Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C₄-HSL (rhlR–rhlI-dependent QS) and 3-oxo-C₁₂-HSL (lasR–lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C₁₂-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.     

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a K_m of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The K_d for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (K_s) for ThDP and (K_c) for Mg²⁺ were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC₅₀ values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (−7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.     

Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity

Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty acids, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level

The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa

Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC₅₀ values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/accumulation assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence assay was established to measure the time-dependent accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM-MexCDOprJ-MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP accumulation was PAO314 > PAO200 > PAO1. Subsequently, the established assay conditions were applied to a radiolabeled assay format using ³H-labeled ciprofloxacin. At the concentration tested, the accumulation of [³H]ciprofloxacin approached a plateau after 15 min and the amount of accumulation in PAO314 was higher (∼2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting.     

Functional characterization of ExFadLO,an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.     

Simultaneous Cr(VI) reduction and phenol degradation in pure cultures of Pseudomonas aeruginosa CCTCC AB91095

Simultaneous Cr(VI) reduction and phenol degradation were investigated in a reactor containing Pseudomonas aeruginosa CCTCC AB91095. Phenol was used as carbon source. P.aeruginosa utilized metabolites formed during phenol degradation as energy source for Cr(VI) reduction. Cr(VI) inhibited both Cr(VI) reduction and phenol degradation when Cr(VI) concentration exceeded the optimum value (20 mg/L), whereas phenol enhanced both Cr(VI) reduction and phenol degradation below the optimum initial concentration of 100 mg/L. Cr(III) was the predominant product of Cr(VI) reduction in cultures after incubation for 24 h. Both Cr(VI) reduction and phenol degradation were influenced by the amount of inocula. The concentration of Cr(VI) and phenol declined quickly from 20, 100 to 3.36, 29.51 mg/L in cultures containing of 5% (v/v) inoculum after incubation for 12 h, respectively. The whole study showed that P. aeruginosa is promising for the reduction of toxic Cr(VI) and degradation of organic pollutants simultaneously in the mineral liquid medium.     

Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers

Pseudomonas aeruginosa K187, a protease- and chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄ and (GlcNAc)₅ were 1.18 mg/mL, 0.76 mg/mL, 1.02 mg/mL, 0.93 mg/mL and 0.90 mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications.     

Identification of Pseudomonas aeruginosa in water-bottling plants on the basis of procedures included in ISO 16266:2006

ISO 16266:2006 provides a standardized procedure for the isolation and identification of Pseudomonas aeruginosa in waters. In some cases the method described in this ISO is not conclusive enough to confirm or discard the presence of this opportunistic human pathogen. In this study the capacity of the procedure described in ISO 16266:2006 to identify presumptive P. aeruginosa isolates was evaluated.Forty-one presumptive P. aeruginosa strains, previously isolated from water-bottling plants following ISO 16266:2006, were submitted to all the tests recommended by ISO 16266:2006 (Cetrimide agar with nalidixic acid, King B agar, Acetamide broth and Oxidase test). Additional tests that have been widely used for the identification of P. aeruginosa were also performed (Asparagine broth and King A agar). Furthermore, we also conducted the non-compulsory ISO 16266:2006 assay to study the capacity of the strains to grow at 4 °C and 42 °C. Finally, all the strains were biochemically phenotyped with PhP-48 plates (Bactus AB, Sweden) and API 20NE galleries (Biomérieux, France), and their 16 rRNA gene was sequenced.ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained.Growth in Asparagine broth should be discarded as a confirmative test as it showed false negatives and false positives. In contrast, API 20NE galleries correctly identified all the confirmed isolates.King A medium and growth tests at 4 °C and 42 °C correctly discriminated all the studied strains, even the two that were not identified with the basic ISO 16266:2006 tests.Given that King A medium and growth tests at 4 °C and 42 °C are straightforward, rapid, and inexpensive, it is strongly recommended that they be used for routine confirmation of P. aeruginosa when applying ISO 16266:2006.     

Monovalent and bivalent N-fucosyl amides as high affinity ligands for Pseudomonas aeruginosa PA-IIL lectin

The adhesion of bacteria to human glycoconjugates can be inhibited by soluble glycomimetics that compete with the natural target. Four monovalent and one divalent α-fucosyl amides have been tested for their affinity for a fucose-binding lectin from Pseudomonas aeruginosa. Isothermal calorimetric titrations demonstrated that they bind to the lectin in the micromolar range, with highest affinity for the divalent ligand. Molecular modelling established that, compared to Ο-fucoside compounds, the glycomimetic amide group resulted in the loss of water-bridged hydrogen bonds that could be partially compensated by additional contact of the aglycone with the protein surface.     

Duality of the carbohydrate-recognition system of Pseudomonas aeruginosa-II lectin (PA-IIL)

The study of Pseudomonas aeruginosa-II lectin (PA-IIL) complexes with Man derivatives as a recognition factor has been neglected since its monomer is a very weak ligand. Here, the roles of Man oligomers and complexes in PA-IIL carbohydrate-recognition were studied by both enzyme-linked lectinosorbent and inhibition assays. From the results obtained, it is proposed that high density weak -OH conformation as seen in yeast mannan is also an important PA-IIL recognition factor. This finding provides a peculiar concept of the duality of PA-IIL recognition system for lFucα1→ and related complexes and for high density Manα1→ complexes present in polymannosylated target macromolecules.     

Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament

Pseudomonas aeruginosa is one of leading opportunistic pathogens in humans and its movement is driven by a flagellar filament that is constituted through the polymerization of a single protein, FliC flagellin (paFliC). paFliC is an essential virulence factor for the colonization of P. aeruginosa. paFliC activates innate immune responses via its recognition by Toll-like receptor 5 (TLR5) and adaptive immunity in the host. Thus, paFliC has been a vaccine candidate to prevent P. aeruginosa infection, particularly for cystic fibrosis patients. To provide structural information on paFliC and its flagellar filament, we have determined the crystal structure of paFliC, which contains the conserved D1 and variable D2 domains, at 2.1 Å resolution. As observed for Salmonella FliC, the paFliC D1 domain is folded into a rod-shaped structure, and paFliC was demonstrated by gel filtration and native PAGE analyses to directly interact with TLR5. Moreover, a structural model of the paFliC-TLR5 complex suggests that paFliC D1 would provide major TLR5-binding sites, similar to Salmonella FliC. In contrast to the D1 domain, the paFliC D2 domain exhibits a unique structure of two β-sheets and one α-helix that has not been found in other flagellins. An in silico construction of a flagellar filament based on the packing of paFliC in the crystal suggests that the D2 domain would be exposed to solution and could play an important role in immunogenicity. Our biophysical and structure-based modeling study on paFliC, the paFliC-TLR5 complex, and the paFliC filament could contribute to the improvement of vaccine design to control P. aeruginosa infection.     

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na⁺, 2Na⁺ and K⁺), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 μM). This effect was not detected for tolaasin I at the concentrations tested (< 28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co²⁺ rather than Zn²⁺: the k_cat (s⁻¹) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co²⁺, and Zn²⁺ conditions, respectively. Consistently, addition of low concentrations of Co²⁺ to PaAP previously saturated with Zn²⁺ greatly enhanced the enzymatic activity, suggesting that Co²⁺ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co²⁺ or Zn²⁺ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co²⁺- and Zn²⁺-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co²⁺ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.     

The junctional integrity of epithelial cells is modulated by Pseudomonas aeruginosa quorum sensing molecule through phosphorylation-dependent mechanisms

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We report that 3O-C₁₂-HSL can disrupt adherens junctions in human epithelial Caco-2 cells as evidenced by a reduction of the expression and distribution of E-cadherin and β-catenin. Using co-immunoprecipitation we also found that P. aeruginosa 3O-C₁₂-HSL-treatment resulted in tyrosine hyperphosphorylation of E-cadherin, β-catenin, occludin and ZO-1. Similarly, serine and threonine residues of E-cadherin and ZO-1 became more phosphorylated after 3O-C₁₂-HSL treatment. On the contrary, occludin and β-catenin underwent dephosphorylation on serine and threonine residues after exposition of 3O-C₁₂-HSL. These changes in the phosphorylation state were paralleled by alteration in the structure of junction complexes and increased paracellular permeability. Moreover, pre-treatment of the Caco-2 cells with protein phosphatase and kinase inhibitors prevented 3O-C₁₂-HSL-induced changes in paracellular permeability and interactions between occludin-ZO-1 and the E-cadherin-β-catenin. These findings clearly suggest that an alteration in the phosphorylation status of junction proteins are involved in the changes in cell junction associations and enhanced paracellular permeability, and that bacterial signals are indeed sensed by the host cells.     

A predicted S-type pyocin shows a bactericidal activity against clinical Pseudomonas aeruginosa isolates through membrane damage

The nucleic acid sequence at the positions 1067817-1066321 of Pseudomonas aeruginosa PAO1 genome was predicted to encode a novel S-type pyocin, designated S5, based on the genome sequence. However, its antimicrobial spectrum, activity and mechanism have not been investigated. Herein, we report that pyocin S5 has an antimicrobial activity against seven clinical P. aeruginosa isolates (DWW3, InA, InB, In3, In4, In7, and In8). Among them, DWW3 is most sensitive with a minimum inhibitory concentration of 12.6 μg/ml and a killing percentage of 95.7 at 225 μg/ml. Further, we demonstrated that the antimicrobial mechanism of pyocin S5 is membrane damage, evidenced by the leakage of intracellular materials, the increase of membrane permeability, and cell surface disruption.