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Introduction: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential., Methods: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml -1 ). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene., Result: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR., Conclusion: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries., (© 2024. The Author(s).)

Pyocin S2 and S4 in Pseudomonas aeruginosa use the same uptake channels as the pyoverdine does in bacteria, indicating a possible connection between them. In this study, we characterized the single bacterial gene expression distribution of three S-type pyocins (Pys2, PA3866, and PyoS5) and examined the impact of pyocin S2 on bacterial uptake of pyoverdine. The findings demonstrated that the expression of the S-type pyocin genes was highly differentiated in bacterial population under DNAdamage stress. Moreover, exogenous addition of pyocin S2 reduces the bacterial uptake of pyoverdine so that the presence of pyocin S2 prevents the uptake of environmental pyoverdine by non-pyoverdine synthesizing 'cheaters', thereby reducing their resistance to oxidative stress. Furthermore, we discovered that overexpression of the SOS response regulator PrtN in bacteria significantly decreased the expression of genes involved in the synthesis of pyoverdine, significantly decreasing the overall synthesis and exocytosis of pyoverdine. These findings imply a connection between the function of the iron absorption system and the SOS stress response mechanism in bacteria.

Pyocins are interbacterial killing complexes made by Pseudomonas aeruginosa primarily to enact intraspecific competition. DNA damage and the ensuing activation of RecA initiate canonical pyocin expression. We recently discovered that deletion of xerC , which encodes a tyrosine recombinase involved in chromosome decatenation, markedly elevates basal pyocin production independently of RecA. Interestingly, the already-elevated basal pyocin expression in Δ xerC cells is substantially further increased by ciprofloxacin treatment. Here, we asked whether this further increase is due to DNA damage additionally activating the canonical RecA-dependent pyocin expression pathway. We also interrogated the relationship between XerC recombinase activity and pyocin expression. Surprisingly, we find that DNA damage-induced pyocin stimulation in Δ xerC cells is independent of RecA but dependent on PrtN, implying a RecA-independent means of DNA damage sensing that activates pyocin expression via PrtN. In sharp contrast to the RecA independence of pyocin expression in Δ xerC strains, specific mutational inactivation of XerC recombinase activity (XerC Y272F ) caused modestly elevated basal pyocin expression and was further stimulated by DNA-damaging drugs, but both effects were fully RecA dependent. To test whether pyocins could be induced by chemically inactivating XerC, we deployed a previously characterized bacterial tyrosine recombinase inhibitor. However, the inhibitor did not activate pyocin expression even at growth-inhibitory concentrations, suggesting that its principal inhibitory activity resembles neither XerC absence nor enzymatic inactivation. Collectively, our results imply a second function of XerC, separate from its recombinase activity, whose absence permits RecA-independent but DNA damage-inducible pyocin expression. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces pyocins-intraspecific, interbacterial killing complexes. The canonical pathway for pyocin production involves DNA damage and RecA activation. Pyocins are released by cell lysis, making production costly. We previously showed that cells lacking the tyrosine recombinase XerC produce pyocins independently of RecA. Here, we show that DNA-damaging agents stimulate pyocin expression in Δ xerC strains without involving RecA. However, strains mutated for XerC recombinase activity display strictly RecA-dependent pyocin production, and a known bacterial tyrosine recombinase inhibitor does not elicit pyocin expression. Our results collectively suggest that the use of XerC inhibition as an antipseudomonal strategy will require targeting the second function of XerC in regulating noncanonical pyocin production rather than targeting its recombinase activity.

In Pseudomonas aeruginosa , the second messenger cyclic-di-GMP and Gac/Rsm signaling pathways are associated with the transition from acute to chronic infection. Therefore, identification of the molecular mechanisms that govern lifestyle choice in bacteria is very important. Here, we identified a novel cyclic-di-GMP modulator, PrtR, which was shown to repress pyocin production by inhibition of PrtN and activate the type III secretion system (T3SS) through PtrB. Compared to a wild-type strain or a prtN mutant, the prtR prtN double mutant exhibited a wrinkly colony and hyperbiofilm phenotype, as well as an increase in intracellular c-di-GMP levels. Interestingly, a diguanylate cyclase (DGC) gene, siaD , was repressed by PrtR. Further experiments revealed that PrtR directly interacts with SiaD and facilitates the accumulation of c-di-GMP in cells. We also demonstrated that PrtR regulates the activity of the Gac/Rsm system, thus affecting expression of the T3SS and type VI secretion system (T6SS) and the formation of biofilm. Taken together, the present findings indicate that PrtR, as a c-di-GMP modulator, plays key roles in the adaptation to opportunistic infection of P. aeruginosa Additionally, this study revealed a novel mechanism for PrtR-mediated regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks., (Copyright © 2021 American Society for Microbiology.)

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics 1-4 . Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold 1,2 . Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage 5 . Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria., (Copyright © 2020 Behrens et al.)

R pyocins are ɸCTX-like myophage tailocins of Pseudomonas sp. Adsorption of R pyocins to target strains occurs by the interaction of tail fiber proteins with core lipopolysaccharide (LPS). Here, we demonstrate that N-terminally truncated R pyocin tail fibers corresponding to a region of variation between R-subtypes are sufficient to bind target strains according to R-subtype. We also report the crystal structures of these tail fiber proteins and show that they form an elongated helical trimer composed of three domains arranged linearly from N- to C-terminus: a baseplate proximal head, medial shaft, and distal foot. The head and shaft domains contain novel structural motifs. The foot domain, however, is composed of a conserved jellyroll fold and shares high structural similarity to the tail fiber of myophage AP22, podophage tailspike C-terminal domains (LKA-1 and ɸ297), and several eukaryotic adhesins (discoidin I/II, agglutinin, and octocoral lectin). Many of these proteins bind polysaccharides by means of their distal loop network, a series of highly variable loops at one end of the conserved jellyroll fold backbone. Our structures reveal that the majority of R-subtype specific polymorphisms cluster in patches covering a cleft formed at the oligomeric interface of the head domain and in a large patch covering much of the foot domain, including the distal loop network. Based on the structural variation in distal loops within the foot region, we propose that the foot is the primary sugar-binding domain of R pyocins and R-subtype specific structural differences in the foot domain distal loop network are responsible for binding target strains in an R-subtype dependent manner., Competing Interests: The authors have declared that no competing interests exist.

Pseudomonas aeruginosa is an opportunistic pathogen and the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa infections are difficult to treat due to a number of antibiotic resistance mechanisms and the organism's propensity to form multicellular biofilms. Epidemic strains of P. aeruginosa often dominate within the lungs of individual CF patients, but how they achieve this is poorly understood. One way that strains of P. aeruginosa can compete is by producing chromosomally encoded bacteriocins, called pyocins. Three major classes of pyocin have been identified in P. aeruginosa : soluble pyocins (S types) and tailocins (R and F types). In this study, we investigated the distribution of S- and R-type pyocins in 24 clinical strains isolated from individual CF patients and then focused on understanding their roles in interstrain competition. We found that (i) each strain produced only one R-pyocin type, but the number of S-pyocins varied between strains, (ii) R-pyocins were generally important for strain dominance during competition assays in planktonic cultures and biofilm communities in strains with both disparate R- and S-pyocin subtypes, and (iii) purified R-pyocins demonstrated significant antimicrobial activity against established biofilms. Our work provides support for a role played by R-pyocins in the competition between P. aeruginosa strains and helps explain why certain strains and lineages of P. aeruginosa dominate and displace others during CF infection. Furthermore, we demonstrate the potential of exploiting R-pyocins for therapeutic gains in an era when antibiotic resistance is a global concern. IMPORTANCE A major clinical problem caused by Pseudomonas aeruginosa , is chronic biofilm infection of the lungs in individuals with cystic fibrosis (CF). Epidemic P. aeruginosa strains dominate and displace others during CF infection, but these intraspecies interactions remain poorly understood. Here we demonstrate that R-pyocins (bacteriocins) are important factors in driving competitive interactions in biofilms between P. aeruginosa strains isolated from different CF patients. In addition, we found that these phage-like pyocins are inhibitory against mature biofilms of susceptible strains. This highlights the potential of R-pyocins as antimicrobial and antibiofilm agents at a time when new antimicrobial therapies are desperately needed., (Copyright © 2019 Oluyombo et al.)

Background: The appearance and dissemination of MDR among pathogenic bacteria has forced the search for new antimicrobials. Bacteriocins have been proposed as potential alternatives for the treatment of infections due to multiresistant strains., Objectives: To analyse the activity of R-pyocins against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis and other sources and evaluate them as a potential adjuvant or alternative to the current antibiotic treatment., Methods: The activity of R-pyocins against 150 strains of P. aeruginosa isolated from patients with cystic fibrosis or bacteraemia was studied through spot assay. Interactions between R-pyocins and antipseudomonal agents were quantitatively studied by the chequerboard method., Results: The proportion of P. aeruginosa isolates susceptible to R-pyocins was found to be higher in cystic fibrosis isolates compared with bacteraemia isolates (79.41% versus 50%). Moreover, no interactions were found between common antipseudomonal agents and R-pyocin susceptibility, except for the ST175 high-risk clone., Conclusions: Our results highlight the possibility of using R-pyocins as therapeutic agents, alone or as adjuvants, against P. aeruginosa in cystic fibrosis.

Pyocins are bacteriocins secreted by Pseudomonas aeruginosa , and they assist in the colonization of different niches. A major subset of these antibacterial proteins adopt a modular organization characteristic of polymorphic toxins. They include a receptor-binding domain, a segment enabling membrane passage, and a toxin module at the carboxy terminus, which eventually kills the target cells. To protect themselves from their own products, bacteriocin-producing strains express an immunity gene concomitantly with the bacteriocin. We show here that a pyocin equipped with a phylogenetically distinct ColM toxin domain, PaeM4, mediates antagonism against a large set of P. aeruginosa isolates. Immunity to PaeM4 is provided by the inner membrane protein PmiC, which is equipped with a transmembrane topology not previously described for the ColM family. Given that strains lacking a pmiC gene are killed by PaeM4, the presence of such an immunity partner likely is a key criterion for escaping cellular death mediated by PaeM4. The presence of a TonB box in PaeM4 and enhanced bacteriocin activity under iron-poor conditions strongly suggested the targeting of a TonB-dependent receptor. Evaluation of PaeM4 activities against TonB-dependent receptor knockout mutants in P. aeruginosa PAO1 revealed that the heme receptor HxuC (PA1302) serves as a PaeM4 target at the cellular surface. Because other ColM-type pyocins may target the ferrichrome receptor FiuA, our results illustrate the versatility in target recognition conferred by the polymorphic nature of ColM-type bacteriocins. IMPORTANCE The antimicrobial armamentarium of a bacterium is a major asset for colonizing competitive environments. Bacteriocins comprise a subset of these compounds. Pyocins are an example of such antibacterial proteins produced by Pseudomonas aeruginosa , killing other P. aeruginosa strains. A large group of these molecules show a modular protein architecture that includes a receptor-binding domain for initial target cell attachment and a killer domain. In this study, we have shown that a novel modular pyocin (PaeM4) that kills target bacteria via interference with peptidoglycan assembly takes advantage of the HxuC heme receptor. Cells can protect themselves from killing by the presence of a dedicated immunity partner, an integral inner membrane protein that adopts a transmembrane topology distinct from that of proteins currently known to provide immunity against such toxin activity. Understanding the receptors with which pyocins interact and how immunity to pyocins is achieved is a pivotal step toward the rational design of bacteriocin cocktails for the treatment of P. aeruginosa infections., (Copyright © 2018 American Society for Microbiology.)

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to "simple" contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains-two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.

A novel transposon belonging to the Tn 3 -like family was identified on the chromosome of a commensal strain of Pseudomonas aeruginosa sequence type 2343 (ET02). Tn 6350 is 7,367 bp long and harbors eight open reading frames (ORFs), an ATPase (IS 481 family), a transposase (DDE catalytic type), a Tn 3 resolvase, three hypothetical proteins, and genes encoding the new pyocin S8 with its immunity protein. We show that pyocin S8 displays activity against carbapenemase-producing P. aeruginosa , including IMP-1, SPM-1, VIM-1, GES-5, and KPC-2 producers., (Copyright © 2017 American Society for Microbiology.)

Bacterial species exhibit biogeographical patterns like those observed in larger organisms. The distribution of bacterial species is driven by environmental selection through abiotic and biotic factors as well dispersal limitations. We asked whether interference competition, a biotic factor, could explain variability in habitat use by Pseudomonas species in the human home. To answer this question, we screened almost 8000 directional, pairwise interactions between 89 Pseudomonas strains including members of the Pseudomonas aeruginosa (n = 29), Pseudomonas fluorescens (n = 21), and Pseudomonas putida (n = 39) species groups for the presence of killing. This diverse set of Pseudomonas strains includes those isolated from several different habitats within the home environment and includes combinations of strains that were isolated from different spatial scales. The use of this strain set not only allowed us to analyze the commonality and phylogenetic scale of interference competition within the genus Pseudomonas but also allowed us to investigate the influence of spatial scale on this trait. Overall, the probability of killing was found to decrease with increasing phylogenetic distance, making it unlikely that interference competition accounts for previously observed differential habitat use among Pseudomonas species and species groups. Strikingly, conspecific P. aeruginosa killing accounted for the vast majority of the observed killing, and this killing was found to differ across the habitat type and spatial scale of the strains' isolation. These data suggest that interference competition likely plays a large role in the within-species dynamics of P. aeruginosa but not other household Pseudomonas species.

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations,especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that(i) isolates from later infection stages inhibited earlier infecting strains less,but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage overtime; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.

How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups., (Copyright © 2015. Published by Elsevier Ltd.)

LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosa that are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtR results in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tac promoter repressed the endogenous prtR promoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Pseudomonas aeruginosa is an opportunistic human pathogen producing a variety of virulence factors. One of them is lipopolysaccharide, consisting of endotoxic lipid A and long-chain O-antigen polysaccharide, which are connected together through a short linker region, called core oligosaccharide. Chemical structures of the core oligosaccharide are well conserved, with one exception, in that certain strains of P. aeruginosa add a terminal glucose residue (Glc(IV) ) to core by a transferase reaction, due to the activity of a glucosyltransferase, WapB. Here, we investigated the regulation of wapB expression. Our results showed that while the majority of analysed genomes of P. aeruginosa contain wapB, many of these have a conserved identical 5-nucleotide deletion in the upstream region that inactivated the promoter. This deletion is within the -10 hexamer that is recognized by a principle sigma factor (RpoD, or σ70) as proven by data from an electromobility shift assay. These results provide the molecular basis of why LPS core of many P. aeruginosa strains is lacking Glc(IV) . In addition, we show that absence of Glc(IV) due to an inactive wapB promoter confers resistance to killing by R3-pyocin, a phage tail-like bacteriocin of P. aeruginosa., (© 2013 John Wiley & Sons Ltd.)

The ubiquitous production of antibacterial toxins, such as bacteriocins, is an ecologically significant class of interbacterial interactions that have primarily evolved through their indirect fitness benefits to the producer. Bacteria release bacteriocins into the environment at a cost to individual cell, but individual bacteriocin-producing cells are unlikely to gain any direct benefit from their own toxin; indeed, cell lysis is required in many species. There is a growing body of research describing the ecological conditions that can favour the evolution of bacteriocin production. However, an important aspect of many bacteriocins has yet to be investigated: the ability of bacteriocin-producing cells to neutralize toxin ('soaking') produced by other clonemates. By competing Pseudomonas aeruginosa bacteriocin-producing wild-type and 'non-soaking' strains against a bacteriocin-susceptible strain, we find that soaking markedly reduces the fitness of a bacteriocin-producing strain at both high and low frequencies.

Social interactions have been shown to play an important role in bacterial evolution and virulence. The majority of empirical studies conducted have only considered social traits in isolation, yet numerous social traits, such as the production of spiteful bacteriocins (anticompetitor toxins) and iron-scavenging siderophores (a public good) by the opportunistic pathogen Pseudomonas aeruginosa, are frequently expressed simultaneously. Crucially, both bacteriocin production and siderophore cheating can be favored under the same competitive conditions, and we develop theory and carry out experiments to determine how the success of a bacteriocin-producing genotype is influenced by social cheating of susceptible competitors and the resultant impact on disease severity (virulence). Consistent with our theoretical predictions, we find that the spiteful genotype is favored at higher local frequencies when competing against public good cheats. Furthermore, the relationship between spite frequency and virulence is significantly altered when the spiteful genotype is competed against cheats compared with cooperators. These results confirm the ecological and evolutionary importance of considering multiple social traits simultaneously. Moreover, our results are consistent with recent theory regarding the invasion conditions for strong reciprocity (helping cooperators and harming noncooperators)., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97 % of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections.

Pseudomonas aeruginosa produces three different types of bacteriocins: the soluble S-pyocins and the bacteriophage-like F- and R-pyocins. R-pyocins kill susceptible bacteria of the same or closely related species with high efficiency. Five different types of R-pyocins (R1- to R5-pyocins) have been described based on their killing spectra and tail fiber protein sequences. We analyzed the distribution of R-pyocin genes in a collection of clinical P. aeruginosa isolates. We found similar percentages of isolates not containing R-pyocins (28%) and isolates containing genes encoding R1-pyocins (25%), R2-pyocins (17%), and R5-pyocins (29%). The R-pyocin-deficient isolates were susceptible to R1-, R2-, and R5-pyocins, while most R2- and R5- pyocin producers were resistant. Determination of the O serotypes revealed that the R-pyocin-susceptible isolates belonged to serotypes O1, O3, and O6, while the R-pyocin-resistant isolates were serotype O10, O11, and O12 isolates. We hypothesized that O-serotype-specific lipopolysaccharide (LPS) packaging densities may account for the distinct accessibilities of R-pyocins to their receptors at the cell surface. Using genetically defined LPS mutants, we showed that the l-Rha residue and two distinct d-Glc residues of the outer core are part of the receptor sites for R1-, R2-, and R5-pyocins, respectively. To illustrate R-pyocin-mediated intraspecies biological warfare, we monitored the population dynamics of two different R-pyocin-producing P. aeruginosa clones of sequential respiratory isolates obtained from a colonized patient. The results of this study highlight the potential role of R-pyocins in shaping bacterial populations during host colonization and support use of these molecules as specific and potent bactericidal agents.

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.

R-type pyocin is a bacteriophage tail-shaped bacteriocin produced by Pseudomonas aeruginosa, but its physiological roles are relatively unknown. Here we describe a role of R-type pyocin in the competitive growth advantages between P. aeruginosa strains. Partial purification and gene disruption revealed that the major killing activity from the culture supernatant of PA14 is attributed to R-type pyocin, neither F-type nor S-type pyocins. These findings may provide insight into the forces governing P. aeruginosa population dynamics to promote and maintain its biodiversity.

Adaptive evolution depends on both the genetic variability in a population of organisms and the selection of the better adapted genotypes. However, for the fittest variants to be selected they must survive over a sufficient period under the new conditions. Bacteria are often exposed to different types of stress in nature, including antibiotics. We analysed the global expression profiles of the opportunistic pathogen Pseudomonas aeruginosa in response to ceftazidime, a PBP3 inhibitor, at different concentrations and times. PBP3 inhibition exerts a global impact on the transcription of a large number of genes. From an adaptive perspective, it is noteworthy the induction of several SOS genes, as well as adaptation, protection and antibiotic resistance genes. Intriguingly, transcription of pyocin genes, previously described as SOS-regulated, was repressed upon PBP3 inhibition. Ciprofloxacin, an SOS inducer, produced transcriptional induction of pyocins. Our results indicate that: (i) the SOS responses resulting from treatments with these two antibiotics cause only partially overlapping transcription profiles; (ii) PBP3 and DNA-gyrase inhibition produce opposite effects on transcription of pyocin genes. Consequently, ceftazidime decreases ciprofloxacin toxicity; (iii) error-prone DNA-polymerase DinB is induced by PBP3 inhibition but not by DNA-gyrase inhibition; (iv) PBP3 inhibition causes induced mutagenesis; (v) ceftazidime upregulates several antibiotic-resistance and adaptation genes; and (vi) ceftazidime concentrations thought previously to be lethal are not, as most cells treated with ceftazidime remain alive and recover their capacity to form colonies. Thus, transcriptional changes demonstrated in this work are likely to be adaptively relevant to cells that survive.

With few novel antimicrobials in development, resistance to the current selection of antibiotics increasingly encroaches on our ability to control microbial infections. One limitation in our understanding of the basis of the constraints on current therapies is our poor understanding of antibiotic interactions with bacteria on a global scale. Custom DNA microarrays were used to characterize the response of Pseudomonas aeruginosa to ciprofloxacin, a fluoroquinolone commonly used in therapy against chronic infections by this intrinsically resistant bacterium. Of the approximately 5,300 open reading frames (ORFs) on the array, 941 genes showed statistically significant (P /=8-fold-increased resistance to ciprofloxacin and other fluoroquinolones, demonstrating that this region is a susceptibility determinant. Since this region is known to be variably present in the genomes of clinical isolates of P. aeruginosa (R. K. Ernst et al., Environ. Microbiol. 5:1341-1349, 2003, and M. C. Wolfgang et al., Proc. Natl. Acad. Sci. USA 100:8484-8489, 2003), these findings demonstrate that the R2/F2 pyocin region is a "loaded gun" that can mediate fluoroquinolone susceptibility in P. aeruginosa.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C. Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5). It interacts with the small component (immunity protein). The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN. R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively. The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.

Pyocin typing may be unstable under certain circumstances. In the early seventies, it was shown that the IncP-2 plasmids pMG1 and pMG2 interfered with the pyocin type of some Pseudomonas aeruginosa strains. Up to now, details of the influence of these plasmids on the pyocins are unknown. In this study, the effect of these plasmids on the R, F, and S pyocins, respectively, was examined. In distinct strains, S pyocin activity was detected in the presence of these plasmids, whereas R and F pyocin activities could not be observed. So the effect of the IncP-2 plasmids pMG1 and pMG2 may be limited to the R and/or F pyocins.

The serotype variants were formed in some isolates of Pseudomonas aeruginosa in the presence of anti-pseudomonal drugs in vitro. Pyocin type, various biochemical properties and drug susceptibility of these variants were compared with those of the parent cells. Furthermore, the patterns of outer membrane proteins were also examined for some respective parent and variant cells by the SDS-PAGE and compared with their drug susceptibilities. In P. aeruginosa No. 1-S (serotype E) and No. 1-R (serotype C), simultaneously isolated from the identical specimen of the infectious patient, the serotype variants were formed by anti-pseudomonal drugs and accompanied by changes in pyocin types, some biochemical properties and susceptibilities to various kinds of anti-pseudomonal drugs. When the profiles of outer membrane proteins of P. aeruginosa No. 1-R parent and two variant cells were evaluated by the SDS-PAGE, the quantitative differences of the porin proteins between the parent and variant cells were well related to their drug susceptibilities. Although eight variant cells with other serotype were formed from the parent cells (serotype A) of P. aeruginosa No. 13, four variants with serotype M and one non-typable variant showed changes in drug susceptibility such as resistance to imipenem only and no changes in biochemical properties. On the other hand, three other variants showed marked changes in two kinds of biochemical properties and in susceptibilities to various kinds of anti-pseudomonal drugs. From the results of SDS-PAGE, it was found that the porin protein profiles of outer membranes of these parent and variant cells except a few cells which corresponded to their drug susceptibility.

Functional domain structures of pyocins AP41, S1, and S2 were assigned by examining the functions of chimeric pyocins and deletion derivatives. Pyocins AP41, S1, and S2 are essentially composed of three domains, the receptor-binding domain, the translocation domain, and the DNase domain, in that order from the N terminus to the C terminus. The alignment of these domains is distinct from that in E2-group colicins with functions similar to those of these pyocins. Pyocins AP41 and S2 have a fourth domain between the receptor-binding and the translocation domains, which is dispensable for their killing functions.

Most strains of Pseudomonas aeruginosa produce various types of bacteriocins (pyocins), namely, R-, F-, and S-type pyocins. The production of all types of pyocins was shown to be regulated by positive (prtN) and negative (prtR) regulatory genes. The prtN gene activates the expression of various pyocin genes, probably by the interaction of its product with the DNA sequences conserved in the 5' noncoding regions of the pyocin genes. The prtR gene represses the expression of the prtN gene, and its product, predicted from the nucleotide sequence, has a structure characteristic of phage repressors and seems to be inactivated by the RecA protein activated by DNA damage. A model for the regulation of the pyocin genes is proposed.

Pyocin AP41 degrades the chromosomal DNA in sensitive strains of Pseudomonas aeruginosa but has little effect on RNA, protein, and lipid syntheses. In vitro experiments showed that the carboxyl-terminal part of the large subunit of pyocin AP41 carries an inherent DNase that is responsible for its killing action.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.

Pyocin typing and serotyping of 433 strains of Pseudomonas aeruginosa from children with cystic fibrosis (CF) showed that pyocin type 9 was predominant, particularly in association with polyagglutinating serotype. The common pyocin groups, 1, 5 and 10, made up only 20% of these isolates in contrast to reported rates of up to 89% in other studies using non-CF strains. No strains of pyocin type 3 were found. Polyagglutinating strains made up 72% of strains from patients colonized with P. aeruginosa for more than 12 mths. Pyocin type 9 was associated with 93% of polyagglutinating strains. The parallel between pyocin type 9 and polyagglutinating serotype suggests that these may both be characteristics acquired by P. aeruginosa colonizing patients with CF. Because of confounding between duration of colonization and exposure to cross-infection, this study does not allow definition of the role of cross-infection in determining the characteristics of these strains in most patients. In siblings, however, evidence supports a role for cross-infection either between siblings or from a common source. In 6 pairs of siblings studied, each pair had at least 1 pyocin group in common concurrently, either at entry to the study or after an interval of several months. Identical and unusual pyocin groups were recognized in samples obtained on the same day from pairs of siblings. More studies are needed to compare results of pyocin typing with methods such as genome fingerprinting to characterize these strains and determine whether the observed distribution of pyocin groups in CF isolates is related to cross-infection or whether the combination of pyocin type 9 with polyagglutinating serotype is a characteristic of CF strains.

Typing of Pseudomonas aeruginosa strains is the necessary precondition for the study and control of intrahospital infection caused by this microorganism. Since O-serotype is considered to be the basic epidemiological marker we have studied presence and distribution of some O-serotypes of 235 Pseudomonas aeruginosa strains isolated from 131 patients. The following O-serotypes have been most frequently found: O11 (21%), O6 (18%) and O12 (16%). The combination of sero and pyocin typing proved the presence of epidemic strains at departments of burns and orthopedics.

240 Ps. aeruginosa strains were isolated from patients in Diyarbakir, 198 (82.5%) were typed, 42 (17.5%) could not been typed with Gillies-Govan Method. Pyocine type 3, 5, 6, 9, 10, 11, 14, 16, 18, 24, 27, 31, 37 were found. Pyocine type 3 (23.8%) was more frequent than others.

A new pyocin typing method for the identification of specific strains of Pseudomonas aeruginosa is described. The method involves a bilayer agar technique in which the pyocin produced by the organism diffuses from the top agar layer number 2 through a filter paper into the bottom agar layer number 1. The filter paper permits the removal of agar layer number 2 and exposes layer number 1 which is then streaked with the indicator strains. Pyocin typing of fifty clinical isolates using both the bi-layer method and the scrap-chloroform reference method of Gillies and Govan produced identical typing results. A total of 375 clinical isolates has been typed by this inexpensive and time-saving bi-layer agar method.

We investigated the effect of FeSO4 on phagocytosis-associated, increased oxidative metabolism via the hexose monophosphate shunt, with special attention to its effect on H2O2 levels. The availability of glutathione peroxidase and glutathione reductase for H2O2 disposal and hexose monophosphate shunt stimulation also are evaluated. The results show an impairment of phagocytosis-associated hexose monophosphate shunt activity together with an increase both of resting and phagocytosing formate oxidation. These apparently paradoxical findings are resolved by demonstrating a direct enhancement of formate oxidation by FeSO4 in a cell-free system. In addition, measurement of H2O2 concentrations via scopoletin fluorescence shows reduction of H2O2 by FeSO4. There is no effect on either glutathione peroxidase or glutathione reductase activities. These data suggest that one mechanism of FeSO4 impairment of microbicidal activity is by its removal of H2O2.