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Tetracycline repressor (TetR) family regulators (TFRs) are the largest group of DNA-binding transcription factors and are widely distributed in bacteria and archaea. TFRs play vital roles in controlling the expression of various genes and regulating diverse physiological processes. Recently, a TFR protein Pseudomonas virulence regulator A (PvrA), was identified from Pseudomonas aeruginosa as the transcriptional activator of genes involved in fatty acid utilization and bacterial virulence. Here, we show that PvrA can simultaneously bind to multiple pseudo-palindromic sites and upregulate the expression levels of target genes. Cryo-electron microscopy (cryo-EM) analysis indicates the simultaneous DNA recognition mechanism of PvrA and suggests that the bound DNA fragments consist of a distorted B-DNA double helix. The crystal structure and functional analysis of PvrA reveal a hinge region that secures the correct domain motion for recognition of the promiscuous promoter. Additionally, our results showed that mutations disrupting the regulatory hinge region have differential effects on biofilm formation and pyocyanin biosynthesis, resulting in attenuated bacterial virulence. Collectively, these findings will improve the understanding of the relationship between the structure and function of the TetR family and provide new insights into the mechanism of regulation of P. aeruginosa virulence., (© 2023. Science China Press.)

The regulation of carbon metabolism and virulence is critical for the rapid adaptation of pathogenic bacteria to host conditions. In Pseudomonas aeruginosa, RccR is a transcriptional regulator of genes involved in primary carbon metabolism and is associated with bacterial resistance and virulence, although the exact mechanism is unclear. Our study demonstrates that PaRccR is a direct repressor of the transcriptional regulator genes mvaU and algU. Biochemical and structural analyses reveal that PaRccR can switch its DNA recognition mode through conformational changes triggered by KDPG binding or release. Mutagenesis and functional analysis underscore the significance of allosteric communication between the SIS domain and the DBD domain. Our findings suggest that, despite its overall structural similarity to other bacterial RpiR-type regulators, RccR displays a more complex regulatory element binding mode induced by ligands and a unique regulatory mechanism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudomonas aeruginosa is a highly pathogenic bacterium known for its ability to sense and coordinate the production of virulence factors in response to host immune responses. However, the regulatory mechanisms underlying this process have remained largely elusive. In this study, we investigate the two-component system CprRS in P. aeruginosa and unveil the crucial role of the sensor protein CprS in sensing the human host defense peptide LL-37, thereby modulating bacterial virulence. We demonstrate that CprS acts as a phosphatase in the presence of LL-37, leading to the phosphorylation and activation of the response regulator CprR. The results prove that CprR directly recognizes a specific sequence within the promoter region of the HigBA toxin-antitoxin system, resulting in enhanced expression of the toxin HigB. Importantly, LL-37-induced HigB expression promotes the production of type III secretion system effectors, leading to reduced expression of proinflammatory cytokines and increased cytotoxicity towards macrophages. Moreover, mutations in cprS or cprR significantly impair bacterial survival in both macrophage and insect infection models. This study uncovers the regulatory mechanism of the CprRS system, enabling P. aeruginosa to detect and respond to human innate immune responses while maintaining a balanced virulence gene expression profile. Additionally, this study provides new evidence and insights into the complex regulatory system of T3SS in P. aeruginosa within the host environment, contributing to a better understanding of host-microbe communication and the development of novel strategies to combat bacterial infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

AlgW, a membrane-bound periplasmic serine protease belonging to the HtrA protein family, is a key regulator of the regulated intramembrane proteolysis (RIP) pathway and is responsible for transmitting the envelope stress signals in Pseudomonas aeruginosa The AlgW PDZ domain senses and binds the C-terminal of mis-localized outer membrane proteins (OMPs) or periplasmic protein MucE, leading to catalytic activation of the protease domain. While AlgW is functionally well studied, its exact activation mechanism remains to be elucidated. Here, we show that AlgW is a novel HtrA protease that can be biochemically activated by both peptide and lipid signals. Compared with the corresponding homologue DegS in Escherichia coli , AlgW exhibits a distinct substrate specificity and regulation mechanism. Structural, biochemical, and mutagenic analyses revealed that, by specifically binding to the C-terminal decapeptide of MucE, AlgW could adopt more relaxed conformation and obtain higher activity than with tripeptide activation. We also investigated the regulatory mechanism of the L A loop in AlgW and proved that the unique structural feature of this region was responsible for the distinct enzymatic property of AlgW. These results demonstrate the unique and diverse activation mechanism of AlgW, which P. aeruginosa may utilize to enhance its adaptability to environmental stress. IMPORTANCE HtrA-family proteases are commonly employed to sense the protein folding stress and activate the regulated intramembrane proteolysis (RIP) cascade in Gram-negative bacteria. Here, we reveal the unique dual-signal activation and dynamic regulation properties of AlgW, an HtrA-type protease triggering the AlgU stress-response pathway, which controls alginate production and mucoid conversion in Pseudomonas aeruginosa The structural and functional data offer insights into the molecular basis underlying the transition of different activation states of AlgW in response to different effectors. Probing these unique features provides an opportunity to correlate the diverse regulation mechanism of AlgW with the high adaptability of P. aeruginosa to environmental changes during infection., (Copyright © 2021 Li et al.)

MucA and MucB are critical negative modulators of sigma factor AlgU and regulate the mucoid conversion of Pseudomonas aeruginosa. Previous studies have revealed that lipid signals antagonize MucA-MucB binding. Here we report the crystal structure of MucB in complex with the periplasmic domain of MucA and polyethylene glycol (PEG), which unveiled an intermediate state preceding the MucA-MucB dissociation. Based on the biochemical experiments, the aliphatic side chain with a polar group was found to be of primary importance for inducing MucA cleavage. These results provide evidence that the hydrophobic cavity of MucB is a primary site for sensing lipid molecules and illustrates the detailed control of conformational switching within MucA-MucB in response to lipophilic effectors.

Pseudomonas aeruginosa, a common nosocomial pathogen, relies on siderophores to acquire iron, crucial for its survival in various environments and during host infections. However, understanding the molecular mechanisms of siderophore regulation remains incomplete. In this study, we found that the BfmRS two-component system, previously associated with biofilm formation and quorum sensing, is essential for siderophore regulation under high osmolality stress. Activated BfmR directly bound to the promoter regions of pvd, fpv, and femARI gene clusters, thereby activating their transcription and promoting siderophore production. Subsequent proteomic and phenotypic analyses confirmed that deletion of BfmRS reduces siderophore-related proteins and impairs bacterial survival in iron-deficient conditions. Furthermore, phylogenetic analysis demonstrated the high conservation of the BfmRS system across Pseudomonas species, functional evidences also indicated that BfmR homologues from Pseudomonas putida KT2440 and Pseudomonas sp. MRSN12121 could bind to the promoter regions of key siderophore genes and osmolality-mediated increases in siderophore production were observed. This work illuminates a novel signaling pathway for siderophore regulation and enhances our understanding of siderophore-mediated bacterial interactions and community establishment. A comprehensive study reveals that the two-component system BfmRS senses high osmotic stress and transcriptionally activates the expression of siderophore genes, which is essential for environmental adaption of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Summary: Type II toxin‐antitoxin (TA) systems modulate many essential cellular processes in prokaryotic organisms. Recent studies indicate certain type II antitoxins also transcriptionally regulate other genes, besides neutralizing toxin activity. Herein, we investigated the diverse transcriptional repression properties of type II TA antitoxin PaHigA from Pseudomonas aeruginosa. Biochemical and functional analyses showed that PaHigA recognized variable pseudopalindromic DNA sequences and repressed expression of multiple genes. Furthermore, we presented high resolution structures of apo‐PaHigA, PaHigA‐PhigBA and PaHigA‐Ppa2440 complex, describing how the rearrangements of the HTH domain accounted for the different DNA‐binding patterns among HigA homologues. Moreover, we demonstrated that the N‐terminal loop motion of PaHigA was associated with its apo and DNA‐bound states, reflecting a switch mechanism regulating HigA antitoxin function. Collectively, this work extends our understanding of how the PaHigB/HigA system regulates multiple metabolic pathways to balance the growth and stress response in P. aeruginosa and could guide further development of anti‐TA oriented strategies for pathogen treatment. [ABSTRACT FROM AUTHOR]