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The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ , which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis. Escherichia coli secretes high-affinity Fe 3+ chelators to solubilize and transport chelated Fe 3+ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe 2+ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe 2+ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB . In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon. IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ , which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Unlike eukaryotes, bacteria undergo large changes in osmolality and cytoplasmic pH. It has been described that during acid stress, bacteria internal pH promptly acidifies, followed by recovery. Here, using pH imaging in single living cells, we show that following acid stress, bacteria maintain an acidic cytoplasm and the osmotic stress transcription factor OmpR is required for acidification. The activation of this response is non-canonical, involving a regulatory mechanism requiring the OmpR cognate kinase EnvZ, but not OmpR phosphorylation. Single cell analysis further identifies an intracellular pH threshold ~6.5. Acid stress reduces the internal pH below this threshold, increasing OmpR dimerization and DNA binding. During osmotic stress, the internal pH is above the threshold, triggering distinct OmpR-related pathways. Preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. These results further emphasize the advantages of single cell analysis over studies of population averages. OmpR is a transcription factor activated in acid and osmotic responses of Gram-negative bacteria, leading to acidification of the bacterial cytoplasm. Here the authors use single cell pH imaging to define the role of OmpR-regulated genes in the acidification response to osmotic and acid stress of Salmonella and E. coli .

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks. Phosphorylation networks are frequently at the heart of complex cellular decision making. Here the authors engineer synthetic phosphorylation devices with feedback regulation in mammalian cells and demonstrate how to use these to achieve tunable and robust control of cell behaviours.

PurposeTo characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates.MethodsCPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing.ResultsA reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers.ConclusionsThe reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

ABSTRACT Acidovorax citrulli, the causal pathogen of bacterial fruit blotch of cucurbits, relies on a functional type III secretion system (T3SS) for pathogenicity. Two‐component systems (TCSs) are primary signal transduction mechanisms for bacteria to detect and adapt to various environmental conditions. However, the role of TCS on regulating T3SS and other virulence factors in response to environmental stimuli is still poorly understood in A. citrulli. Here, we report the identification of a conserved TCS, OmpR/EnvZ, involved in hypersensitive response (HR) induction in Nicotiana benthamiana by screening a transposon‐insertion library in the group II strain xjL12 of A. citrulli. Transcription analysis confirmed that OmpRAc/EnvZAc was upregulated in response to elevated osmotic pressure, low and high pH conditions, and host environment. Deletions of envZAc, ompRAc, or both envZAc and ompRAc in A. citrulli attenuated virulence to melon seedlings and mature leaf tissues, and delayed HR in N. benthamiana. OmpRAc was activated by EnvZAc and directly bound to the promoter region of hrpG, a major regulator of T3SS. This binding activated hrpG transcription and promoted T3SS assembly in T3SS‐inducing medium, XVM2. Additionally, the OmpRAc/EnvZAc mutants of A. citrulli displayed reduced swimming motility due to impaired flagella formation, but also had enhanced biofilm formation and exopolysaccharide production. OmpRAc/EnvZAc regulation of these virulence factors in A. citrulli depended on its own conserved phosphorylation sites. This work illuminates a signalling pathway for regulating the T3SS and provides insights into the OmpR/EnvZ‐mediated virulence regulatory network in A. citrulli. Acidovorax citrulli OmpR/EnvZ responds to extracellular pH and osmolarity, controls the T3SS by binding to the hrpG promoter, and is involved in regulating flagellar biogenesis, biofilm, and exopolysaccharide production.

Soil-inhabiting streptomycetes are nature’s medicine makers, producing over half of all known antibiotics and many other bioactive natural products. However, these bacteria also produce many volatiles, molecules that disperse through the soil matrix and may impact other (micro)organisms from a distance. Here, we show that soil- and surface-grown streptomycetes have the ability to kill bacteria over long distances via air-borne antibiosis. Our research shows that streptomycetes do so by producing surprisingly high amounts of the low-cost volatile ammonia, dispersing over long distances to inhibit the growth of Gram-positive and Gram-negative bacteria. Glycine is required as precursor to produce ammonia, and inactivation of the glycine cleavage system nullified ammonia biosynthesis and concomitantly air-borne antibiosis. Reduced expression of the porin master regulator OmpR and its cognate kinase EnvZ is used as a resistance strategy by E. coli cells to survive ammonia-mediated antibiosis. Finally, ammonia was shown to enhance the activity of canonical antibiotics, suggesting that streptomycetes adopt a low-cost strategy to sensitize competitors for antibiosis from a distance.

Cefiderocol, a novel siderophore cephalosporin, demonstrates promising in vitro activity against multidrug-resistant Gram-negative bacteria, including carbapenemase-producing strains. Nonetheless, only a few reports are available regarding the acquisition of resistance in clinical settings, primarily due to its recent usage. This study aimed to investigate cefiderocol resistance using an in vitro resistance development model to gain insights into the underlying molecular resistance mechanisms. Cefiderocol susceptible reference strains ( Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa ) and a clinical Acinetobacter baumannii complex isolate were exposed to increasing cefiderocol concentrations using a high-throughput resistance development model. Cefiderocol susceptibility testing was performed using broth microdilution. Whole-genome sequencing was employed to identify newly acquired resistance mutations. Our in vitro resistance development model led to several clones of strains exhibiting cefiderocol resistance, with MIC values 8-fold to 512-fold higher than initial levels. In total, we found 42 different mutations in 26 genes, of which 35 could be described for the first time. Putative loss-of-function mutations were detected in the envZ , tonB , and cirA genes in 13 out of 17 isolates, leading to a decrease in cefiderocol influx. Other potential resistance mechanisms included multidrug efflux pumps ( baeS , czcS , nalC ), antibiotic-inactivating enzymes ( ampR , dacB ), and target mutations in penicillin-binding-protein genes ( mrcB ). This study reveals new insights into underlying molecular resistance mechanisms against cefiderocol. While mutations leading to reduced influx via iron transporters was the most frequent resistance mechanism, we also detected several other novel resistance mutations causing cefiderocol resistance.

Histidine kinases are key regulators in the bacterial two-component systems that mediate the cellular response to environmental changes. The vast majority of the sensor histidine kinases belong to the bifunctional HisKA family, displaying both kinase and phosphatase activities toward their substrates. The molecular mechanisms regulating the opposing activities of these enzymes are not well understood. Through a combined NMR and crystallographic study on the histidine kinase HK853 and its response regulator RR468 from Thermotoga maritima , here we report a pH-mediated conformational switch of HK853 that shuts off its phosphatase activity under acidic conditions. Such a pH-sensing mechanism is further demonstrated in the EnvZ-OmpR two-component system from Salmonella enterica in vitro and in vivo, which directly contributes to the bacterial infectivity. Our finding reveals a broadly conserved mechanism that regulates the phosphatase activity of the largest family of bifunctional histidine kinases in response to the change of environmental pH. Bacteria adapt to changing environmental conditions through signal transduction mediated by the two-component system (TCS). Here, the authors combine X-ray crystallography and NMR studies to characterize a pH-gated conformational switch that regulates the phosphatase activity of TCS bifunctional histidine kinases.

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853–RR468 and EnvZ–OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH. Bacterial two-component systems are composed of a sensor histidine kinase (HK) and an effector response regulator and upon signal detection, the HK autophosphorylates a conserved His residue. Here the authors structurally and functionally characterise two HKs, HK853–RR468 and EnvZ–OmpR, and find that the rotamer of the phosphorylatable catalytic His is not influenced by the environmental pH, ruling out an earlier proposed pH-gated model.

A thorough understanding of the mechanisms of Rcs and EnvZ/OmpR phosphorelay systems that allow Yersinia enterocolitica to thrive in various environments is crucial to prevent and control Y. enterocolitica infections. In this study, we showed that RcsB and OmpR have the ability to function differently in modulating a diverse array of physiological processes in Y. enterocolitica. The rcsB mutant stimulated flagella biosynthesis and increased motility, biofilm formation and c-di-GMP production by upregulating flhDC, hmsHFRS and hmsT. However, mutation in ompR exhibited a non-motile phenotype due to the lack of flagella. Biofilm formation was reduced and less c-di-GMP was produced through the downregulation of flhDC, hmsHFRS and hmsT expression when Y. enterocolitica was exposed to low osmolarity conditions. Furthermore, OmpR was identified to be important for Y. enterocolitica to grow in extreme temperature conditions. Importantly, ompR mutations in Y. enterocolitica were more sensitive to polymyxin B and sodium dodecyl sulfate than rcsB mutations. Since motility, biofilm formation and environmental tolerance are critical for bacterial colonization of the host, these findings indicated that OmpR is more critical than RcsB in shaping the pathogenic phenotype of Y. enterocolitica.