poorly understood. Working with Escherichia coli , we show that activating the Rcs phosphorelay, an envelope stress-signaling system, in the absence of external stresses slows growth, shortens cells, and increases the concentration of the key division protein FtsZ, leading to more closely spaced division sites. Depleting the levels of IgaA, a regulator of the Rcs pathway, yielded similar phenotypes. However, activating Rcs via drug-induced cell-wall disruption did not affect growth rate, indicating that the physiological impact of this pathway depends on the context of activation. Our findings reveal links among cell growth, shape homeostasis, and cell envelope stress. Understanding this coupling further will provide new avenues to predict and modulate bacterial growth and physiology during stress.

The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors. The β-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE , but not bamB , completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE . Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex. IMPORTANCE The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, and its integrity is monitored by various stress response systems. Although the Rcs system is involved in the envelope stress response and regulates genes controlling numerous bacterial cell functions of Yersinia enterocolitica, whether it can sense the truncated LPS in Y. enterocolitica remains unclear. In this study, the deletion of the Y. enterocolitica waaF gene truncated the structure of LPS and produced a deep rough LPS. The truncated LPS increased the cell surface hydrophobicity and outer membrane permeability, generating cell envelope stress. The truncated LPS also directly exposed the smooth outer membrane to the external environment and attenuated the resistance to adverse conditions, such as impaired survival under polymyxin B and sodium dodecyl sulfate (SDS) exposure. Further phenotypic experiment and gene expression analysis indicated that the truncated LPS was correlated with the activation of the Rcs phosphorelay, thereby repressing cell motility and biofilm formation. Our findings highlight the importance of LPS integrity in maintaining membrane function and broaden the understanding of Rcs phosphorelay signaling in response to cell envelope stress, thus opening new avenues to develop effective antimicrobial agents for combating Y. enterocolitica infections.

The overall role of the Rcs phosphorelay in Proteus mirabilis is largely unknown. Previous work had demonstrated that the Rcs phosphorelay represses the flhDC operon and activates the minCDE cell division inhibition system. To identify additional cellular functions regulated by the Rcs phosphorelay, an analysis of RNA-seq data was undertaken. In this report, the results of the RNA-sequencing are discussed with an emphasis on the predicted roles of the Rcs phosphorelay in swarmer cell differentiation, motility, biofilm formation, and virulence. RcsB is shown to activate genes important for differentiation and fimbriae formation, while repressing the expression of genes important for motility and virulence. Additionally, to follow up on the RNA-Seq data, we demonstrate that an rcsB mutant is deficient in its ability to form biofilm and exhibits enhanced virulence in a Galleria mellonella waxworm model. Overall, these results indicate the Rcs regulon in P. mirabilis extends beyond flagellar genes to include those involved in biofilm formation and virulence. Furthermore, the information presented in this study may provide clues to additional roles of the Rcs phosphorelay in other members of the Enterobacteriaceae .

The Rcs phosphorelay is a complex signaling pathway found in many, but not all, members of the Enterobacteriaceae. The complexity of this pathway is due to the direct involvement of three proteins (RcsC, RcsD and RcsB) in the phosphorelay and the presence of multiple accessory proteins with important roles in modulating the inputs and outputs associated with this signaling pathway. This article will discuss the various inputs and outputs associated with the Rcs phosphorelay and also present a model suggesting an important role for this signaling pathway in the temporal control of virulence in Salmonella enterica and biofilm formation in Escherichia coli.

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsCPcc and RcsDPcc, and a response regulator RcsBPcc as key components of this system, and an additional small lipoprotein RcsFPcc. This study indicates that inactivation of rcsCPcc, rcsDPcc and rcsBPcc enhances production of virulence factors with the highest effect detected for rcsBPcc. Interestingly, mutation of rcsFPcc has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsBPcc response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.