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This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus , Enterococcus , Streptococcus , Micrococcus , Lactobacillus , Leuconostoc, and Listeria , in addition to a pronounced inhibitory effect against Lactococcus and Clostridium . It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter ( rafCDE ), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry., Competing Interests: The authors declare no conflict of interest.

Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli , followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro . An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.

Objective: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics., Methods: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay., Results: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100˚C and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4˚C. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells., Conclusion: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium , has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-class III bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli , but it became soluble using phosphate buffer (pH 7.4) supplemented with 8 M urea. Purification using immobilized-Ni 2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with >95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori , a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100 mg per liter of culture. To the best of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H. pylori . This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.

Pediococcus pentosaceus ST65ACC is a bacteriocinogenic lactic acid bacteria (LAB) isolated from Brazilian artisanal cheese that is capable of inhibiting different food pathogens, mainly Listeria monocytogenes. The production of bacteriocins can be influenced by several growth conditions, such as temperature, pH, and medium composition. This study aimed to evaluate the effect of different culture media on the production of bacteriocins and antimicrobial activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The strains were inoculated alone and in coculture in four different media: BHI broth, MRS broth, meat broth, and reconstituted skim milk (RSM) 10% (w/v). The culture media were then incubated at 37 °C for 96 h, and count analysis, pH measurement, and bacteriocin production were performed at 0, 24, 48, 72 and 96 h. L. monocytogenes was inhibited to nondetectable levels in coculture with P. pentosaceus ST65ACC in MRS broth within 96 h, consistent with the high production of bacteriocin throughout the analysis period (3,200-12,800 AU/mL). However, lower inhibitory activities of P. pentosaceus ST65ACC on L. monocytogenes Scott A were recorded in BHI, RSM, and meat broth, with low or no production of bacteriocins at the analyzed times. The composition of these culture media may have repressed the production and activity of bacteriocins and, consequently, the antagonist activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The results showed that the antimicrobial activity was more effective in MRS broth, presenting greater production of bacteriocins and less variability when compared to the other media analyzed., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic mining and modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials., (© 2024. Springer Nature Limited.)

Campylobacter jejuni ( C. jejuni ) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni ., Importance: Campylobacter jejuni ( C. jejuni ) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni . These findings hold promise for the application of probiotic EVs for pathogen control., Competing Interests: R.H., S.M., and W.M. are co-inventors and co-applicants with the University of Ottawa on a patent application (US Patent Application Number 63/607,682, filed 08 December 2023) entitled "LIGILACTOBACILLUS PROBIOTICS, LIGILACTOBACILLUS EXTRACELLULAR VESICLES AND METHODS OF USING SAME" that covers the inhibitory activity of probiotic extracellular vesicles. The remaining authors declare no competing interests.

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics., (© 2024. The Author(s).)

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes . Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C 18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH 2 - KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A bacteriocin paracin wx3 was investigated as a candidate of natural preservative to control green pepper soft rot. Firstly, paracin wx3 was heterologously expressed in Pichia pastoris X33 with an improved yield of 0.537 g/L. Its size and amino acid sequence were confirmed by Tricine-SDS-PAGE and LC-MS/MS. Then, result of antibacterial activity showed that its MIC value against Pectobacterium carotovorum was 16 μg/mL. In vitro, paracin wx3 completely killed the pathogen at high concentrations ≥8 × MIC. In vivo, disease incidence of green pepper soft rot was decreased from 90% (control) to <2% (8 × MIC). Subsequently, results of action mode showed that paracin wx3 inhibited the growth of pathogen by pore-formation on cell membrane. Last, paracin wx3 treatment reduced losses of weight, firmness, total soluble solid, Vc of green pepper during storage. It also inhibited the production of soft rot volatile p-xylene, 1-butanol, 2-methyl-2-propanol, 3-hydroxybutan-2-one-D, 2-pentyl furan, butanal, etc., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacteriocins are antimicrobial peptides that are naturally produced by many bacteria. They hold great potential in the fight against antibiotic resistant bacteria, including ESKAPE pathogens. Engineered live biotherapeutic products (eLBPs) that secrete bacteriocins can be created to deliver targeted bacteriocin production. Here we develop a modular bacteriocin secretion platform that can be used to express and secrete multiple bacteriocins from non-pathogenic Escherichia coli host strains. As a proof of concept we create Enterocin A (EntA) and Enterocin B (EntB) secreting strains that show strong antimicrobial activity against Enterococcus faecalis and Enterococcus faecium in vitro, and characterise this activity in both solid culture and liquid co-culture. We then develop a Lotka-Volterra model that can be used to capture the interactions of these competitor strains. We show that simultaneous exposure to EntA and EntB can delay Enterococcus growth. Our system has the potential to be used as an eLBP to secrete additional bacteriocins for the targeted killing of pathogenic bacteria., (© 2024. The Author(s).)

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti- S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the Δ graRS mutant exhibited increased susceptibility to Pep5, while the Δ srrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG , and Pep5 susceptibility was significantly increased in the Δ dltABCD, Δ mprF, and Δ vraFG mutants. Regarding the Δ srrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the Δ srrAB mutant. We constructed Δ qoxABCD and Δ cydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the Δ srrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti- S . aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics., Competing Interests: The authors declare no conflict of interest.

Background: Using bacterial (probiotic) strains can influence beneficial health statuses, e.g., through dairy products; however, they must be assessed as safe before consumption; broiler rabbits represent a suitable model for this purpose. This study evaluated the safety and effect (functionality) of the dairy-derived, plantaricin-producing strain Lactiplantibacillus plantarum LP17L/1 using broiler rabbits. The following parameters were assessed to evaluate safety and functionality: microbiota, phagocytic activity (non-specific immunity parameter), blood serum biochemistry, oxidative stress enzyme, growth performance, cecal hydrolytic activity, and jejunal morphometry., Methods: Previously validated methods were used for the analyses. The appropriate administrations approved the experiments., Results: Although only up to 1.0 colony forming unit (CFU)/g (log10) LP17L/1 reached the gastrointestinal tract of the rabbits, the total lactic acid bacteria and amylolytic streptococci were significantly increased ( p < 0.001). The other microbiota were not influenced, meaning there was no negative influence on phagocytic activity, growth parameters, and biochemistry in the analyzed blood serum. Conversely, lower GPx values were measured in the experimental group than in the control group, meaning LP17L/1 did not induce oxidative stress. LP17L/1 caused an increase in villi length to crypt depth ratio, while hydrolytic activity was also significantly increased ( p < 0.01, p < 0.001)., Conclusions: L. plantarum LP17L/1 was confirmed as safe. Regarding the functionality, no negative influence on the microbiota was noted, and the tested parameters were not negatively influenced. Demonstrating that the LP17L/1 strain is safe provides further chances for its industrial application. Additional studies are being conducted., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

The lasso peptide microcin J25 (MccJ25) possesses strong antibacterial properties and is considered a potential effective component of bacterial disease treatment drugs and safe food preservatives. Although MccJ25 can be heterologously expressed in Bacillus subtilis as we have previously reported, its regulation and accumulation are yet to be understood. Here, we investigated the expression level and stability of MccJ25 in B. subtilis strains with disruption in peptidase genes pepA , pepF, and pepT . Oligoendopeptidase F (PepF) was found to be involved in reduction of the production of MccJ25 by degradation of its precursor peptide. In the pepF mutant, the MccJ25 reached a concentration of 1.68 µM after a cultivation time exceeding 60 hours, while the wild-type strain exhibited a concentration of only 0.14 µM. Moreover, the production of MccJ25 in B. subtilis downregulated the genes associated with sporulation, and this may contribute to its accumulation. Finally, this study provides a strategy to improve the stability and production of MccJ25 in B. subtilis ., Importance: MccJ25 displays significant antibacterial activity, a well-defined mode of action, exceptional safety, and remarkable stability. Hence, it presents itself as a compelling candidate for an optimal antibacterial or anti-endotoxin medication. The successful establishment of exogenous production of MccJ25 in Bacillus subtilis provides a strategy for reducing its production cost and diversifying its utilization. In this study, we have provided evidence indicating that both peptidase PepF and sporulation are significant factors that limit the expression of MccJ25 in B. subtilis. The Δ pepF and Δ sigF mutants of B. subtilis express MccJ25 with higher production yield and enhanced stability. To sum up, this study developed several better engineered strains of B. subtilis , which greatly reduced the consumption of MccJ25 during the nutrient depletion stage of the host strain, improved its production, and elucidated factors that may be involved in reducing MccJ25 accumulation in B. subtilis ., Competing Interests: The authors declare no conflict of interest.

Wild-type Lactococcus lactis strain LAC460 secretes prophage-encoded bacteriocin-like lysin LysL, which kills some Lactococcus strains, but has no lytic effect on the producer. LysL carries two N-terminal enzymatic active domains (EAD), and an unknown C-terminus without homology to known domains. This study aimed to determine whether the C-terminus of LysL carries a cell wall binding domain (CBD) for target specificity of LysL. The C-terminal putative CBD region of LysL was fused with His-tagged green fluorescent protein (HGFPuv). The HGFPuv&#95;CBDlysL gene fusion was ligated into the pASG-IBA4 vector, and introduced into Escherichia coli. The fusion protein was produced and purified with affinity chromatography. To analyse the binding of HGFPuv&#95;CBDLysL to Lactococcus cells, the protein was mixed with LysL-sensitive and LysL-resistant strains, including the LysL-producer LAC460, and the fluorescence of the cells was analysed. As seen in fluorescence microscope, HGFPuv&#95;CBDLysL decorated the cell surface of LysL-sensitive L. cremoris MG1614 with green fluorescence, whereas the resistant L. lactis strains LM0230 and LAC460 remained unfluorescent. The fluorescence plate reader confirmed the microscopy results detecting fluorescence only from four tested LysL-sensitive strains but not from 11 tested LysL-resistant strains. Specific binding of HGFPuv&#95;CBDLysL onto the LysL-sensitive cells but not onto the LysL-resistant strains indicates that the C-terminus of LysL contains specific CBD. In conclusion, this report presents experimental evidence of the presence of a CBD in a lactococcal phage lysin. Moreover, the inability of HGFPuv&#95;CBDLysL to bind to the LysL producer LAC460 may partly explain the host's resistance to its own prophage lysin., (© 2024. The Author(s).)

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacteriocins are peptides or proteins produced by bacteria to kill or inhibit the growth of other bacteria inhabiting the same ecological niche. The growing interest in bacteriocins reflects their potential use in food preservation and treatment of infections caused by antibiotic-resistant pathogenic bacteria, among other applications. The number of published studies on the identification of new bacteriocin-producing strains is constantly increasing. At the same time, there is a noticeable lack of research describing the mechanisms of action of most newly identified bacteriocins, as well as the mechanisms leading to the development of resistance to these bacteriocins and cross-resistance to antibiotics. Detailed understanding of these issues will allow to develop guidelines ensuring the most effective, safe and long-term use of bacteriocins without the risk of resistance development. This work describes the main assumptions of the doctoral dissertation of Aleksandra Tymoszewska, which objectives were to characterize the mechanisms of action and of resistance to class II bacteriocins of Gram-positive bacteria. Using the model bacterium Lactococcus lactis, two groups of bacteriocins were examined: (i) garvicins Q, A, B and C, and BacSJ; and (ii) aureocin A53 (AurA53)- and enterocin L50 (EntL50)-like bacteriocins. Bacteriocins of group (i) have been shown to recognize susceptible cells and form pores in the cell membrane using a specific receptor, the mannose-specific phosphotransferase system (Man-PTS), and sensitive bacteria have been shown to acquire resistance to the these bacteriocins by modifying the structure of Man-PTS. On the other hand, the acquisition of resistance to group (ii) membrane-targeting and receptor-independent bacteriocins occurs through changes in the structure of the bacterial cell wall and membrane, which are induced by changes in the expression of proteins involved in lipid metabolism or components of the YsaCB-KinG-LlrG regulatory system. The results shed new light on previous views on the mechanisms of action of bacteriocins and open up opportunities for their further study.

Cheese is a food in which toxic concentrations of biogenic amines (BA) may be reached, mainly as a consequence of the decarboxylation of determined amino acids by certain lactic acid bacteria (LAB). To maintain the food safety of cheese, environmentally friendly strategies are needed that specifically prevent the growth of BA-producing LAB and the accumulation of BA. The bacteriocins produced by LAB are natural compounds with great potential as food biopreservatives. This work examines the antimicrobial potential of 7 bacteriocin-containing, cell-free supernatants (CFS: coagulin A-CFS, enterocin A-CFS, enterocin P-CFS, lacticin 481-CFS, nisin A-CFS, nisin Z-CFS and plantaricin A-CFS) produced by LAB against 48 strains of the LAB species largely responsible for the accumulation of the most important BA in cheese, that is, histamine, tyramine, and putrescine. Susceptibility to the different CFS was strain-dependent. The histamine-producing species with the broadest sensitivity spectrum were Lentilactobacillus parabuchneri (the species mainly responsible for the accumulation of histamine in cheese) and Pediococcus parvulus. The tyramine-producing species with the broadest sensitivity spectrum was Enterococcus faecium, and Enterococcus faecalis and Enterococcus hirae were among the most sensitive putrescine producers. Nisin A-CFS was active against 31 of the 48 BA-producing strains (the broadest antimicrobial spectrum recorded). Moreover, commercial nisin A prevented biofilm formation by 67% of the BA-producing, biofilm-forming LAB strains. These findings underscore the potential of bacteriocins in the control of BA-producing LAB and support the use of nisin A as a food-grade biopreservative for keeping BA-producing LAB in check and reducing BA accumulation in cheese., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core., Importance: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin., Competing Interests: The authors declare no conflict of interest.

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control.

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).)

The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cherrak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Members of the genus Lysinibacillus attract attention for their mosquitocidal, bioremediation, and plant growth-promoting abilities. Despite this interest, comprehensive studies focusing on genomic traits governing plant growth and stress resilience in this genus using whole-genome sequencing are still scarce. Therefore, we sequenced and compared the genomes of three endophytic Lysinibacillus irui strains isolated from Canary Island date palms with the ex-type strain IRB4-01. Overall, the genomes of these strains consist of a circular chromosome with an average size of 4.6 Mb and a GC content of 37.2%. Comparative analysis identified conserved gene clusters within the core genome involved in iron acquisition, phosphate solubilization, indole-3-acetic acid biosynthesis, and volatile compounds. In addition, genome analysis revealed the presence of genes encoding carbohydrate-active enzymes, and proteins that confer resistance to oxidative, osmotic, and salinity stresses. Furthermore, pathways of putative novel bacteriocins were identified in all genomes. This illustrates possible common plant growth-promoting traits shared among all strains of L. irui. Our findings highlight a rich repertoire of genes associated with plant lifestyles, suggesting significant potential for developing inoculants to enhance plant growth and resilience. This study is the first to provide insights into the overall genomic signatures and mechanisms of plant growth promotion and biocontrol in the genus Lysinibacillus. KEY POINTS: • Pioneer study in elucidating plant growth promoting in L. irui through comparative genomics. • Genome mining identified biosynthetic pathways of putative novel bacteriocins. • Future research directions to develop L. irui-based biofertilizers for sustainable agriculture., (© 2024. The Author(s).)

Background: Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated., Results: The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms., Conclusions: Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix., (© 2024. The Author(s).)

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa , respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK&#95;0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis . Next, we sought to determine its identity and characterize its toxicity. We found that YPK&#95;0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis . We further identified YPK&#95;0952 as a DNase dependent on Mg 2+ , Ni 2+ , Mn 2+ , and Co 2+ bivalent metal ions, and the homologous immune protein YPK&#95;0953 can inhibit its activity. In summary, YPK&#95;0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK&#95;0953 prevents self-attack in Y. pseudotuberculosis .IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK&#95;0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis . YPK&#95;0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli ) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation., Competing Interests: The authors declare no conflict of interest.

Ice cream manufacture commonly results in the accumulation of wasted product that contains valuable food-grade quality components, including fat, carbohydrates, and protein. Methods have been developed for recovering the fat from this waste stream, but this results in the generation of a co-product rich in fermentable carbohydrates. This study aimed to investigate the potential for using this co-product as a fermentation substrate for production of antimicrobial peptides, called bacteriocins, by dairy starter cultures. Results showed that Streptococcus thermophilus B59671 and Lactococcus lactis 11454 produced the broad-spectrum bacteriocins thermophilin 110 and nisin, respectively, when the fermentation substrate was melted ice cream, or a co-product generated by a modified butter churning technique. Bacteriocin production varied depending on the brand and variety of vanilla ice cream used in this study. When an alternate enzyme-assisted fat extraction technique was used, S. thermophilus metabolism was impaired within the resulting co-product, and thermophilin 110 production was not observed. Lactococcus lactis was still able to grow in this co-product, but antimicrobial activity was not observed. Results from this study suggest the co-product generated when using the churning technique is a better choice to use as a base medium for future studies to optimize bacteriocin production., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99m Tc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted., Methods and Results: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99m Tc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99m Tc-duramycin + areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99m Tc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm 2 ; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm 2 ), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99m Tc-duramycin + tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P =0.008). Compared with infarct, gene expression in the 99m Tc-duramycin + -noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P <0.05; circumferential strain -52.9%, P <0.05)., Conclusions: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.

Food preservation is a schematic and scientific procedure employed for the maintenance and improvement of food's quality, shelf life, and nutritional value. Although, on one hand, ancient conventional methods such as freezing, pasteurization, canning, and chemical methods have the potential to lengthen the shelf life of edible substances, but on the other hand, they can deteriorate its nutritional value as well. Present research focuses on the identification of promising bacteriocins against Pseudomonas fragi via subtractive proteomics pipeline as an alternative approach for food preservation. Bacteriocins are small peptides produced by certain microbes to naturally defend themselves by destroying other closely related bacteria residing in their neighborhood. P. fragi lies among the most notable microbes responsible for the elicitation of food spoilage. Due to increasing emergence and prevalence of multidrug resistance bacteria, there is a need to unravel novel drug targets, crucially involved in food decay process. Based on subtractive scrutinization, UDP-N-acetylglucosamine O-acyltransferase (LpxA) was chosen as promising therapeutic protein target that could play a significant role in progression of food spoilage. Subtilosin A, thuricin-CD, and mutacin B-NY266 were found as the most robust inhibitors of LpxA according to the molecular docking assay results. Molecular dynamic simulations and binding energy calculations via MM/PBSA method of LpxA and three top hit docked complexes, i.e., LpxA-subtilosin A, LpxA-thuricin-CD, and LpxA-mutacin B-NY266, revealed stability throughout simulations and ensured that shortlisted bacteriocins had strong affinity for LpxA., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteriocins are highly diverse, abundant, and heterogeneous antimicrobial peptides that are ribosomally synthesized by bacteria and archaea. Since their discovery about a century ago, there has been a growing interest in bacteriocin research and applications. This is mainly due to their high antimicrobial properties, narrow or broad spectrum of activity, specificity, low cytotoxicity, and stability. Though initially used to improve food quality and safety, bacteriocins are now globally exploited for innovative applications in human, animal, and food systems as sustainable alternatives to antibiotics. Bacteriocins have the potential to beneficially modulate microbiota, providing viable microbiome-based solutions for the treatment, management, and non-invasive bio-diagnosis of infectious and non-infectious diseases. The use of bacteriocins holds great promise in the modulation of food microbiomes, antimicrobial food packaging, bio-sanitizers and antibiofilm, pre/post-harvest biocontrol, functional food, growth promotion, and sustainable aquaculture. This can undoubtedly improve food security, safety, and quality globally. This review highlights the current trends in bacteriocin research, especially the increasing research outputs and funding, which we believe may proportionate the soaring global interest in bacteriocins. The use of cutting-edge technologies, such as bioengineering, can further enhance the exploitation of bacteriocins for innovative applications in human, animal, and food systems., (© 2024. The Author(s).)

Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.

It has become a tradition for the BAMP (Bacteriocins and Antimicrobial Peptides) symposium to be a part of the IPC (International Probiotic Conference). In 2024, IPC/BAMP will be held on the 18th-20th of June in Prague, Czech Republic ( www.probiotic-conference.net ) and will reunite scientists, students, and representatives from industry and regulations agencies from all around the world. The meeting will serve as a platform for the exchange of knowledge and ideas regarding the past, present, and future of beneficial microbes, probiotics, antimicrobials, and proteins, and their influence on a prosperous and healthier future., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Lactobacillus acidophilus is lactic acid bacteria that produce bacteriocins. Bacteriocins are antimicrobial peptides or proteins that exhibit activity against closely related bacteria. The aim of this study was to determine the effect of L. acidophilus ATCC 4356 bacteriocin against Staphylococcus aureus. Material and Methods . We used four different phenotypic methods for antimicrobial activities against two standard strains: methicillin-resistant S. aureus (MRSA) ATCC 33591 and methicillin-susceptible S. aureus (MSSA) ATCC 25923. The methods were (1) agar well diffusion, (2) overlay soft agar, (3) paper disk, and (4) modification of punch hole. The ammonium sulfate method was used to concentrate crude bacteriocin, and ultrafiltration and dialysis tubes were used to remove ammonium sulfate from the bacteriocins. Each method was repeated in triplicate., Result: L. acidophilus ATCC 4356 showed antimicrobial activity against both MRSA and MSSA standard strains only by the overlay soft agar method and not by the agar well diffusion, punch hole modification, and paper disk methods. No antimicrobial effects were observed in crude bacteriocins concentrated., Conclusion: The growth inhibition of S. aureus in overlay soft agar method may be due to the production of bacteriocin-like substances. The overlay soft agar method is a qualitative test, so there is a need for further study to optimize the conditions for the production of bacteriocin-like substances in the culture supernatant and precise comparison between the inhibitory activity and pheromone secretion of different strains., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Zeinab Fagheei Aghmiyuni et al.)

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S . mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG , an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG , 19/108 strains (17.6%) carried only mukFEG , and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG . We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type β). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type β (MukFβ). Then, we constructed a mukFEG -deletion mutant complemented with MukFαEG or MukFβEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes., Importance: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans . This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution., Competing Interests: The authors declare no conflict of interest.

Multidrug-resistant (MDR) bacteria are widespread in the environment and pose a serious threat to public health. It has been shown that bacteriocins have a great potential in controlling MDR pathogens, including Staphylococcus aureus. A previously reported Lactobacillus salivarius bacteriocin XJS01 exhibited good antibacterial activity against MDR S. aureus 2612:1606BL1486 (henceforth referred to as S. aureus&#95;26), but its molecular mechanism remains unknown. Herein, we investigated the antibacterial mechanism of XJS01 on S. aureus&#95;26 using an approach combining transcriptomics and metabolomics. The results showed that XJS01 induced significant changes at both transcriptional and metabolic levels in S. aureus&#95;26. In total, 231 differentially expressed genes (DEGs) and 206 differentially abundance metabolites (DAMs) were identified in S. aureus&#95;26 treated with 1 × MIC (minimum inhibition concentration) XJS01 compared with untreated (XJS01-free) cells (control). Functional analysis revealed that these DEGs and DAMs, alone with the related pathways and biological processes, were typically involved in stress response, being primarily related to metal uptake, cell virulence, self-help mechanism, amino acid and energy metabolism, bacterial stress response (e.g., two-component system), and membrane transport (e.g., phosphotransferase system). Overall, this study uncovered the multi-target effects of bacteriocins against MDR S. aureus at the genome-wide transcriptional and metabolic levels. These findings might be useful in the development of bacteriocins for the control of MDR S. aureus and other drug-resistant bacteria., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections., (© 2024. The Author(s).)

The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation of the lipid bilayer. Helical unpacking is driven by disruption of the peptide hydrophobic core when in contact with membranes. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of Gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with the membranes, a series of arginine single-site mutants were constructed that destabilize the hydrophobic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations analysis, and release kinetics of calcein, show that decreased stabilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes and in killing bacteria. Finally, using the computational technique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants, whose trends correlate well with our experiment.

Biofilm, a microbial community formed by especially pathogenic and spoilage bacterial species, is a critical problem in the food industries. It is an important cause of continued contamination by foodborne pathogenic bacteria. Therefore, removing biofilm is the key to solving the high pollution caused by foodborne pathogenic bacteria in the food industry. Lactobacillus, a commonly recognized probiotic that is healthy for consumer, have been proven useful for isolating the potential biofilm inhibitors. However, the addition of surface components and metabolites of Lactobacillus is not a current widely adopted biofilm control strategy at present. This review focuses on the effects and preliminary mechanism of action on biofilm inhibition of Lactobacillus-derived components including lipoteichoic acid, exopolysaccharides, bacteriocins, secreted protein, organic acids and some new identified molecules. Further, the review discusses several modern biofilm identification techniques and particularly interesting new technology of biofilm inhibition molecules. These molecules exhibit stronger inhibition of biofilm formation, playing a pivotal role in food preservation and storage. Overall, this review article discusses the application of biofilm inhibitors produced by Lactobacillus, which would greatly aid efforts to eradicate undesirable bacteria from environment in the food industries., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth., (© 2024. The Author(s).)

Natural competence is the principal driver of streptococcal evolution. While acquisition of new traits could facilitate rapid fitness improvement for bacteria, entry into the competent state is a highly orchestrated event, involving an interplay between various pathways. We present a new type of competence-predation coordination mechanism in Streptococcus sanguinis. Unlike other streptococci that mediate competence through the ComABCDE regulon, several key components are missing in the S. sanguinis ComCDE circuitry. We assembled two synthetic biology devices linking competence-stimulating peptide (CSP) cleavage and export with a quantifiable readout to unravel the unique features of the S. sanguinis circuitry. Our results revealed the ComC precursor cleavage pattern and the two host ABC transporters implicated in the export of the S. sanguinis CSP. Moreover, we discovered a ComCDE-dependent bacteriocin locus. Overall, this study presents a mechanism for commensal streptococci to maximize transformation outcome in a fluid environment through extensive circuitry rewiring., Competing Interests: Declaration of interests The authors declare no competing financial interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The PsdRSAB and ApsRSAB detoxification modules, together with the antimicrobial peptides (AMPs)-resistance determinants Dlt system and MprF protein, play major roles in the response to AMPs in Lacticaseibacillus paracasei BL23. Sensitivity assays with a collection of mutants showed that the PsdAB ABC transporter and the Dlt system are the main subtilin resistance determinants. Quantification of the transcriptional response to subtilin indicate that this response is exclusively regulated by the two paralogous systems PsdRSAB and ApsRSAB. Remarkably, a cross-regulation of the derAB, mprF and dlt-operon genes-usually under control of ApsR-by PsdR in response to subtilin was unveiled. The high similarity of the predicted structures of both response regulators (RR), and of the RR-binding sites support this possibility, which we experimentally verified by protein-DNA binding studies. ApsR-P shows a preferential binding in the order P derA  > P dlt  > P mprF  > P psdA . However, PsdR-P bound with similar apparent affinity constants to the four promoters. This supports the cross-regulation of derAB, mprF and the dlt-operon by PsdR. The possibility of cross-regulation at the level of RR-promoter interaction allows some regulatory overlap with two RRs controlling the expression of systems involved in maintenance of critical cell membrane functions in response to lantibiotics., (© 2024. The Author(s).)

Prophages integrated into bacterial genomes can become cryptic or defective prophages, which may evolve to provide various traits to bacterial cells. Previous research on Marinomonas mediterranea MMB-1 demonstrated the production of defective particles. In this study, an analysis of the genomes of three different strains (MMB-1, MMB-2, and MMB-3) revealed the presence of a region named MEDPRO1, spanning approximately 52 kb, coding for a defective prophage in strains MMB-1 and MMB-2. This prophage seems to have been lost in strain MMB-3, possibly due to the presence of spacers recognizing this region in an I-F CRISPR array in this strain. However, all three strains produce remarkably similar defective particles. Using strain MMB-1 as a model, mass spectrometry analyses indicated that the structural proteins of the defective particles are encoded by a second defective prophage situated within the MEDPRO2 region, spanning approximately 13 kb. This finding was further validated through the deletion of this second defective prophage. Genomic region analyses and the detection of antimicrobial activity of the defective prophage against other Marinomonas species suggest that it is an R-type bacteriocin. Marinomonas mediterranea synthesizes antimicrobial proteins with lysine oxidase activity, and the synthesis of an R-type bacteriocin constitutes an additional mechanism in microbial competition for the colonization of habitats such as the surface of marine plants.IMPORTANCEThe interactions between bacterial strains inhabiting the same environment determine the final composition of the microbiome. In this study, it is shown that some extracellular defective phage particles previously observed in Marinomonas mediterranea are in fact R-type bacteriocins showing antimicrobial activity against other Marinomonas strains. The operon coding for the R-type bacteriocin has been identified., Competing Interests: The authors declare no conflict of interest.

Bacillus probiotics exhibit considerable economic potential owing to their heightened resilience to external stressors and relatively lower costs related to production and preservation. Although Bacillus paralicheniformis has been acknowledged as a plant-promoting bacterium for a long time, understanding its potential as a probiotic is still in its nascent stages. In this study, the safety and probiotic characteristics of a strain of HMPM220325, isolated from artisanal fruit dairy products, were examined through whole-genome sequencing and phenotypic analysis. The whole genome of HMPM220325 was analyzed for antimicrobial resistance genes, pathogenicity factors, and genes associated with probiotic traits including stress resistance, spore formation, gut adhesion, competitive exclusion of pathogens, bacteriocin expression, and carbohydrate metabolism related to prebiotic utilization. Also, wet lab experiments were conducted for the characterization of probiotics. The identification of the organism as B. paralicheniformis was verified. Its safety was assessed through in silico analysis, the haemolytic activity test, and the acute oral toxicity test. B. paralicheniformis HMPM220325 demonstrated its ability to survive in the pH range of 4-10 and bile salt concentrations of 0-0.9% (w/v), tolerate temperatures between 20 and 60 °C, and exhibit a robust antioxidant capacity. Moreover, B. paralicheniformis HMPM220325 demonstrated a moderate level of hydrophobicity, had the ability to form biofilms, achieved a self-aggregation rate of 51.77 ± 1.01% within 6 hours, and successfully colonized the mouse intestine for a duration of up to 17 days. Additionally, the genome of B. paralicheniformis HMPM220325 contains three gene clusters associated with the biosynthesis of bacteriocins and exhibits co-aggregation with Staphylococcus aureus , Pseudomonas aeruginosa , and Salmonella enterica serovar Typhimurium. The findings of the genomic analysis align with those obtained from the experimental investigation, thereby substantiating the potential of B. paralicheniformis HMPM220325 as a probiotic suitable for incorporation in dairy functional foods and feed applications.

Streptococcus intermedius secretes the human-specific cytolysin intermedilysin (ILY), a crucial factor in the pathogenicity of this bacterium. Previously, we reported that a lactose phosphotransferase repressor (LacR) represses ily expression, and that its mutation increases ILY production. Interestingly, UNS40, a strain isolated from a liver abscess, produces high levels of ILY despite the absence of mutations in the lacR promoter and coding regions. Our results showed that a G > A mutation at the -90th position from the transcription start point in the UNS40 ily promoter region increased hemolytic activity and decreased the binding ability to LacR. To elucidate the regions involved in the repression of ily expression, we generated mutant strains, in which point or deletion mutations were introduced into the ily promoter region, and then compared their hemolytic activity. Among the point mutations, -120 C > A and -90 G > A and their flanking mutations increased hemolytic activity. These results indicated that these mutations may increase the virulence of S. intermedius., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Here, we describe hitherto unknown shape-function of S/O-HexNActransferase SvGT (ORF AQF52&#95;3101) instrumental in glycosylation of bacteriocin SvC (ORF AQF52&#95;3099) in Streptomyces venezuelae ATCC 15439. Data from gel filtration, mass spectrometry, analytical ultracentrifugation, and Small Angle X-ray Scattering (SAXS), experiments confirmed elongated dimeric shape in solution for SvGT protein. Enzyme assays confirmed the dependence of SvGT on the availability of Mg 2+ ions to be functionally activated. SAXS data analysis provided that apo and Mg 2+ -activated protein adopt a shape characterized by a radius of gyration and maximum linear dimension of 5.2 and 17.0 nm, and 5.3 and 17.8 nm, respectively. Alphafold2 server was used to model the monomeric chain of this protein which was docked on self to obtain different poses of the dimeric entity. Experimental SAXS data was used to select and refine the structure of SvGT dimer. Results showed that Mg 2+ ions induce reorientation of the GT domain of one chain leading to a dimer with C2 symmetry, and the C-terminal portion entangles with each other in all states. Mutation-rendered alteration in activity profiles confirmed the role of conserved residues around catalytic motif. Global structure analysis puts forth the need to understand the role of constitutionally diverse C-terminal portion in regulating substrate selectivity.Communicated by Ramaswamy H. Sarma.

Gut bacteria are known to produce bacteriocins to inhibit the growth of other bacteria. Consequently, bacteriocins have attracted increased attention as potential microbiome-editing tools. In this study we examine the inhibitory spectrum of 75 class II bacteriocins against 48 representative gut microbiota species. The bacteriocins were heterologously expressed in Escherichia coli and evaluated in vitro, ex vivo and in vivo. In vitro assays revealed 22 bacteriocins to inhibit at least one species and showed selective inhibition patterns against species implicated in certain disorders and diseases. Three bacteriocins were selected for ex vivo assessment on mouse feces. Based on 16S rRNA sequencing of the cultivated feces we showed that the two bacteriocins: Actifencin (#13) and Bacteroidetocin A (#22) selectively inhibited the growth of Lactobacillus and Bacteroides , respectively. The probiotic: E. coli Nissle 1917 was engineered to express these two bacteriocins in mice. However, the selective inhibitory patterns found in the in vitro and ex vivo experiments could not be observed in vivo . Our study describes a methodology for heterologous high throughput bacteriocin expression and screening and elucidates the inhibitory patterns of class II bacteriocins on the gut microbiota.

Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections and associated with lung infections in cystic fibrosis (CF) patients (Lyczak et al., Microbes Infect 2:1051-1060, 2000). Multiple drug-resistant P. aeruginosa strains pose a serious problem because of antibiotic treatment failure. There is therefore a need for alternative anti-Pseudomonas molecules. Soluble pyocins (S-pyocins) are bacteriocins produced by P. aeruginosa strains that kill sensitive strains of the same species. These bacteriocins and their immunity gene are easily cloned and expressed in E. coli and their activity spectrum against different P. aeruginosa strains can be tested. In this chapter, we describe the procedures for cloning, expression, and sensitivity testing of two different S-pyocins. We also describe how to identify their receptor binding domain in sensitive strains, how to construct chimeric pyocins with extended activity spectra, and how to identify new pyocins in genomes by multiplex PCR., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)