Unraveling the regulation of pyocyanin synthesis by RsmA through MvaU and RpoS in Pseudomonas aeruginosa ID4365

Abstract

Pyocyanin is a phenazine with redox activity produced by Pseudomonas aeruginosa that is harmful to other bacteria and eukaryotic organisms by generating reactive oxygen species. Gene regulation of pyocyanin synthesis has been addressed in the PAO1 and PA14 strains and involves the three‐quorum sensing systems Las, Rhl, and Pqs; the regulators RsaL, MvaU, and RpoS, and the posttranscriptional Rsm system, among others. Here, we determined how RsmA regulates pyocyanin synthesis in P. aeruginosa ID4365, an overproducer strain. We found that, in the protease peptone glucose ammonium salts medium, rsmA inactivation increases pyocyanin production compared with the wild‐type strains ID4365, PAO, and PA14. We showed that RsmA regulates inversely the expression of both phz operons involved in pyocyanin synthesis; particularly the phz2 operon is positively regulated at the transcriptional level indirectly through MvaU. In addition, we found that the phz1 operon contributes mainly to pyocyanin synthesis and that RsmA negatively regulates phzM and phzS expression. Finally, we showed that translation of the sigma factor RpoS is positively regulated by RsmA, and the expression of rpoS under an independent promoter decreases pyocyanin production in the IDrsmA strain. These results indicate that RsmA regulates not only the genes for pyocyanin production but also their regulators.

The alternative sigma factor RpoS regulates Pseudomonas aeruginosa quorum sensing response by repressing the pqsABCDE operon and activating vfr

“…To explore the effect of different concentrations of RpoS on the QS network in P. aeruginosa , we constructed a derivative of PAO1 with rpoS gene fused to the arabinose‐inducible promoter P BAD of E. coli (Figure S1). The fusion lacks the rpoS 5′ untranslated region (Montelongo‐Martínez et al., 2023) as it is the regulatory target of several small RNAs (Carloni et al., 2017; Lu et al., 2018). The P BAD rpoS construction was inserted in the rpoS native locus, leaving the upstream nlpD gene intact (which holds the native rpoS promoter in its coding region).…”

“…By densitometry analyses of Western blot assays, we determined that 0.05% and 1% of arabinose induced rpoS expression to reach 26% and 150% of RpoS protein levels compared to the wild‐type PAO1 strain (100%), respectively (Figure S2). A Δ rpoS mutant (Montelongo‐Martínez et al., 2023) was used as the condition of 0% RpoS to replicate the effects of lacking rpoS gene on the QS response reported in previous publications (Schuster et al., 2004; Suh et al., 1999; Whiteley et al., 2000). The different RpoS levels produced in P. aeruginosa did not affect its growth and cell viability (Figure S3a,b).…”

“…The resulting plasmid was named pJETApr‐nlpD. The plasmid pHERD20T‐rpoS (Montelongo‐Martínez et al., 2023), which contains the P. aeruginosa rpoS gene fused to the E. coli P BAD promoter, was used as a template to amplify a 2376 DNA fragment (which contains the araC gene and the P BAD :: rpoS fusion) using the oligonucleotides araCTHindIIIFw and rpoSHindIIIRv. The PCR product was digested with HindIII, gel purified, and cloned into the respective restriction site of the pJETnlpD‐Apr plasmid (downstream of the Apr cassette).…”

“…The protein concentration of soluble fractions was determined by the Bradford method (Bradford, 1976), and 40 μg of total protein were used for Western blot assays. RpoS protein was detected as previously reported (Montelongo-Martínez et al, 2023), using a polyclonal antiserum anti-RpoS of A. vinelandii (Muriel-Millán et al, 2017). LasR and RhlR proteins were detected using purified antibodies raised in rabbits previously reported (Grosso-Becerra et al, 2014;Medina et al, 2003) at dilutions 1:1000 and 1:200, respectively.…”

Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa

“…Detection of RpoS levels in A. vinelandii was carried out as previously described (Muriel‐Millán et al, 2017) from cells grown at 8 h in PY medium. For detection of RpoS in P. aeruginosa strains, the antiserum anti‐RpoS of A. vinelandii was used, as previously reported (Montelongo‐Martínez et al, 2022) in cells grown in LB medium at OD 600nm 0.2–0.3 (exponential phase). For the determination of in vivo stability of RpoS, the strains were grown as mentioned above and protein synthesis was stopped by adding spectinomycin or streptomycin (1 mg/mL) to cultures of A. vinelandii or P. aeruginosa , respectively.…”