ATTREE, I. | bioRxiv Science

bioRxiv2025

Avirulent Pseudomonas aeruginosa T3SS-negative strains belonging to Clade 5 produce variable quantities of secondary metabolites

Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. P. aeruginosa secretes metalloproteases such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites -- pyocyanin, rhamnolipids, and pyoverdine -- varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including monorhamnolipids, highlighting their potential for biotechnological applications.

microbiology↗

bioRxiv2024

Functional and pangenomic exploration of Roc two-component regulatory systems identifies novel players across Pseudomonas species

The opportunistic pathogen Pseudomonas aeruginosa counts on a large collection of two-component regulatory systems (TCSs) to sense and adapt to changing environments. Among them, the Roc (Regulation of cup) system is a one-of-a-kind network of branched TCSs, composed of two histidine kinases (HKs) (RocS1 and RocS2) interacting with three response regulators (RRs) (RocA1, RocR and RocA2), which regulate virulence, antibiotic resistance and biofilm formation. Based on extensive work on the Roc system, previous data suggested the existence of other key regulators yet to be discovered. In this work, we identified PA4080, renamed RocA3, as a fourth RR that is activated by RocS1 and RocS2 and that positively controls the expression of the cupB operon. Comparative genomic analysis of the locus identified a gene - rocR3 - adjacent to rocA3 in a subpopulation of strains which encodes a protein with structural and functional similarity to the c-di-GMP phosphodiesterase RocR. Furthermore, we identified a fourth branch of the Roc system consisting of the PA2583 HK, renamed RocS4, and of the Hpt protein HptA. Using a bacterial two-hybrid system, we showed that RocS4 interacts with HptA, which in turn interacts with RocA1, RocA2 and RocR3. Finally, we mapped the pangenomic RRs repertoire establishing a comprehensive view of the plasticity of such regulators among clades of the species. Overall, our work provides a comprehensive inter-species definition of the Roc system, nearly doubling the number of proteins known to be involved in this interconnected network of TCSs controlling pathogenicity in Pseudomonas species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/618891v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1059b56org.highwire.dtl.DTLVardef@1195eeaorg.highwire.dtl.DTLVardef@a67f45org.highwire.dtl.DTLVardef@ed9ed5_HPS_FORMAT_FIGEXP M_FIG C_FIG ABBREVIATED SUMMARYRoc system account for a particularly interconnected yet incomplete network of two-component regulatory system involved in the virulence of Pseudomonas aeruginosa. Our work identified the missing RocA3 regulator and propose new players of the system delineating their conservation between the clade of the species.

microbiology↗