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Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22-6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems., (© 2021. The Author(s).)

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli , Listeria monocytogenes , and Staphylococcus aureus . Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0-7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3-10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04-0.62 mg·mL -1 and 0.08-1.25 mg·mL -1 , respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975  μ g·mL -1 (i.e., moderate toxicity) to Artemia salina . Identification of the peptide sequences of this fraction by liquid chromatography-tandem mass spectrometry yielded 130 proteins with retention times of 15.21-19.57 min. Eleven proteins with MWs of 1345.66-2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34-26.22 kcal·mol -1 ) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces , especially BLIS SCA-8, as antibacterial agents., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2021 Muhammad A. Kurnianto et al.)

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ , where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (Δ ddE and Δ ddF ), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

Lactic acid bacteria (LAB) are Gram-positive and catalase-negative microorganisms used to produce fermented foods. They appear morphologically as cocci or rods and they do not form spores. LAB used in food fermentation are from the Lactobacillus and Bifidobacterium genera and are useful in controlling spoilage and pathogenic microbes, due to the bacteriocins and acids that they produce. Consequently, LAB and their bacteriocins have emerged as viable alternatives to chemical food preservatives, curtesy of their qualified presumption of safety (QPS) status. There is growing interest regarding updated literature on the applications of LAB and their products in food safety, inhibition of the proliferation of food spoilage microbes and foodborne pathogens, and the mitigation of viral infections associated with food, as well as in the development of creative food packaging materials. Therefore, this review explores empirical studies, documenting applications and the extent to which LAB isolates and their bacteriocins have been used in the food industry against food spoilage microorganisms and foodborne pathogens including viruses; as well as to highlight the prospects of their numerous novel applications as components of hurdle technology to provide safe and quality food products.

Lactobacillus paragasseri was identified as a novel sister taxon of L. gasseri in 2018. Since the reclassification of L. paragasseri, there has been hardly any report describing the probiotic properties of this species. In this study, an L. paragasseri strain UBLG-36 was sequenced and analyzed to determine the molecular basis that may confer the bacteria with probiotic potential. UBLG-36 was previously documented as an L. gasseri strain. Average nucleotide identity and phylogenomic analysis allowed accurate taxonomic identification of UBLG-36 as an L. paragasseri strain. Analysis of the draft genome (~1.94 Mb) showed that UBLG-36 contains 5 contigs with an average G+C content of 34.85%. Genes essential for the biosynthesis of bacteriocins, adhesion to host epithelium, stress resistance, host immunomodulation, defense, and carbohydrate metabolism were identified in the genome. Interestingly, L. paragasseri UBLG-36 also harbored genes that code for enzymes involved in oxalate catabolism, such as formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). In vitro oxalate degradation assay showed that UBLG-36 is highly effective in degrading oxalate (averaging more than 45% degradation), a feature that has not been reported before. As a recently identified bacterium, there are limited genomic reports on L. paragasseri, and our draft genome sequence analysis is the first to describe and emphasize the probiotic potential and oxalate degrading ability of this species. With results supporting the probiotic functionalities and oxalate catabolism of UBLG-36, we propose that this strain is likely to have immense biotechnological applications upon appropriate characterization., Competing Interests: The authors have declared that no competing interests exist.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

The microbiomes on human body surfaces affect health in multiple ways. They include not only commensal or mutualistic bacteria but also potentially pathogenic bacteria, which can enter sterile tissues to cause invasive infection. Many commensal bacteria produce small antibacterial molecules termed bacteriocins that have the capacity to eliminate specific colonizing pathogens; as such, bacteriocins have attracted increased attention as potential microbiome-editing tools. Metagenome-based and activity-based screening approaches have strongly expanded our knowledge of the abundance and diversity of bacteriocin biosynthetic gene clusters and the properties of a continuously growing list of bacteriocin classes. The dynamic acquisition, diversification or loss of bacteriocin genes can shape the fitness of a bacterial strain that is in competition with bacteriocin-susceptible bacteria. However, a bacteriocin can only provide a competitive advantage if its fitness benefit exceeds the metabolic cost of production, if it spares crucial mutualistic partner strains and if major competitors cannot develop resistance. In contrast to most currently available antibiotics, many bacteriocins have only narrow activity ranges and could be attractive agents for precision therapy and prevention of infections. A common scientific strategy involving multiple disciplines is needed to uncover the immense potential of microbiome-shaping bacteriocins., (© 2021. Springer Nature Limited.)

Lactiplantibacillus plantarum, previously named "Lactobacillus plantarum," is found in a wide variety of environments exhibiting a high level of intraspecies genetic diversity. To investigate the strain diversity, we performed comparative genomic analyses of the 54 complete genome sequences. The results revealed that L. plantarum subsp. plantarum was split into three lineages, A, B and C. Of the genes beneficial for probiotic activity, only those associated with the biosynthesis of plantaricin (Pln), an L. plantarum-specific bacteriocin, were found to be significantly different among the lineages. The genes related to the biosynthesis of plnE/F were conserved throughout the three lineages, whereas the outgroups did not possess any Pln-producing genes. In lineage C, the deepest and ancestral type branch, plnE/F genes, were well conserved. In lineage B, loss of gene function was observed due to mobile elements in the pln loci. In lineage A, most strains were predicted to produce more than one type of Pln by possessing diverse Pln-encoding genes. These results showed the presence of functional diversity arising from the trifurcating evolution in L. plantarum subsp. plantarum and demonstrated that Pln is an indicator for differentiating the three lineages., (© 2021. The Author(s).)

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC 50 around 1 μM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genome evolution is an essential and stringently regulated aspect of biological fitness. For bacteria, natural competence is one of the principal mechanisms of genome evolution and is frequently subject to multiple layers of regulation derived from a plethora of environmental and physiological stimuli. Here, we present a regulatory mechanism that illustrates how such disparate stimuli can be integrated into the Streptococcus mutans natural competence phenotype. S. mutans possesses an intriguing, but poorly understood ability to coordinately control its independently regulated natural competence and bacteriocin genetic pathways as a means to acquire DNA released from closely related, bacteriocin-susceptible streptococci. Our results reveal how the bacteriocin-specific transcription activator BrsR directly mediates this coordination by serving as an anti-adaptor protein responsible for antagonizing the proteolysis of the inherently unstable, natural competence-specific alternative sigma factor ComX. This BrsR ability functions entirely independent of its transcription regulator function and directly modulates the timing and severity of the natural competence phenotype. Additionally, many of the DNA uptake proteins produced by the competence system were surprisingly found to possess adaptor abilities, which are employed to terminate the BrsR regulatory circuit via negative feedback. BrsR-competence protein heteromeric complexes directly inhibit nascent brsR transcription as well as stimulate the Clp-dependent proteolysis of extant BrsR proteins. This study illustrates how critical genetic regulatory abilities can evolve in a potentially limitless variety of proteins without disrupting their conserved ancestral functions. These unrecognized regulatory abilities are likely fundamental for transducing information through complex genetic networks., Competing Interests: The authors declare no competing interest.

Plantaricin Q7 is a bacteriocin produced by Lactobacillus plantarum Q7 with food preservation potential. Low yield is one of the bottlenecks of the wide application of plantaricin Q7. Nontargeted metabolomics was performed to reveal the mechanism of plantaricin Q7 biosynthesis. The results showed that the composition and abundance of intracellular metabolites varied significantly at key time points of plantaricin Q7 synthesis. Differential metabolic pathways were purine metabolism; pyrimidine metabolism; alanine, aspartate, and glutamate metabolism; amino acid biosynthesis; aminoacyl-tRNA biosynthesis; and ABC transporters. Differential metabolites were xanthine, deoxyadenosine, uracil, 5-methylcytosine, α-ketoglutarate, γ-aminobutyric acid, glutamate, glutamine, and tryptophan. Based on metabolomics information, the putative metabolic synthesis pathway of plantaricin Q7 was proposed. Glutamine, glutamate, and 5-methylcytosine could be critical metabolites and simulate plantaricin Q7 biosynthesis significantly ( P < 0.05). Bacteriocin production was investigated by comparative metabolomics in this report, which could help to achieve higher plantaricin Q7 yield by metabolic regulation.

Bacteria inhibit and kill one another with a diverse array of compounds, including bacteriocins and antibiotics. These attacks are highly regulated, but we lack a clear understanding of the evolutionary logic underlying this regulation. Here, we combine a detailed dynamic model of bacterial competition with evolutionary game theory to study the rules of bacterial warfare. We model a large range of possible combat strategies based upon the molecular biology of bacterial regulatory networks. Our model predicts that regulated strategies, which use quorum sensing or stress responses to regulate toxin production, will readily evolve as they outcompete constitutive toxin production. Amongst regulated strategies, we show that a particularly successful strategy is to upregulate toxin production in response to an incoming competitor's toxin, which can be achieved via stress responses that detect cell damage (competition sensing). Mirroring classical game theory, our work suggests a fundamental advantage to reciprocation. However, in contrast to classical results, we argue that reciprocation in bacteria serves not to promote peaceful outcomes but to enable efficient and effective attacks., Competing Interests: RN, NO, AL, AF, KF No competing interests declared, (© 2021, Niehus et al.)

Lytic bacteriophages are expected as effective tools to control infectious bacteria in human and pathogenic or spoilage bacteria in foods. Leaderless bacteriocins (LLBs) are simple bacteriocins produced by Gram-positive bacteria. LLBs do not possess an N-terminal leader peptide in the precursor, which means that they are active immediately after translation. In this study, we constructed a novel antimicrobial agent, an LLB-producing phage (LLB-phage), by genetic engineering to introduce the LLB structural gene into the lytic phage genome. To this end, lnqQ (structure gene of an LLB, lacticin Q) and trxA , an essential gene for T7 phage genome replication, were integrated in tandem into T7 phage genome using homologous recombination in Escherichia coli host strain. The recombinant lnqQ -T7 phage was isolated by a screening method using Δ trxA host strain. lnqQ -T7 phage formed a clear halo in agar plates containing both E. coli and lacticin Q-susceptible Bacillus coagulans, indicating that lnqQ -T7 phage could produce a significant amount of lacticin Q. Lacticin Q production did not exert a significant effect on the lytic cycle of T7 phage. In fact, the production of lacticin Q enhanced T7 phage lytic activity and helped to prevent the emergence of bacterial populations resistant against this phage. These results serve as a proof of principle for LLB-phages. There are different types of LLBs and phages, meaning that in the future, it may be possible to produce any number of LLB-phages which can be designed to efficiently control different types of bacterial contamination in different settings. IMPORTANCE We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ -T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain. There are several different types of LLBs and phages, suggesting that we may be able to design a battery of LLB-phages by selecting novel combinations of LLBs and phages. These constructs could be tailored to control various bacterial contaminations and infectious diseases.

A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo . The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.

Human gut microbes exhibit a spectrum of cooperative and antagonistic interactions with their host and also with other microbes. The major Bacteroides host-targeting virulence factor, Bacteroides fragilis toxin (BFT), is produced as an inactive protoxin by enterotoxigenic B. fragilis strains. BFT is processed by the conserved bacterial cysteine protease fragipain (Fpn), which is also encoded in B. fragilis strains that lack BFT. In this report, we identify a secreted antibacterial protein (fragipain-activated bacteriocin 1 [Fab1]) and its cognate immunity protein (resistance to fragipain-activated bacteriocin 1 [RFab1]) in enterotoxigenic and nontoxigenic strains of B. fragilis. Although BFT and Fab1 share no sequence identity, Fpn also activates the Fab1 protoxin, resulting in its secretion and antibacterial activity. These findings highlight commonalities between host- and bacterium-targeting toxins in intestinal bacteria and suggest that antibacterial antagonism may promote the conservation of pathways that activate host-targeting virulence factors. IMPORTANCE The human intestine harbors a highly complex microbial community; interpersonal variation in this community can impact pathogen susceptibility, metabolism, and other aspects of health. Here, we identified and characterized a commensal-targeting antibacterial protein encoded in the gut microbiome. Notably, a shared pathway activates this antibacterial toxin and a host-targeting toxin. These findings highlight unexpected commonalities between host- and bacterium-targeting toxins in intestinal bacteria.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA Arg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNA Arg s; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNA Arg s from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNA Arg ICG , the most abundant species of E. coli tRNA Arg s, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNA Arg ICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNA Arg ICG dominant-negatively impairs translation in vitro . Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNA Arg ICG , which is consistent with our structural model. Finally, elevation of cleaved tRNA Arg ICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNA Arg ICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops. Abbreviations: mnm 5 U: 5-methylaminomethyluridine; mcm 5 s 2 U: 5-methoxycarbonylmethyl-2-thiouridine.

Lacticin 3147 is a dual-acting two-peptide bacteriocin which is generally active against Gram-positive bacteria, including Listeria monocytogenes and antimicrobial-resistant bacteria such as Closteroides difficile in the colon. L. monocytogenes infections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. difficile causes one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Although lacticin 3147 is degraded by intestinal proteases and has poor aqueous solubility, encapsulation of the bacteriocin could enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 should be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present. Thus, the feasibility of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 was investigated. Bacteriocin activity was observed after encapsulation and release from a lipid matrix. Moreover, activity was seen after exposure to degrading enzymes. Further optimization of SLN delivery systems could enable the successful pharmaceutical development of active lacticin 3147 as an alternative to traditional antibiotics.

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins., (© 2021. The Author(s).)

A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata , the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus , respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.

Fermentation is one of if not the oldest food processing technique, yet it is still an emerging field when it comes to its numerous mechanisms of action and potential applications. The effect of microbial activity on the taste, bioavailability and preservation of the nutrients and the different food matrices has been deciphered by the insights of molecular microbiology. Among those roles of fermentation in the food chain, biopreservation remains the one most debated. Presumably because it has been underestimated for quite a while, and only considered - based on a food safety and technological approach - from the toxicological and chemical perspective. Biopreservation is not considered as a traditional use, where it has been by design - but forgotten - as the initial goal of fermentation. The 'modern' use of biopreservation is also slightly different from the traditional use, due mainly to changes in cooling of food and other ways of preservation, Extending shelf life is considered to be one of the properties of food additives, classifying - from our perspective - biopreservation wrongly and forgetting the role of fermentation and food cultures. The present review will summarize the current approaches of fermentation as a way to preserve and protect the food, considering the different way in which food cultures and this application could help tackle food waste as an additional control measure to ensure the safety of the food., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0-8.0) and preservation (- 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe 2+ and Mn 2+ . Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin's action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02-0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production., (© 2021 John Wiley & Sons Ltd.)

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) strains are the causative agents of severe foodborne diseases in both humans and animals. In this study, porcine pathogenic E. coli strains ( n  = 277) as well as porcine commensal strains ( n  = 188) were tested for their susceptibilities to 34 bacteriocin monoproducers to identify the most suitable bacteriocin types inhibiting porcine pathogens. Under in vitro conditions, the set of pathogenic E. coli strains was found to be significantly more susceptible to the majority of tested bacteriocins than commensal E. coli. Based on the production of bacteriocins with specific activity against pathogens, three potentially probiotic commensal E. coli strains of human origin were selected. These strains were found to be able to outcompete ETEC strains expressing F4 or F18 fimbriae in liquid culture and also decreased the severity and duration of diarrhea in piglets during experimental ETEC infection as well as pathogen numbers on the last day of in vivo experimentation. While the extents of the probiotic effect were different for each strain, the cocktail of all three strains showed the most pronounced beneficial effects, suggesting synergy between the tested E. coli strains. IMPORTANCE Increasing levels of antibiotic resistance among bacteria also increase the need for alternatives to conventional antibiotic treatment. Pathogenic Escherichia coli represents a major diarrheic infectious agent of piglets in their postweaning period; however, available measures to control these infections are limited. This study describes three novel E. coli strains producing antimicrobial compounds (bacteriocins) that actively inhibit a majority of toxigenic E. coli strains. The beneficial effect of three potentially probiotic E. coli strains was demonstrated under both in vitro and in vivo conditions. The novel probiotic candidates may be used as prophylaxis during piglets' postweaning period to overcome common infections caused by E. coli.

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20Mo T (DSM 11483 T ), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5 T (DSM 28587 T ) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5 T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20Mo T isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide natural products characterized by the presence of lanthionine and methyllanthionine cross-linked amino acids formed by dehydration of Ser/Thr residues followed by conjugate addition of Cys to the resulting dehydroamino acids. Class I lanthipeptide dehydratases utilize glutamyl-tRNA Glu as a cosubstrate to glutamylate Ser/Thr followed by glutamate elimination. A vast majority of lanthipeptides identified from class I synthase systems have been from Gram-positive bacteria. Herein, we report the heterologous expression and modification in Escherichia coli of two lanthipeptides from the Gram-negative Bacteroidetes Pedobacter lusitanus NL19. These peptides are representative of a group of compounds frequently encoded in Pedobacter genomes. Structural characterization of the lanthipeptides revealed a novel ring pattern as well as an unusual ll-lanthionine stereochemical configuration and a cyclase that lacks the canonical zinc ligands found in most LanC enzymes.

Infections caused by Gram-negative bacteria can be challenging to treat due to the outer membrane permeability barrier and the increasing emergence of antibiotic resistance. During infection, Gram-negative pathogens must acquire iron, an essential nutrient, in the host. Many Gram-negative bacteria utilize sophisticated iron acquisition machineries based on siderophores, small molecules that bind iron with high affinity. In this review, we provide an overview of siderophore-mediated iron acquisition in Enterobacteriaceae and show how these systems provide a foundation for the conceptualization and development of approaches to prevent and/or treat bacterial infections. Differences between the siderophore-based iron uptake machineries of pathogenic Enterobacteriaceae and commensal microbes may lead to the development of selective "Trojan-horse" antimicrobials and immunization strategies that will not harm the host microbiota., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human β-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

New ecosystems are being actively mined for new bioactive compounds. Because of the large amount of unexplored biodiversity, bacteria from marine environments are especially promising. Further, host-associated microbes are of special interest because of their low toxicity and compatibility with host health. Here, we identified and characterized biosynthetic gene clusters encoding antimicrobial compounds in host-associated enterococci recovered from fecal samples of wild marine animals remote from human-affected ecosystems. Putative biosynthetic gene clusters in the genomes of 22 Enterococcus strains of marine origin were predicted using antiSMASH5 and Bagel4 bioinformatic software. At least one gene cluster encoding a putative bioactive compound precursor was identified in each genome. Collectively, 73 putative antimicrobial compounds were identified, including 61 bacteriocins (83.56%), 10 terpenes (13.70%), and 2 (2.74%) related to putative nonribosomal peptides (NRPs). Two of the species studied, Enterococcus avium and Enterococcus mundtti , are rare causes of human disease and were found to lack any known pathogenic determinants but yet possessed bacteriocin biosynthetic genes, suggesting possible additional utility as probiotics. Wild marine animal-associated enterococci from human-remote ecosystems provide a potentially rich source for new antimicrobial compounds of therapeutic and industrial value and potential probiotic application.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These genetically encoded peptides are biosynthesized by multifunctional enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity and catalyze iterative rounds of post-translational modification. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native mass spectrometry coupled to ion mobility (native IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its noncovalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes native IM-MS as a versatile approach for characterizing intermolecular interactions and for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

The human skin microbiota forms a key barrier against skin pathogens and is important in modulating immune responses. Recent studies identify lactobacilli as endogenous inhabitants of healthy skin, while inflammatory skin conditions are often associated with a disturbed skin microbiome. Consequently, lactobacilli-based probiotics are explored as a novel treatment of inflammatory skin conditions through their topical skin application. This review focuses on the potential beneficial role of lactobacilli (family Lactobacillaceae) in the skin habitat, where they can exert multifactorial local mechanisms of action against pathogens and inflammation. On one hand, lactobacilli have been shown to directly compete with skin pathogens through adhesion inhibition, production of antimicrobial metabolites, and by influencing pathogen metabolism. The competitive anti-pathogenic action of lactobacilli has already been described mechanistically for common different skin pathogens, such as Staphylococcus aureus, Cutibacterium acnes, and Candida albicans. On the other hand, lactobacilli also have an immunomodulatory capacity associated with a reduction in excessive skin inflammation. Their influence on the immune system is mediated by bacterial metabolites and cell wall-associated or excreted microbe-associated molecular patterns (MAMPs). In addition, lactobacilli can also enhance the skin barrier function, which is often disrupted as a result of infection or in inflammatory skin diseases. Some clinical trials have already translated these mechanistic insights into beneficial clinical outcomes, showing that topically applied lactobacilli can temporarily colonize the skin and promote skin health, but more and larger clinical trials are required to generate in vivo mechanistic insights and in-depth skin microbiome analysis., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The study aimed to evaluate qualitatively and quantitatively the antimicrobial capacity of 10 potential probiotic Lactobacillus strains against model enteropathogens and spoilage microorganisms. The probiotic strains (live and heat-killed forms) were also assessed for their ability to inhibit adhesion of selected pathogens to Caco-2 cells. The largest inhibition zones (the diffusion method) were connected with the usage of whole bacteria cultures (WBC), also high and moderate with cell-free supernatant (CFS) and the lowest with cell-free neutralized supernatant (CNS). The highest antagonistic activity of Lactobacillus strains was observed against L. monocytogenes strains, moderate activity against Salmonella, Shigella, Escherichia coli, Pseudomonas and, the lowest against S.aureus, Bacillus and Enterococcus. The inhibition of adhesion to Caco-2 cells was very high in the case of E. coli, Salmonella and L. monocytogenes, and moderate in the case of S.aureus. On average, the inhibition effect was higher when pathogenic bacteria were treated by WBC, than heat-killed Lactobacillus. Although, in most samples, the effect was not significantly different (P> 0.05). The strains Lb. brevis O24 and Lb. rhamnosus K3 showed the biggest overall antimicrobial properties, and were most effective in adherence inhibition of investigated indicator strains. These bacteria or their metabolites can be used for the production of various foods or pharmaceutical products., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Plantaricin EF, a kind of natural antibacterial substance, has shown inhibitory effect on most pathogen and spoilage microorganisms, which possessed great potential in food preservation. However, the lower production of plantaricin EF has limited its large-scale production and application. In this study, the effect of maltose on plantaricin EF production and its regulation mechanism in Lactobacillus plantarum 163 were investigated. Maltose significantly improved the biomass and plantaricin EF production, which increased by 3.35 and 3.99 times comparing to the control without maltose, respectively. The maximum production of plantaricin E and F in fed-batch fermentation were 10.55 mg/L and 22.94 mg/L, respectively. Besides, qPCR results showed that maltose remarkably improved transcription of plnA, plnB, plnD, plnE, plnF, plnG1 and plnH, and heighten transcription of lamR, lamK, hpk6 and rrp6. These results provided an effective method to enhance plantaricin EF production and revealed a possible regulatory mechanism from transcriptome results that hpk6, rrp6, lamK and lamR were relative to plantaricin EF production. Genes, hpk6 and rrp6, promote transcription of plnG1, whereas lamK and lamR enhance transcription of plnA, plnB and plnD, which increased plantaricin EF production. KEYPOINTS: • Maltose was proved to be effective in promoting the biosynthesis of plantaricin EF. • Maltose promoted the transcription of biosynthesis and secretion genes of plantaricin EF. • Up-regulation of genes lamR, lamK, hpk6 and rrp6 heightened the plantaricin EF production.

Ready-to-eat (RTE) sliced emulsion type sausages are sensitive to recontamination with Listeria (L.) monocytogenes during processing and packaging steps. Since Listeria spp. are able to grow on those products under cold storage conditions, taking steps to reduce the recontamination risk and implementing antibacterial hurdles contribute to consumer safety and increase the product quality. With this study data about the suitability of culture broth, cell-free supernatant (CFS) or concentrated bacteriocin preparations (CFS conc ) of bacteriocin-producing lactic acid bacteria (LAB) obtained from fermented sausages from Germany as protective culture or antibacterial additive were provided. In different challenge tests, the potential of selected LAB or their preparations were investigated for their potential to reduce growth of L. monocytogenes and/or Brochothrix (B.) thermosphacta on sliced RTE emulsion type sausages under modified atmosphere or vacuum during refrigerated storage for a 21-day period. Applied LAB culture broth and CFS could not reduce the growth of L. monocytogenes or B. thermosphacta. On the other hand, samples treated with CFS conc obtained from Pediococcus spp. strains showed a significant inhibition (p < 0.05) of more than 1.5 log 10 of the applied L. monocytogenes strains during the storage period. The growth of B. thermosphacta could not be influenced. Thereby, the need for concentrating preparations was shown to be important to obtain a suitable antibacterial preparation that would contribute to consumer safety and food quality when applied as a protective additive., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glycosylated bacteriocins (glycocins) are potential clean label food preservatives and new alternatives to antibiotics. Further development requires the availability of a method for laboratory evolution of glycocins, wherein the challenges to overcome include ensuring glycosylation in a heterologous host, avoiding potential toxicity of active glycocins to the host, and provisioning of a one-pot screening assay for active mutants. Employing EntS, a sequential O/S- di-glycosyltransferase from Enterococcus faecalis TX0104, a proof of the concept microbial system and high throughput screening assay (SELECT-GLYCOCIN) is developed for generation of O/S- linked glycopeptide libraries and screening of glycocins for desired activity/property. The method enabled enzyme-dependent in vivo glycosylation in the heterologous host and rapid screening of mutants of enterocin 96 (Ent96)- a glycocin active against food-borne pathogen L. monocytogenes. Using SELECT-GLYCOCIN, a library of random (1.5 X 10^3) and rational (17) mutants of Ent96 was generated. The mutants were screened for bioactivity to identify a total of 376 random and 14 rational mutants as bioactive. Downstream detailed analysis of 16 random and 14 rational mutants led to the identification of sequence- and or glyco-variants namely, G16E-H24Q, C13T, and Ent96-K4&#95;K5insYYGNGV (PedioEnt96) as improved antimicrobials. To summaries, SELECT-GLYCOCIN provides a system and a generic method for discovery and screening of glycocins that can further be adapted to any known/unknown glycocins and can be employed in food preservatives' and drug discovery programs.

Natural methods to control food pathogens are required and bacteriocins have received much interest in this regard. The aim of this study was to investigate the ability of the novel bacteriocin capidermicin to inhibit Listeria monocytogenes. Agar-based deferred antagonism assays were carried out with the capidermicin producer against 17 L. monocytogenes strains and large zones of inhibition were observed for 12 strains. Minimal inhibitory concentration assays performed with purified capidermicin peptide revealed MIC values between 680 nM and 11 μM. Biofilm assays were performed with five L. monocytogenes strains. Addition of capidermicin prevented biofilm formation by one strain and could remove pre-established biofilms of all five strains. Broth based growth experiments demonstrated that addition of capidermicin resulted in an extended lag phase of both L. monocytogenes strains tested. Kill-curve experiments showed that capidermicin was able to potentiate the anti-Listeria effects of the lantibiotic nisin. This enhanced killing by the combination of both peptides was also observed in model food systems (cottage cheese and chocolate milk). In summary, we show that capidermicin can inhibit L. monocytogenes and warrants further investigation as a potential natural agent for the control of this pathogen., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The scope of bioengineering is expanding from the creation of single strains to the design of microbial communities, allowing for division-of-labour, specialised sub-populations and interaction with "wild" microbiomes. However, in the absence of stabilising interactions, competition between microbes inevitably leads to the removal of less fit community members over time. Here, we leverage amensalism and competitive exclusion to stabilise a two-strain community by engineering a strain of Escherichia coli which secretes a toxin in response to competition. We show experimentally and mathematically that such a system can produce stable populations with a composition that is tunable by easily controllable parameters. This system creates a tunable, stable two-strain consortia while only requiring the engineering of a single strain.

The lanthipeptide mersacidin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus amyloliquefaciens . It has antimicrobial activity against a range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus , giving it potential therapeutic relevance. The structure and bioactivity of mersacidin are derived from a unique combination of lanthionine ring structures, which makes mersacidin also interesting from a lantibiotic-engineering point of view. Until now, mersacidin and its derivatives have exclusively been produced in Bacillus strains and purified from the supernatant in their bioactive form. However, to fully exploit its potential in lanthipeptide-engineering, mersacidin would have to be expressed in a standardized expression system and obtained in its inactive prepeptide form. In such a system, the mersacidin biosynthetic enzymes could be employed to create novel peptides, enhanced by the recent advancements in RiPP engineering, while the leader peptide prevents activity against the expression host. This system would however need a means of postpurification in vitro leader processing to activate the obtained precursor peptides. While mersacidin's native leader processing mechanism has not been confirmed, the bifunctional transporter MrsT and extracellular Bacillus proteases have been suggested to be responsible. Here, a modular system is presented for the heterologous expression of mersacidin in Escherichia coli , which was successfully used to produce and purify inactive premersacidin. The purified product was used to determine the cleavage site of MrsT. Additionally, it was concluded from antimicrobial activity tests that in a second processing step mersacidin is activated by specific extracellular proteases from Bacillus amyloliquefaciens .

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL 1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU , which encodes UTP-glucose-1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis Spontaneous mutants that developed resistance to the antimicrobial effects of BacL 1 and BacA were revealed to carry a truncation deletion of the C-terminal amino acid (aa) region 288 to 298 of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL 1 Interestingly, these mutants had reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalis IMPORTANCE Cell wall-associated polysaccharides of bacteria are involved in various physiological characteristics. Recent studies demonstrated that the cell wall-associated polysaccharide of Enterococcus faecalis is required for susceptibility to bactericidal antibiotic agents. Here, we demonstrated that a galU mutation resulted in resistance to the enterococcal lytic bacteriocin Bac41. The galU homologue is reported to be essential for the biosynthesis of species-specific cell wall-associated polysaccharides in other Firmicutes In E. faecalis , the galU mutant lost the E. faecalis -specific cell wall-associated polysaccharide EPA (enterococcal polysaccharide antigen). The mutant also displayed reduced susceptibility to antibacterial agents and an abnormal cell morphology. We demonstrated here that galU was essential for EPA biosynthesis in E. faecalis , and EPA production might underlie susceptibility to lytic bacteriocin and antibiotic agents by undefined mechanisms., (Copyright © 2021 American Society for Microbiology.)

Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism., Competing Interests: The authors have declared that no competing interests exist.

Class II lanthipeptide synthetases (LanM enzymes) catalyze the multistep post-translational modification of genetically encoded precursor peptides into macrocyclic (often antimicrobial) lanthipeptides. The reaction sequence involves dehydration of serine/threonine residues, followed by intramolecular addition of cysteine thiols onto the nascent dehydration sites to construct thioether bridges. LanMs utilize two separate active sites in an iterative yet highly coordinated manner to maintain a remarkable level of regio- and stereochemical control over the multistep maturation. The mechanisms underlying this biosynthetic fidelity remain enigmatic. We recently demonstrated that proper function of the haloduracin β synthetase (HalM2) requires dynamic structural elements scattered across the surface of the enzyme. Here, we perform kinetic simulations, structural analysis of reaction intermediates, hydrogen-deuterium exchange mass spectrometry studies, and molecular dynamics simulations to investigate the contributions of these dynamic HalM2 structural elements to biosynthetic efficiency and fidelity. Our studies demonstrate that a large, conserved loop (HalM2 residues P349-P405) plays essential roles in defining the precursor peptide binding site, facilitating efficient peptide dehydration, and guiding the order of thioether ring formation. Moreover, mutations near the interface of the HalM2 dehydratase and cyclase domains perturb cyclization fidelity and result in aberrant thioether topologies that cannot be corrected by the wild type enzyme, suggesting an element of kinetic control in the normal cyclization sequence. Overall, this work provides the most comprehensive correlation of the structural and functional properties of a LanM enzyme reported to date and should inform mechanistic studies of the biosynthesis of other ribosomally synthesized and post-translationally modified peptide natural products.

Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions.

Streptococcus gallolyticus subsp. gallolyticus is an emerging opportunistic pathogen responsible for septicemia and endocarditis in the elderly. Invasive infections by S. gallolyticus subsp. gallolyticus are strongly linked to the occurrence of colorectal cancer (CRC). It was previously shown that increased secondary bile salts under CRC conditions enhance the bactericidal activity of gallocin, a bacteriocin produced by S. gallolyticus subsp. gallolyticus , enabling it to colonize the mouse colon by outcompeting resident enterococci (L. Aymeric, F. Donnadieu, C. Mulet, L. du Merle, et al., Proc Natl Acad Sci U S A 115:E283-E291, 2018, https://doi.org/10.1073/pnas.1715112115). In a separate study, we showed that S. gallolyticus subsp. gallolyticus produces and secretes a 21-mer peptide that activates bacteriocin production (A. Proutière, L. du Merle, B. Périchon, H. Varet, et al., mBio 11:e03187-20, 2020, https://doi.org/10.1128/mBio.03187-20). This peptide was named CSP because of its sequence similarity with competence-stimulating peptides found in other streptococci. Here, we demonstrate that CSP is a bona fide quorum sensing peptide involved in activation of gallocin gene transcription. We therefore refer to CSP as GSP (gallocin-stimulating peptide). GSP displays some unique features, since its N-terminal amino acid lies three residues after the double glycine leader sequence. Here, we set out to investigate the processing and export pathway that leads to mature GSP. Heterologous expression in Lactococcus lactis of the genes encoding GSP and the BlpAB transporter is sufficient to produce the 21-mer form of GSP in the supernatant, indicating that S. gallolyticus subsp. gallolyticus BlpAB displays an atypical cleavage site. We also conducted the first comprehensive structure-activity relationship (SAR) analysis of S. gallolyticus subsp. gallolyticus GSP to identify its key structural features and found that unlike many other similar streptococci signaling peptides (such as CSPs), nearly half of the mature GSP sequence can be removed (residues 1 to 9) without significantly impacting the peptide activity. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus is an opportunistic pathogen associated with colorectal cancer (CRC) and endocarditis. S. gallolyticus subsp. gallolyticus utilizes quorum sensing (QS) to regulate the production of a bacteriocin (gallocin) and gain a selective advantage in colonizing the colon. In this article, we report (i) the first structure-activity relationship study of the S. gallolyticus subsp. gallolyticus QS pheromone that regulates gallocin production, (ii) evidence that the active QS pheromone is processed to its mature form by a unique ABC transporter and not processed by an extracellular protease, and (iii) supporting evidence of interspecies interactions between streptococcal pheromones. Our results revealed the minimal pheromone scaffold needed for gallocin activation and uncovered unique interactions between two streptococcal QS signals that warrant further study., (Copyright © 2021 Harrington et al.)

Microorganisms have developed quorum sensing (QS) systems to detect small signaling molecules that help to control access to additional nutrients and space in highly competitive polymicrobial niches. Many bacterial processes are QS-regulated; two examples are the highly related traits of the natural genetic competence state and the production of antimicrobial peptides such as bacteriocins. The Streptococcus genus is widely studied for its competence and for its ability to produce bacteriocins, as these antimicrobial peptides have significant potential in the treatment of infections caused by multiple-resistant pathogens, a severe public health issue. The transduction of a two-component system controls competence in streptococci: (1) ComD/E, which controls the competence in the Mitis and Anginosus groups, and (2) ComR/S, which performs the same function in the Bovis, Mutans, Salivarius, and Pyogenic groups. The cell-to-cell communication required for bacteriocin production in the Streptococcus groups is controlled mainly by a paralog of the ComD/E system. The relationships between pheromone signals and induction pathways are related to the bacteriocin production systems. In this review, we discuss the recent advances in the understanding of signaling and the induction of bacteriocin biosynthesis by QS regulation in streptococci. This information could aid in the design of better methods for the development and production of these antimicrobial peptides. It could also contribute to the analysis and emerging applications of bacteriocins in terms of their safety, quality, and human health benefits.

Background: Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5, a locally isolated probiotic from Indonesia. Plantaricin IIA-1A5 exhibits antibacterial activity against wide spectrum of pathogenic bacteria, thus promising to be applied in various food products. Nevertheless, thermal stability of this bacteriocin remains to be fully investigated., Objective: This study aims to determine thermal stability of plantaricin IIA-1A5 through kinetic and thermodynamic parameters., Method: To address, plantaricin IIA-1A5 was purified from Lactobacillus plantarum IIA-1A5, which was growth under whey media, using ammonium sulfate precipitation followed by ionexchange chromatography. Purified plantaricin IIA-IA5 was then subjected to analysis of its bacteriocin activity. The thermal inactivation of bacteriocin from L. plantarum IIA-1A5 was calculated by incubating the bacteriocin at different temperatures ranging from 60-80 °C for 30 to 90 min, which was then used to calculate its kinetic and thermodynamic parameters., Results: The result showed the inactivation rates (k-value) were ranging from 0.008 to 0.013 min-1. Heat resistance of plantaricin IIA-1A5 (D-value) at constant heating temperature of 60, 65, 70, 75, and 80 °C were 311.6, 305.9, 294.5, 198.9, and 180.2 min, which indicated a faster inactivation at higher temperatures. D-value sensitivity for temperature changes (z-value) was calculated to be 75.76 °C. Further, thermodynamic analysis suggested that plantaricin IIA-1A5 is thermostable, with activation energy (Ea) of 29.02 kJ mol-1., Conclusion: This result showed that plantaricin IIA-1A5 is considerably more heat-stable than plantaricin members and promises to be applied in food industries where heat treatments are applied. Furthermore, a possible mechanism by which plantaricin IIA-1A5 maintains its stability was also discussed by referring to its thermodynamic parameters., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Probiotic bacteria are of utmost importance owing to their extensive utilisation in dairy products and in the prevention of various intestinal diseases. The objective of this study was to assess the probiotic properties of bacteriocin-producing isolates of Lactobacillus helveticus and Lactobacillus plantarum isolated from traditional Pakistani yoghurt. In this study, ten bacteriocin-producing isolates were selected to screen for the probiotic property. The isolates showed resistance to acidic pH (6-6.5), bile salt (0.01-1%), and 1-7% NaCl salt and showed good growth at acidic pH and antibacterial activity against ten different foodborne pathogens. Interestingly, these isolates were proved to be effective against Actinobacter baumannii but least effective against Klebsiella pneumoniae and Pseudomonas aeruginosa . A few isolates were found to be resistant to some antibiotics like vancomycim, gentamycin, erythromycin, streptomycin, and clindamycin. Our results provide strong evidence in favour of traditional Pakistani yoghurts as a potential source of bacteriocin-producing bacteria with an added benefit of the probiotic property. Specifically, LBh5 was considered a good probiotic isolate as compared to other isolates used in the study. Further extensive research should be done on isolation and characterisation of probiotic isolates from local fermented foods, and then, these isolates should be used in the development of probiotic enriched food supplements in Pakistan., Competing Interests: The authors report no conflict of interest., (Copyright © 2020 Mahreen Ul Hassan et al.)

Microbial natural products constitute a wide variety of chemical compounds, many which can have antibiotic, antiviral, or anticancer properties that make them interesting for clinical purposes. Natural product classes include polyketides (PKs), nonribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While variants of biosynthetic gene clusters (BGCs) for known classes of natural products are easy to identify in genome sequences, BGCs for new compound classes escape attention. In particular, evidence is accumulating that for RiPPs, subclasses known thus far may only represent the tip of an iceberg. Here, we present decRiPPter (Data-driven Exploratory Class-independent RiPP TrackER), a RiPP genome mining algorithm aimed at the discovery of novel RiPP classes. DecRiPPter combines a Support Vector Machine (SVM) that identifies candidate RiPP precursors with pan-genomic analyses to identify which of these are encoded within operon-like structures that are part of the accessory genome of a genus. Subsequently, it prioritizes such regions based on the presence of new enzymology and based on patterns of gene cluster and precursor peptide conservation across species. We then applied decRiPPter to mine 1,295 Streptomyces genomes, which led to the identification of 42 new candidate RiPP families that could not be found by existing programs. One of these was studied further and elucidated as a representative of a novel subfamily of lanthipeptides, which we designate class V. The 2D structure of the new RiPP, which we name pristinin A3 (1), was solved using nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) data, and chemical labeling. Two previously unidentified modifying enzymes are proposed to create the hallmark lanthionine bridges. Taken together, our work highlights how novel natural product families can be discovered by methods going beyond sequence similarity searches to integrate multiple pathway discovery criteria., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: P.C. is currently an employee of Verily Life Sciences. M.H. is currently an employee of LifeMine Therapeutics. M.S.D. is a member of the Scientific Advisory Board of DeepBiome Therapeutics. M.A.F. is a cofounder and director of Federation Bio. M.H.M. is on the scientific advisory board of Hexagon Bio and co-founder of Design Pharmaceuticals.

Applying a circular economy approach, this research explores the use of cheese whey permeate (CWP), by-product of whey ultrafiltration, as cheap substrate for the production of bacterial cellulose (BC) and Sakacin-A, to be used in an antimicrobial packaging material. BC from the acetic acid bacterium Komagataeibacter xylinus was boosted up to 6.77 g/L by supplementing CWP with β-galactosidase. BC was then reduced to nanocrystals (BCNCs, 70% conversion yield), which were then conjugated with Sakacin-A, an anti-Listeria bacteriocin produced by Lactobacillus sakei in a CWP based broth. Active conjugates (75 Activity Units (AU)/mg), an innovative solution for bacteriocin delivery, were then included in a coating mixture applied onto paper sheets at 25 AU/cm 2 . The obtained antimicrobial food package was found effective in reducing Listeria population in storage trials carried out on a fresh Italian soft cheese (named "stracchino") intentionally inoculated with Listeria. Production costs of the active material have been mainly found to be associated (90%) to the purification steps. Setting a maximum prudential 50% cost reduction during process up-scaling, conjugates coating formulation would cost around 0.89 €/A4 sheet. Results represent a practical example of a circular economy production procedure by using a food industry by-product to produce antimicrobials for food preservation.