Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity.

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.

Characterization of the bacteriocins and the PrtR regulator in a plant-associated Pseudomonas strain.

The emergence of antibiotic resistant bacterial strains demands the development of new antimicrobial agents. In the last decades, bacteriocins have gained significant interest due to their potential application as biopreservatives in the food industry and as therapeutic agents in medicine. Recent studies project the use of these antimicrobials in agriculture as biocontrol agents. The characterization of bacteriocins and their genetic regulation, however, have been scarcely studied in plant-associated bacteria. In this report, an in-silico and proteomic analysis was performed to identify the bacteriocins produced by Pseudomonas fluorescens SF4c. More than one functional bacteriocin was detected in this strain (S-type bacteriocins and phage-tail-like bacteriocins [tailocins]). It is known that the regulator PrtR represses bacteriocin production in P. aeruginosa under normal condition. However, the mechanism for tailocin regulation remains unknown in plant-associated pseudomonads. In this work, an orthologue of the prtR of P. aeruginosa was identified in the SF4c-tailocin cluster and a prtR null mutant constructed. The expression and production of tailocins was abolished in this mutant; thus evidencing that, unlike P. aeruginosa, PrtR is a positive regulator of tailocins expression in P. fluorescens.

Effect of a Pseudomonas fluorescens tailocin against phytopathogenic Xanthomonas observed by atomic force microscopy.

Phage tail-like bacteriocins, called tailocins, represent a class of protein complexes produced by a multitude of bacteria. Pseudomonas fluorescens SF4c, a strain isolated from wheat rhizosphere, produces a bacteriocin similar to phage tail-like pyocins of Pseudomonas aeruginosa. This tailocin has antimicrobial activity against several phytopathogenic strains of the genus Xanthomonas and Pseudomonas. In this work, the effect of the SF4c tailocin on the phytopathogenic strain X. axonopodis pv vesicatoria Xcv Bv5-4a was analyzed through Atomic Force Microscopy (AFM). We demonstrated that tailocins adhere and cause damage to the cell envelope of strain Xcv Bv5-4a. This results in a rapid leakage of intracellular materials, with the subsequent decrease of cell volume. Finally, lysis of sensitive bacteria occurs. This study provides, to our knowledge, the first evidence about the effect of a tailocin analyzed by AFM. Further studies are in progress to evaluate the use of SF4c tailocin in the biocontrol of bacterial spot on tomato.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189

Abstract Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189

Abstract Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.(AU)