Genomic and phenotypic characterization of Pseudomonas sp. GOM7, a novel marine bacterial species with antimicrobial activity against multidrug-resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) represents a serious threat to global health. The development of new drugs to combat infections caused by bacteria resistant to multiple or even all available antibiotics is urgent. Most antibiotics used up to date have been identified from soil microorganisms. The marine environment represents an alternative source with great potential for the identification of microorganisms that produce bioactive molecules, including antibiotics. In this study, we analyzed the antibacterial activity of a collection of 82 bacterial strains isolated from marine water and sediment samples collected from the Southwestern Gulf of Mexico. Eight of the marine isolates inhibited the growth of different pathogenic bacteria, seven of which were identified as presumptive Pseudomonas aeruginosa. Interestingly, genome sequencing and phylogenetic analysis revealed that the remaining marine isolate showing antibacterial activity is a novel Pseudomonas species that we denominated Pseudomonas sp. GOM7, which was not pathogenic in the Galleria mellonella infection model in the conditions tested. Notably, Pseudomonas sp. GOM7 inhibited the growth of multidrug and methicillin-resistant strains of the priority pathogen Staphylococcus aureus. Our results show that the anti-S. aureus compound(s) produced by Pseudomonas sp. GOM7 can be extracted from the culture supernatant of this bacterium with the organic solvent ethyl acetate. Annotation of the Pseudomonas sp. GOM7 genome revealed the presence of several biosynthetic gene clusters predicted to code for possible antimicrobial compounds. Our results further highlight the potential of bacteria from the Gulf of Mexico as a source of novel antimicrobials.

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

In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

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

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.

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins.

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.