Molecular mechanism of the one-component regulator RccR on bacterial metabolism and virulence.

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.

Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution.

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree of recognition of small molecules and the complex composition of biofluids present significant obstacles for current detection technologies. Although nanopore sensing excels in the analysis of small molecules, the direct detection of small molecules in complex biofluids remains a challenge. In this study, we present a method for sensing the small molecule drug gentamicin in whole blood based on the mechanosensitive channel of small conductance in Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect gentamicin and distinguish its main components with only a monomethyl difference. The 'molecular sieve' structure of PaMscS enables the direct measurement of gentamicin in human whole blood within 10 min. Furthermore, a continuous monitoring device constructed based on PaMscS achieved continuous monitoring of gentamicin in live rats for approximately 2.5 h without blood consumption, while the drug components can be analyzed in situ. This approach enables rapid and convenient drug monitoring with single-molecule level resolution, which can significantly lower the threshold for drug concentration monitoring and promote more efficient drug use. Moreover, this work also lays the foundation for the future development of continuous monitoring technology with single-molecule level resolution in the living body.

Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.