A viral ADP-ribosyltransferase attaches RNA chains to host proteins.

The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1.

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

During heat stress in Myxococcus xanthus, the CdbS PilZ domain protein, in concert with two PilZ-DnaK chaperones, perturbs chromosome organization and accelerates cell death.

C-di-GMP is a bacterial second messenger that regulates diverse processes in response to environmental or cellular cues. The nucleoid-associated protein (NAP) CdbA in Myxococcus xanthus binds c-di-GMP and DNA in a mutually exclusive manner in vitro. CdbA is essential for viability, and CdbA depletion causes defects in chromosome organization, leading to a block in cell division and, ultimately, cell death. Most NAPs are not essential; therefore, to explore the paradoxical cdbA essentiality, we isolated suppressor mutations that restored cell viability without CdbA. Most mutations mapped to cdbS, which encodes a stand-alone c-di-GMP binding PilZ domain protein, and caused loss-of-function of cdbS. Cells lacking CdbA and CdbS or only CdbS were fully viable and had no defects in chromosome organization. CdbA depletion caused post-transcriptional upregulation of CdbS accumulation, and this CdbS over-accumulation was sufficient to disrupt chromosome organization and cause cell death. CdbA depletion also caused increased accumulation of CsdK1 and CsdK2, two unusual PilZ-DnaK chaperones. During CdbA depletion, CsdK1 and CsdK2, in turn, enabled the increased accumulation and toxicity of CdbS, likely by stabilizing CdbS. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induced the CdbA/CsdK1/CsdK2/CdbS system, causing a CsdK1- and CsdK2-dependent increase in CdbS accumulation. Thereby this system accelerates heat stress-induced chromosome mis-organization and cell death. Collectively, this work describes a unique system that contributes to regulated cell death in M. xanthus and suggests a link between c-di-GMP signaling and regulated cell death in bacteria.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Memory CD8(+) T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function.

How systemic metabolic alterations during acute infections impact immune cell function remains poorly understood. We found that acetate accumulates in the serum within hours of systemic bacterial infections and that these increased acetate concentrations are required for optimal memory CD8(+) T cell function in vitro and in vivo. Mechanistically, upon uptake by memory CD8(+) T cells, stress levels of acetate expanded the cellular acetyl-coenzyme A pool via ATP citrate lyase and promoted acetylation of the enzyme GAPDH. This context-dependent post-translational modification enhanced GAPDH activity, catalyzing glycolysis and thus boosting rapid memory CD8(+) T cell responses. Accordingly, in a murine Listeria monocytogenes model, transfer of acetate-augmented memory CD8(+) T cells exerted superior immune control compared to control cells. Our results demonstrate that increased systemic acetate concentrations are functionally integrated by CD8(+) T cells and translate into increased glycolytic and functional capacity. The immune system thus directly relates systemic metabolism with immune alertness.

A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

Marine Proteobacteria metabolize glycolate via the ß-hydroxyaspartate cycle.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments1. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria2-4, the further fate of this C2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the ß-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago5. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing-to our knowledge-the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

A noncanonical cytochrome c stimulates calcium binding by PilY1 for type IVa pili formation.

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many several other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on calcium and a noncanonical cytochrome c (TfcP) with an unusual His/Cys heme ligation. We provide evidence that TfcP is unlikely to participate in electron transport and instead stimulates calcium binding by PilY1.1 at low-calcium concentrations, thereby stabilizing PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results not only identify a previously undescribed function of cytochromes c but also illustrate how incorporation of an accessory factor expands the environmental range under which the T4aP system functions.

FlrA-independent production of flagellar proteins is required for proper flagellation in Shewanella putrefaciens.

Flagella are multiprotein complexes whose assembly and positioning require complex spatiotemporal control. Flagellar assembly is thought to be controlled by several transcriptional tiers, which are mediated through various master regulators. Here, we revisited the regulation of flagellar genes in polarly flagellated gammaproteobacteria by the regulators FlrA, RpoN (σ54 ) and FliA (σ28 ) in Shewanella putrefaciens CN-32 at the transcript and protein level. We found that a number of regulatory and structural proteins were present in the absence of the main regulators, suggesting that initiation of flagella assembly and motor activation relies on the abundance control of only a few structural key components that are required for the formation of the MS- and C-ring and the flagellar type III secretion system. We identified FlrA-independent promoters driving expression of the regulators of flagellar number and positioning, FlhF and FlhG. Reduction of the gene expression levels from these promoters resulted in the emergence of hyperflagellation. This finding indicates that basal expression is required to adjust the flagellar counter in Shewanella. This is adding a deeper layer to the regulation of flagellar synthesis and assembly.

Implementation of the ß-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol.

Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized ß-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as 'plug-and-play' module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.

Asparagine Uptake: a Cellular Strategy of Methylocystis to Combat Severe Salt Stress.

Methylocystis spp. are known to have a low salt tolerance (≤1.0% NaCl). Therefore, we tested various amino acids and other well-known osmolytes for their potential to act as an osmoprotectant under otherwise growth-inhibiting NaCl conditions. Adjustment of the medium to 10 mM asparagine had the greatest osmoprotective effect under severe salinity (1.50% NaCl), leading to partial growth recovery of strain SC2. The intracellular concentration of asparagine increased to 264 ± 57 mM, with a certain portion hydrolyzed to aspartate (4.20 ± 1.41 mM). In addition to general and oxidative stress responses, the uptake of asparagine specifically induced major proteome rearrangements related to the KEGG level 3 categories of "methane metabolism," "pyruvate metabolism," "amino acid turnover," and "cell division." In particular, various proteins involved in cell division (e.g., ChpT, CtrA, PleC, FtsA, FtsH1) and peptidoglycan synthesis showed a positive expression response. Asparagine-derived 13C-carbon was incorporated into nearly all amino acids. Both the exometabolome and the 13C-labeling pattern suggest that in addition to aspartate, the amino acids glutamate, glycine, serine, and alanine, but also pyruvate and malate, were most crucially involved in the osmoprotective effect of asparagine, with glutamate being a major hub between the central carbon and amino acid pathways. In summary, asparagine induced significant proteome rearrangements, leading to major changes in central metabolic pathway activity and the sizes of free amino acid pools. In consequence, asparagine acted, in part, as a carbon source for the growth recovery of strain SC2 under severe salinity. IMPORTANCE Methylocystis spp. play a major role in reducing methane emissions into the atmosphere from methanogenic wetlands. In addition, they contribute to atmospheric methane oxidation in upland soils. Although these bacteria are typical soil inhabitants, Methylocystis spp. are thought to have limited capacity to acclimate to salt stress. This called for a thorough study into potential osmoprotectants, which revealed asparagine as the most promising candidate. Intriguingly, asparagine was taken up quantitatively and acted, at least in part, as an intracellular carbon source under severe salt stress. The effect of asparagine as an osmoprotectant for Methylocystis spp. is an unexpected finding. It may provide Methylocystis spp. with an ecological advantage in wetlands, where these methanotrophs colonize the roots of submerged vascular plants. Collectively, our study offers a new avenue into research on compounds that may increase the resilience of Methylocystis spp. to environmental change.

A small Ustilago maydis effector acts as a novel adhesin for hyphal aggregation in plant tumors.

The biotrophic basidiomycete fungus Ustilago maydis causes smut disease in maize. Hallmarks of the disease are characteristic large tumors in which dark pigmented spores are formed. Here, we functionally characterized a novel core effector lep1 (late effector protein 1) which is highly expressed during tumor formation and contributes to virulence. We characterize lep1 mutants, localize the protein, determine phenotypic consequences upon deletion as well as constitutive expression, and analyze relationships with the repellent protein Rep1 and hydrophobins. In tumors, lep1 mutants show attenuated hyphal aggregation, fail to undergo massive late proliferation and produce only a few spores. Upon constitutive expression, cell aggregation is induced and the surface of filamentous colonies displays enhanced hydrophobicity. Lep1 is bound to the cell wall of biotrophic hyphae and associates with Rep1 when constitutively expressed in hyphae. We conclude that Lep1 acts as a novel kind of cell adhesin which functions together with other surface-active proteins to allow proliferation of diploid hyphae as well as for induction of the morphological changes associated with spore formation.

Baseplate variability of Vibrio cholerae chemoreceptor arrays.

The chemoreceptor array, a remarkably ordered supramolecular complex, is composed of hexagonally packed trimers of receptor dimers networked by a histidine kinase and one or more coupling proteins. Even though the receptor packing is universal among chemotactic bacteria and archaea, the array architecture has been extensively studied only in selected model organisms. Here, we show that even in the complete absence of the kinase, the cluster II arrays in Vibrio cholerae retain their native spatial localization and the iconic hexagonal packing of the receptors with 12-nm spacing. Our results demonstrate that the chemotaxis array is versatile in composition, a property that allows auxiliary chemotaxis proteins such as ParP and CheV to integrate directly into the assembly. Along with its compositional variability, cluster II arrays exhibit a low degree of structural stability compared with the ultrastable arrays in Escherichia coli We propose that the variability in chemoreceptor arrays is an important mechanism that enables the incorporation of chemotaxis proteins based on their availability.

A Serial Sample Processing Strategy with Improved Performance for in-Depth Quantitative Analysis of Type III Secretion Events in Pseudomonas aeruginosa.

The efficient analysis of secretomes is important to study the mechanisms of bacterial secretion. However, secretome analysis of bacteria that rely on rich media for optimal secretion via modern quantitative shotgun proteomics workflows is often hampered by the higher degree of sample impurities. This may be a reason for the low number of quantitative secretome investigations in such cases. We assessed the efficiency and amenability for rich media secretome analysis of different workflows including precipitation, SP3, and a combined, serial workflow. Using the model organism Pseudomonas aeruginosa, we found that the combined TCA-SP3 strategy outperformed the other tested methods on all monitored qualitative and quantitative levels. This method proved to be most efficient in the recovery of proteins secreted by the type III secretion system (T3SS), including all known effector proteins and secretion machinery components. We monitored the compositional changes of secretome samples over time, and observed a strong increase in the secreted protein fraction by the T3SS 2 to 3 h after T3SS induction. Our study conceptually illustrates how the combination of TCA precipitation and SP3 results in orthogonality in depleting sample impurities accompanied by improved chromatographic peptide separation, and more efficient MS detection with improved quantification parameters.

Specific proteomic adaptation to distinct environments in Vibrio parahaemolyticus includes significant fluctuations in expression of essential proteins.

Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analysing the proteome of our model organism Vibrio parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony, there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programmes that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life-style, environment and differentiation state of the bacterium.

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs.

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H2 to the culture headspace had the most significant positive effect on the growth yield under CH4 (6%) and O2 (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH4, while CH4 consumption was significantly reduced. Regardless of H2 addition, CH4 utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O2/CH4 mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H2 addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H2 (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H2 may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH4, such as the production of high-quality feed protein.

High-throughput enrichment of temperature-sensitive argininosuccinate synthetase for two-stage citrulline production in E. coli.

Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria.

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms.

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface.IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

Osmosensing by the bacterial PhoQ/PhoP two-component system.

The PhoQ/PhoP two-component system plays an essential role in the response of enterobacteria to the environment of their mammalian hosts. It is known to sense several stimuli that are potentially associated with the host, including extracellular magnesium limitation, low pH, and the presence of cationic antimicrobial peptides. Here, we show that the PhoQ/PhoP two-component systems of Escherichia coli and Salmonella can also perceive an osmotic upshift, another key stimulus to which bacteria become exposed within the host. In contrast to most previously established stimuli of PhoQ, the detection of osmotic upshift does not require its periplasmic sensor domain. Instead, we show that the activity of PhoQ is affected by the length of the transmembrane (TM) helix as well as by membrane lateral pressure. We therefore propose that osmosensing relies on a conformational change within the TM domain of PhoQ induced by a perturbation in cell membrane thickness and lateral pressure under hyperosmotic conditions. Furthermore, the response mediated by the PhoQ/PhoP two-component system was found to improve bacterial growth recovery under hyperosmotic stress, partly through stabilization of the sigma factor RpoS. Our findings directly link the PhoQ/PhoP two-component system to bacterial osmosensing, suggesting that this system can mediate a concerted response to most of the established host-related cues.