S-type Pyocins Research Articles

Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents

IntroductionBacterial 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.MethodsThe 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.ResultThe 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.ConclusionThese 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.

Phenotypic and Genotypic Criteria for the Screening of Highly Active S-Type Pyocins Pseudomonas aeruginosa Producers

Bacteriocins of Pseudomonas aeruginosa, especially S-type pyocins, show high efficiency as analogs of antimicrobial drugs. Various screening methods can be used to identify producers of highly active pyocins, but there are no clear criteria for selecting perspective strains. The aim of this work was to determine criteria that can be used during phenotypic and genotypic screening for the selection of perspective highly active S-type pyocin P. aeruginosa producers. Methods. The objects of investigation were 40 P. aeruginosa strains. Pyocins were obtained from each culture, relative coefficients of activity spectrum and sensitivity were determined for all the strains used. The obtained results of the phenotypic screening were compared with the data of the genotypic screening. Results. The use of the proposed method of activity assessment according to the lysis intensity made it possible to phenotypically assess the expression of pyocin genes. It was established that according to the new criteria, only one strain — P. aeruginosa UCM B-333 — can be included in the group of the most active pyocin producers that inhibit the growth of more than 75% of indicator cultures. The majority of representatives of maximally and highly active producers were characterized by high resistance to the action of other pyocins, which can be considered as an additional criterion for the selection of perspective strains. During genotypic screening, it was established that the quantity of pyocin genes in the genome cannot be interpreted as a clear criterion of the producer’s perspective. However, 50% of representatives of maximally and highly active pyocin producers were characterized by the presence of two pyocin genes, while in 47.7% of moderately active and 54.5% of low active producers, one pyocin gene was detected more often. It was established that with widening the bacteriocin activity spectrum, the detection frequency of pyocin S1 and S5 genes increases, and for pyocin S2 and S3 genes — decreases. Thus, among the producers of maximally and highly active bacteriocins, pyocin S1 and S5 genes were identified with the highest frequency — 42.8% and 78.6%, and pyocin S2 and S3 genes — with the lowest one — 28.6% and 7.1%, respectively. Gene of pyocin S4 with tRNase activity were detected with equally high frequency in all groups of producers. Conclusions. The method of activity assessment by the lysis intensity allows not only to determine the presence of pyocins, but also to phenotypically evaluate the level of their expression, which is an important criterion for the selection of perspective producers. Bacteriocins with a wider activity spectrum are synthesized by P. aeruginosa strains with higher resistance to the action of pyocins from other cultures. The most optimal genotypic criterion for the selection of a highly perspective pyocin producer, detection of genes combination of bacteriocins with different mechanisms of action — with DNase activity (pyocin S1) and the ability to pore formation (pyocin S5) — can be considered.

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energise their transport into cells and catalyse their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Functional synergism of pyoverdine and the S-type pyocins of Pseudomonas aeruginosa

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.

Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

Soluble Antimicrobial Peptide Pyocin of Pseudomonas aeruginosa and its Therapeutics: A Review Article

Pyocinsis a bacteriocin produced by a group of Gram-ngative bacteria that belongs to Pseudomonasspecies. Pyocin is classified as a two distinct families of pyocins. (i) S-type pyocins (colicin-like bacteriocins), (ii) Tailocins (high-molecular weight bacteriocins that resemble phage tails).The structure of S-type pyocin is similar to that of colicin except that many S-type pyocins have three domains. Under normal conditions, the expression of prtN is repressed by PrtR. Upon exposure to stress conditions, such as DNA damage by chemicals or ultraviolet irradiation, an activated RecA triggers autoproteolytic cleavage of PrtR, which abrogates prtN repression and leads to pyocinproduction. The outer membrane receptors for three pyocins have been identified. These are FpvAI, FpvAII and FptA, all of which are involved in the uptake of iron-siderophore complexes. Before being translocated through the membrane and killing their target.This is becoming increasingly important as microbial imbalances in the natural gut flora have been suggested to play a role in a range of chronic diseases such as inflammatory bowel disease, diabetes, obesity and rheumatoid arthritis.Pyocin has now been shown to have antimicrobial activity againstbacteria in a biofilm is a limiting factor in the successful treatment of a range of chronic infections

Interaction between S-Type Pyocins and Microcin-II-Like Bacteriocins in Pseudomonas aeruginosa

According to our previous results, S-type bacteriocins of Pseudomonas aeruginosa are characterized by high activity against phytopathogenic Pseudomonas syringae strains. In addition to these pyocins producing strains are able to synthesize microcin-II-like bacteriocins. Presence of interaction between these two killer factors can determine methods of their use and activity increase of bacteriocins with antiphytopathogenic properties. The aim of the work was to test possibility of interaction between S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa. Methods. The objects of the study were pyocins produced by 6 P. aeruginosa strains. Killer factors in composition of induced lysates were concentrated by 70% ammonium sulphate precipitation, dialyzed through dialysis membrane with molecular weight cut-off (MWCO) 3.5 kDa. Then ion-exchange chromatography with DEAE-cellulose, gel filtration with Sephadex G-75 and ultracentrifugation at 215.000 g for 1 and 4 hours were used for their separation. Protein concentration and antimicrobial activity were determined in obtained fractions. Visualization of proteins in active fraction composition was conducted by electrophoresis according to the Laemmli method. Results. Under ion-exchange chromatography with DEAE-cellulose application elution of bacteriocins available in lysate composition occurs simultaneously. The highest indices of activity and protein concentration were in the 4th fraction, containing two protein bands with molecular weight near 58 and 9 kDa, which are typical for S5 pyocin and microcin-II-like bacteriocins of P. aeruginosa. Further gel filtration of sampled fractions through Sephadex G-75 allowed to separate noted killer factors and obtaine purified fraction containing microcin-II-like pyocins only. Application of ultracentrifugation during 1 hour didn’t precipitate studied bacteriocins, whereas during 4 hours – lead to their separation. At the same time a twofold increase of activity indices for S-type pyocins in precipitates and for microcin-IIlike killer factors – in supernatants were observed. However achieved concentration was characterized by short-term effect, since in 14 days activity of supernatants decreased by 4–16 times, and for precipitates – by 80–640 times. Then revealed tendency for activity decrease continued. Conclusions. S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa interact with each other, that ensures their stabilization and protects again destruction. Application of methods that cause separation of these killer factors is inexpedient, since it results into considerable decrease of bacteriocin activity indices.

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.

Structural and biophysical analysis of nuclease protein antibiotics.

Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-angle X-ray scattering data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.

A ptsP deficiency in PGPR Pseudomonas fluorescens SF39a affects bacteriocin production and bacterial fitness in the wheat rhizosphere

Pseudomonas fluorescens SF39a is a plant-growth-promoting bacterium isolated from wheat rhizosphere. In this report, we demonstrate that this native strain secretes bacteriocins that inhibit growth of phytopathogenic strains of the genera Pseudomonas and Xanthomonas. An S-type pyocin gene was detected in the genome of strain SF39a and named pys. A non-polar pys::Km mutant was constructed. The bacteriocin production was impaired in this mutant.To identify genes involved in bacteriocin regulation, random transposon mutagenesis was carried out. A miniTn5Km1 mutant, called P. fluorescens SF39a-451, showed strongly reduced bacteriocin production. This phenotype was caused by inactivation of the ptsP gene which encodes a phosphoenolpyruvate phosphotransferase (EINtr) of the nitrogen-related phosphotransferase system (PTSNtr). In addition, this mutant showed a decrease in biofilm formation and protease production, and an increase in surface motility and pyoverdine production compared with the wild-type strain. Moreover, we investigated the ability of strain SF39a-451 to colonize the wheat rhizosphere under greenhouse conditions. Interestingly, the mutant was less competitive than the wild-type strain in the rhizosphere. To our knowledge, this study provides the first evidence of both the relevance of the ptsP gene in bacteriocin production and functional characterization of a pyocin S in P. fluorescens.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa

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.

The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697–4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI–III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway.

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.

Comparative genomic analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes

BackgroundThe recently described Type VI Secretion System (T6SS) represents a new paradigm of protein secretion in bacteria. A number of bioinformatic studies have been conducted to identify T6SS gene clusters in the available bacterial genome sequences. According to these studies, Salmonella harbors a unique T6SS encoded in the Salmonella Pathogenicity Island 6 (SPI-6). Since these studies only considered few Salmonella genomes, the present work aimed to identify novel T6SS loci by in silico analysis of every genome sequence of Salmonella available.ResultsThe analysis of sequencing data from 44 completed or in progress Salmonella genome projects allowed the identification of 3 novel T6SS loci. These clusters are located in differentially-distributed genomic islands we designated SPI-19, SPI-20 and SPI-21, respectively. SPI-19 was identified in a subset of S. enterica serotypes including Dublin, Weltevreden, Agona, Gallinarum and Enteritidis. In the later, an internal deletion eliminated most of the island. On the other hand, SPI-20 and SPI-21 were restricted to S. enterica subspecies arizonae (IIIa) serotype 62:z4,z23:-. Remarkably, SPI-21 encodes a VgrG protein containing a C-terminal extension similar to S-type pyocins of Pseudomonas aeruginosa. This is not only the first evolved VgrG described in Salmonella, but also the first evolved VgrG including a pyocin domain described so far in the literature. In addition, the data indicate that SPI-6 T6SS is widely distributed in S. enterica and absent in serotypes Enteritidis, Gallinarum, Agona, Javiana, Paratyphi B, Virchow, IIIa 62:z4,z23:- and IIIb 61:1,v:1,5,(7). Interestingly, while some serotypes harbor multiple T6SS (Dublin, Weltvreden and IIIa 62:z4,z23:-) others do not encode for any (Enteritidis, Paratyphi B, Javiana, Virchow and IIIb 61:1,v:1,5,(7)). Comparative and phylogenetic analyses indicate that the 4 T6SS loci in Salmonella have a distinct evolutionary history. Finally, we identified an orphan Hcp-like protein containing the Hcp/COG3157 domain linked to a C-terminal extension. We propose to designate this and related proteins as "evolved Hcps".ConclusionAltogether, our data suggest that (i) the Salmonella T6SS loci were acquired by independent lateral transfer events and (ii) evolved to contribute in the adaptation of the serotypes to different lifestyles and environments, including animal hosts. Notably, the presence of an evolved VgrG protein related to pyocins suggests a novel role for T6SS in bacterial killing. Future studies on the roles of the identified T6SS loci will expand our knowledge on Salmonella pathogenesis and host specificity.

Pyocin S2 (Sa) Kills Pseudomonas aeruginosa Strains via the FpvA Type I Ferripyoverdine Receptor

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.

Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide

BackgroundPseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure.ResultsHerein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated.Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity.ConclusionThis finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells.

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage.

Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.

Molecular Characterization of Pyocin S3, a Novel S-type Pyocin from Pseudomonas aeruginosa

The genetic determinant for the soluble pyocin S3 was isolated from a genomic library constructed in the plasmid pGV1122, of Pseudomonas aeruginosa strain P12 isolated from a cystic fibrosis patient. The nucleotide sequence of a 3270-base pair DNA fragment was determined, and the two structural genes, pyoS3A and pyoS3I, and the 3'- and 5'-flanking regions were localized. Transcription (Northern blot) analysis showed that the two genes were co-transcribed. The genes pyoS3A and pyoS3I code for polypeptides of 767 and 154 amino acids, respectively, with calculated molecular weights of 81,385 and 17,047. Pyocin S3 was produced in Escherichia coli from a plasmid and purified as a complex of two components (S3A and S3I) corresponding to the pyoS3A and pyoS3I gene products, respectively. The S3A component, like pyocin S3, had a killing effect involving DNase activity and was inhibited by the S3I protein. Comparisons of the predicted amino acid sequence of the two components of pyocin S3 to those of pyocins S1, S2, and AP41 indicate that pyocin S3 is a new type of S-type pyocin.

Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes

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.

Purification and properties of an S-type pyocin, pyocin AP41.

Pyocin AP41, a protease-sensitive bacteriocin produced by Pseudomonas aeruginosa PAF41, was purified to a homogeneous state and characterized. The molecular weight of this pyocin was about 95,000 as determined by the combination of gel filtration and sedimentation velocity analysis. This pyocin was a complex of two kinds of polypeptides. Highly purified preparations showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their apparent molecular weights were 90,000 and 6,000 to 7,000, respectively. Two proteins could be separated by gel filtration in the presence of 6 M urea. Amino acid compositions of these components were determined. The large component had pyocin activity similar to the complex, whereas the small component did not. Sensitive cells were killed by this pyocin only under growing conditions and with single-hit kinetics. The pyocin-treated cells lysed in about 30 min with concomitant production of their resident pyocins or phages. The induced production of resident pyocins caused by pyocin AP41 depended on a recA gene function.