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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. The authors report near-atomic resolution structures of the R-type bacteriocin from Pseudomonas aeruginosa in the pre-contraction and post-contraction states, and these structures provide insight into the mechanism of action of molecular syringes.

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture. Key points • Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages • Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth • Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way

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.

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. 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.

Bacteria have evolved numerous strategies to increase their competitiveness and fight against each other. Indeed, a large arsenal of antibacterial weapons is available in order to inhibit the proliferation of competitor cells. Polymorphic toxin systems (PTS), recently identified by bioinformatics in all major bacterial lineages, correspond to such a system primarily involved in conflict between related bacterial strains. They are typically composed of a secreted multidomain toxin, a protective immunity protein, and multiple cassettes encoding alternative toxic domains. The C-terminal domains of polymorphic toxins carry the toxic activity, whereas the N-terminal domains are related to the trafficking mode. In silico analysis of PTS identified over 150 distinct toxin domains, including putative nuclease, deaminase, or peptidase domains. Immunity genes found immediately downstream of the toxin genes encode small proteins that protect bacteria against their own toxins or against toxins secreted by neighboring cells. PTS encompass well-known colicins and pyocins, contact-dependent growth inhibition systems which include CdiA and Rhs toxins and some effectors of type VI secretion systems. We have recently characterized the MafB toxins, a new family of PTS deployed by pathogenic Neisseria spp. Many other putative PTS have been identified by in silico predictions but have yet to be characterized experimentally. However, the high number of these systems suggests that PTS have a fundamental role in bacterial biology that is likely to extend beyond interbacterial competition.

Pseudomonas aeruginosa significantly induces health-associated infections in a variety of species other than humans. Over the years, the opportunistic pathogen has developed resistance against commonly used antibiotics. Since most P. aeruginosa strains are multi-drug resistant, regular antibiotic treatment of its infections is becoming a dire concern, shifting the global focus towards the development of alternate antimicrobial approaches. Pyocins are one of the most diverse antimicrobial peptide combinations produced by bacteria. They have potent antimicrobial properties, mainly against bacteria from the same phylogenetic group. P. aeruginosa , whether from clinical or environmental origins, produce several different pyocins that show inhibitory activity against other multi-drug-resistant strains of P. aeruginosa . They are, therefore, good candidates for alternate therapeutic antimicrobials because they have a unique mode of action that kills antibiotic-resistant bacteria by attacking their biofilms. Here, we review pseudomonas -derived antimicrobial pyocins with great therapeutic potential against multi-drug-resistant P. aeruginosa .

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. 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.

Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa -specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. IMPORTANCE Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

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.

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 over time; (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.