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In urinary tract infections (UTIs), different bacteria can live in a polymicrobial community consisting of different species. It is unknown how community members affect the conjugation efficiency of uropathogenic Escherichia coli . We investigated the influence of individual species often coisolated from urinary infections (UTI) on the conjugation efficiency of E. coli isolates in artificial urine medium. Pairwise conjugation rate experiments were conducted between a donor E. coli strain containing the pOXA-48 plasmid and six uropathogenic E. coli isolates, in the presence and absence of five different species commonly coisolated in polymicrobial UTIs to elucidate their effect on the conjugation efficiency of E. coli . We found that the basal conjugation rates of pOXA-48, in the absence of other species, are dependent on the bacterial host genetic background. Additionally, we found that bacterial interactions have an overall positive effect on the conjugation rate of pOXA-48. Particularly, Gram-positive enterococcal species were found to enhance the conjugation rates towards uropathogenic E. coli isolates. We hypothesize that the nature of the coculture and physical interactions are important for these increased conjugation rates in an artificial urine medium environment., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) have raised significant interest, forming a potential new class of antibiotics in the fight against multi-drug-resistant bacteria. Various AMPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). One post-translational modification found in AMPs is the halogenation of Trp residues. This modification has, for example, been shown to be critical for the activity of the potent AMP NAI-107 from Actinoallomurus . Due to the importance of organohalogens, establishing methods for facile and selective halogen atom installation into AMPs is highly desirable. In this study, we introduce an expression system utilizing the food-grade strain Lactococcus lactis , facilitating the efficient incorporation of bromo-Trp (BrTrp) into (modified) peptides, exemplified by the lantibiotic nisin with a single Trp residue or analogue incorporated at position 1. This provides an alternative to the challenges posed by halogenase enzymes, such as poor substrate selectivity. Our method yields expression levels comparable to that of wild-type nisin, while BrTrp incorporation does not interfere with the post-translational modifications of nisin (dehydration and cyclization). One brominated nisin variant exhibits a 2-fold improvement in antimicrobial activity against two tested pathogens, including a WHO priority pathogen, while maintaining the same lipid II binding and bactericidal activity as wild-type nisin. The work presented here demonstrates the potential of this methodology for peptide halogenation, offering a new avenue for the development of diverse antimicrobial products labeled with BrTrp.

Objectives: Environmental conditions can influence mutation rates in bacteria. Fever is a common response to infection that alters the growth conditions of infecting bacteria. Here we examine how a temperature change, such as is associated with fever, affects the mutation rate towards antibiotic resistance., Methods: We used a fluctuation test to assess the mutation rate towards antibiotic resistance in Escherichia coli at two different temperatures: 37°C (normal temperature) and 40°C (fever temperature). We performed this measurement for three different antibiotics with different modes of action: ciprofloxacin, rifampicin and ampicillin., Results: In all cases, the mutation rate towards antibiotic resistance turned out to be temperature dependent, but in different ways. Fever temperatures led to a reduced mutation rate towards ampicillin resistance and an elevated mutation rate towards ciprofloxacin and rifampicin resistance., Conclusions: This study shows that the mutation rate towards antibiotic resistance is impacted by a small change in temperature, such as associated with fever. This opens a new avenue to mitigate the emergence of antibiotic resistance by coordinating the choice of an antibiotic with the decision of whether or not to suppress fever when treating a patient. Hence, optimized combinations of antibiotics and fever suppression strategies may be a new weapon in the battle against antibiotic resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

As the most abundant gram-negative bacterial order in the gastrointestinal tract, Bacteroidales bacteria have been extensively studied for their contribution to various aspects of gut health. These bacteria are renowned for their involvement in immunomodulation and their remarkable capacity to break down complex carbohydrates and fibers. However, the human gut microbiota is known to produce many metabolites that ultimately mediate important microbe-host and microbe-microbe interactions. To gain further insights into the metabolites produced by the gut commensal strains of this order, we examined the metabolite composition of their bacterial cell cultures in the stationary phase. Based on their abundance in the gastrointestinal tract and their relevance in health and disease, we selected a total of six bacterial strains from the relevant genera Bacteroides, Phocaeicola, Parabacteroides, and Segatella. We grew these strains in modified Gifu anaerobic medium (mGAM) supplemented with mucin, which resembles the gut microbiota's natural environment. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolite profiling revealed 179 annotated metabolites that had significantly differential abundances between the studied bacterial strains and the control growth medium. Most of them belonged to classes such as amino acids and derivatives, organic acids, and nucleot(s)ides. Of particular interest, Segatella copri DSM 18205 (previously referred to as Prevotella copri) produced substantial quantities of the bioactive metabolites phenylethylamine, tyramine, tryptamine, and ornithine. Parabacteroides merdae CL03T12C32 stood out due to its ability to produce cadaverine, histamine, acetylputrescine, and deoxycarnitine. In addition, we found that strains of the genera Bacteroides, Phocaeicola, and Parabacteroides accumulated considerable amounts of proline-hydroxyproline, a collagen-derived bioactive dipeptide. Collectively, these findings offer a more detailed comprehension of the metabolic potential of these Bacteroidales strains, contributing to a better understanding of their role within the human gut microbiome in health and disease., 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 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Antimicrobial peptides (AMPs) show promise as alternatives to traditional antibiotics for treating drug-resistant infections. Their adaptability and diverse sequence possibilities allow for rational design by modulating physicochemical determinants to achieve desired biological properties, transforming them into peptides for potential new therapies. Nisin, one of the best-studied AMPs, is believed to have potential to be used as a therapeutic, particularly against antibiotic-resistant bacteria. However, its instability in physiological conditions limits its use in clinical applications and pharmaceutical development. Exploration of new natural variants of nisin has uncovered diverse properties using different domains. Shuffling peptide modules can fine-tune the chemical properties of these molecules, potentially enhancing stability while maintaining or improving antimicrobial activity. In this study, hybrid AMPs were created by combining domains from three unique nisin variants, i.e. nisin A, cesin and rombocin, leading to the identification of a promising variant, named cerocin A, which harbours only 25 amino acids compared to the typical 31-35 amino acid length of nisin. Cerocin A demonstrates potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), approaching that of nisin itself. Cerocin A's mode of action involves a dual mechanism through the combination of two domains, consisting of a small ring/domain (6 amino acids) from the C-terminal end of rombocin attached to the preceding peptide of cesin, changing it from a bacteriostatic to a bactericidal peptide. Further mutation studies identified a new variant, cerocin V, with significantly improved resistance against trypsin degradation, while maintaining high potency. Importantly, cerocin V showed no undesired toxic effects on human red blood cells and remained stable in human plasma. In conclusion, we demonstrate that peptide construction using domain engineering is an effective strategy for manipulating both biological and physicochemical aspects, leading to the creation of novel bioactive molecules with desired properties. These constructs are appealing candidates for further optimization and development as novel antibiotics., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Due to intramolecular ring structures, the ribosomally produced and post-translationally modified peptide mersacidin shows antimicrobial properties comparable to those of vancomycin without exhibiting cross-resistance. Although the principles of mersacidin biosynthesis are known, there is no information on the molecular control processes for the initial stimulation of mersacidin bioproduction. By using Bacillus subtilis for heterologous biosynthesis, a considerable amount of mersacidin could be produced without the mersacidin-specific immune system and the mersacidin-activating secretory protease. By using the established laboratory strain Bacillus subtilis 168 and strain 3NA, which is used for high cell density fermentation processes, in combination with the construction of reporter strains to determine the promoter strengths within the mersacidin core gene cluster, the molecular regulatory circuit of Spo0A, a master regulator of cell differentiation including sporulation initiation, and the global transcriptional regulator AbrB, which is involved in cell adaptation processes in the transient growth phase, was identified to control the initial stimulation of the mersacidin core gene cluster. In a second downstream regulatory step, the activator MrsR1, encoded in the core gene cluster, acts as a stimulatory element for mersacidin biosynthesis. These findings are important to understand the mechanisms linking environmental conditions and microbial responses with respect to the bioproduction of bioactive metabolites including antimicrobials such as mersacidin. This information will also support the construction of production strains for bioactive metabolites with antimicrobial properties., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We report the efficient and site selective modification of non-canonical dehydroamino acids in ribosomally synthesized and post-transationally modified peptides (RiPPs) by β-amination. The singly modified thiopeptide Thiostrepton showed an up to 35-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity remained good, albeit lower than the unmodified peptide. Also the lanthipeptide nisin could be modified using this method., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Nisin serves as the prototype within the lantibiotic group of antimicrobial peptides, exhibiting a broad-spectrum inhibition against Gram-positive bacteria, including important food-borne pathogens and clinically relevant antibiotic-resistant strains. The gene-encoded nature of nisin allows for gene-based bioengineering, enabling the generation of novel derivatives. It has been demonstrated that nisin mutants can be produced with improved functional properties. Here, we particularly focus on the uncommon amino acid residues dehydroalanine (Dha) and dehydrobutyrin (Dhb), whose functions are not yet fully elucidated. Prior to this study, we developed a new expression system that utilizes the nisin modification machinery NisBTC to advance expression, resulting in enhanced peptide dehydration efficiency. Through this approach, we discovered that the dehydrated amino acid Dhb at position 18 in the peptide rombocin, a short variant of nisin, displayed four times higher activity compared to the non-dehydrated peptide against the strain Lactococcus lactis. Furthermore, we observed that in the peptides nisin and rombocin, the dehydrated amino acid Dha at residue positon 18 exhibited superior activity compared to the dehydrated amino acid Dhb. Upon purifying the wild-type nisin and its variant nisinG18/Dha to homogeneity, the minimum inhibitory concentration (MIC) indicated that the variant exhibited activity similar to that of wild-type nisin in inhibiting the growth of Bacillus cereus but showed twice the MIC values against the other four tested Gram-positive strains. Further stability tests demonstrated that the dehydrated peptide exhibited properties similar to wild-type nisin under different temperatures but displayed higher resistance to proteolytic enzymes compared to wild-type nisin., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins., Competing Interests: OPK and AJvH were employed by Omnicin Therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict., (Copyright © 2024 Liu, van Heel and Kuipers.)

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become a major health risk and a serious worldwide issue. MAFLD typically arises from aberrant lipid metabolism, insulin resistance, oxidative stress, and inflammation. However, subjacent causes are multifactorial. The gut has been proposed as a major factor in health and disease, and over the last decade, bacterial strains with potentially beneficial effects on the host have been identified. In vitro cell models have been commonly used as an early step before in vivo drug assessment and can confer complementary advantages in gut and liver health research. In this study, several selected strains of the order Bacteroidales were used in a three-cell line in vitro analysis (HT-29, Caco-2, and HepG2 cell lines) to investigate their potential as new-generation probiotics and microbiota therapeutics. Antimicrobial activity, a potentially useful trait, was studied, and the results showed that Bacteroidales can be a source of either wide- or narrow-spectrum antimicrobials targeting other closely related strains. Moreover, Bacteroides sp. 4_1_36 induced a significant decrease in gut permeability, as evidenced by the high TEER values in the Caco-2 monolayer assay, as well as a reduction in free fatty acid accumulation and improved fatty acid clearance in a steatosis HepG2 model. These results suggest that Bacteroidales may spearhead the next generation of probiotics to prevent or diminish MAFLD., (© 2024. The Author(s).)

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes . Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes . Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes .

Importance: Genome mining studies have revealed the remarkable combinatorial diversity of ribosomally synthesized and post-translationally modified peptides (RiPPs) in marine bacteria, including prochlorosins. However, mining strategies also prove valuable in investigating the genomic landscape of associated genes within biosynthetic gene cluster (BGC) specific to targeted RiPPs of interest. Our study contributes to the enrichment of knowledge regarding prochlorosin diversity. It offers insights into potential mechanisms involved in their biosynthesis and modification, such as hyper-modification, which may give rise to active lantibiotics. Additionally, our study uncovers putative novel promiscuous post-translational enzymes, thereby expanding the chemical space explored within the Synechococcus genus. Moreover, this research extends the applications of mining techniques beyond the discovery of new RiPP-like clusters, allowing for a deeper understanding of genomics and diversity. Furthermore, it holds the potential to reveal previously unknown functions within the intriguing RiPP families, particularly in the case of prochlorosins., Competing Interests: The authors declare no conflict of interest.

Antimicrobial resistance is a global concern, far from being resolved. The need of new drugs against new targets is imminent. In this work, we present a family of aminoalkyl resveratrol derivatives with antibacterial activity inspired by the properties of cationic amphipathic antimicrobial peptides. Surprisingly, the newly designed molecules display modest activity against aerobically growing bacteria but show surprisingly good antimicrobial activity against anaerobic bacteria (Gram-negative and Gram-positive) suggesting specificity towards this bacterial group. Preliminary studies into the action mechanism suggest that activity takes place at the membrane level, while no cross-resistance with traditional antibiotics is observed. Actually, some good synergistic relations with existing antibiotics were found against Gram-negative pathogens. However, some cytotoxicity was observed, despite their low haemolytic activity. Our results show the importance of the balance between positively charged moieties and hydrophobicity to improve antimicrobial activity, setting the stage for the design of new drugs based on these molecules.

Development of natural, broad-spectrum, and eco-friendly bio-fungicides is of high interest in the agriculture and food industries. In this context, Bacillus genus has shown great potential for producing a wide range of antimicrobial metabolites against various pathogens. A Bacillus velezensis strain FB2 was isolated from an agricultural field of National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan, exhibiting good antifungal properties. The complete genome of this strain was sequenced, and its antifungal potential was assayed by dual culture method. Moreover, structural characterization of its antifungal metabolites, produced in vitro, were studied. Genome analysis and mining revealed the secondary metabolite gene clusters, encoding non-ribosomal peptides (NRPs) production (e.g., surfactin, iturin and fengycin) and polyketide (PK) synthesis (e.g., difficidin, bacillaene and macrolactin). Furthermore, the Bacillus velezensis FB2 strain was observed to possess in vitro antifungal activity; 41.64, 40.38 and 26% growth inhibition against major fungal pathogens i.e. Alternaria alternata, Fusarium oxysporum and Fusarium solani respectively. Its lipopeptide extract obtained by acid precipitation method was also found effective against the above-mentioned fungal pathogens. The ESI-MS/MS analysis indicated various homologs of surfactin and iturin-A, responsible for their antifungal activities. Overall, this study provides a better understanding of Bacillus velezensis FB2, as a promising candidate for biocontrol purposes, acting in a safe and sustainable way, to control plant pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hammad 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.)

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface ( https://apps.embl.de/combact/ )., (© 2023. The Author(s).)

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum . Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis , the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum . However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

Mass spectrometry (MS) is widely used in proteomic analysis but cannot differentiate between molecules with the same mass-to-charge ratio. Nanopore technology might provide an alternative method for the rapid and cost-effective analysis and sequencing of proteins. In this study, we demonstrate that nanopore currents can distinguish between diastereomeric and enantiomeric differences in l- and d-peptides, not observed by conventional MS analysis, down to individual d-amino acids in small opioid peptides. Molecular dynamics simulations suggest that similar to chiral chromatography the resolution likely arises from multiple chiral interactions during peptide transport across the nanopore. Additionally, we used nanopore recordings to rapidly assess 4- and 11-amino acid ring formation in lanthipeptides, a process used in the synthesis of pharmaceutical peptides. The cyclization step requires distinguishing between constitutional isomers, which have identical MS signals and typically involve numerous tedious experiments to confirm. Hence, nanopore technology offers new possibilities for the rapid and cost-effective analysis of peptides, including those that cannot be easily differentiated by mass spectrometry.

The Bacteroidales order, widely distributed among diverse human populations, constitutes a key component of the human microbiota. Members of this Gram-negative order have been shown to modulate the host immune system, play a fundamental role in the gut's microbial food webs, or be involved in pathogenesis. Bacteria inhabiting such a complex environment as the human microbiome are expected to display social behaviors and, hence, possess factors that mediate cooperative and competitive interactions. Different types of molecules can mediate interference competition, including non-ribosomal peptides (NRPs), polyketides, and bacteriocins. The present study investigates the potential of Bacteroidales bacteria to biosynthesize class I bacteriocins, which are ribosomally synthesized and post-translationally modified peptides (RiPPs). For this purpose, 1,136 genome-sequenced strains from this order were mined using BAGEL4. A total of 1,340 areas of interest (AOIs) were detected. The most commonly identified enzymes involved in RiPP biosynthesis were radical S-adenosylmethionine (rSAM), either alone or in combination with other biosynthetic enzymes such as YcaO. A more comprehensive analysis of a subset of 9 biosynthetic gene clusters (BGCs) revealed a consistent association in Bacteroidales BGCs between peptidase-containing ATP-binding transporters (PCATs) and precursor peptides with GG-motifs. This finding suggests a possibly shared mechanism for leader peptide cleavage and transport of mature products. Notably, human metagenomic studies showed a high prevalence and abundance of the RiPP BGCs from Phocaeicola vulgatus and Porphyromonas gulae . The mature product of P. gulae BGC is hypothesized to display γ-thioether linkages and a C-terminal backbone amidine, a potential new combination of post-translational modifications (PTM). All these findings highlight the RiPP biosynthetic potential of Bacteroidales bacteria, as a rich source of novel peptide structures of possible relevance in the human microbiome context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fernandez-Cantos, Garcia-Morena, Yi, Liang, Gómez-Vázquez and Kuipers.)

The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. The incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as a host for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extend the toolbox for (lanthi)peptide drug improvement and discovery., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural bioactive peptide discovery is a challenging and time-consuming process. However, advances in synthetic biology are providing promising new avenues in peptide engineering that allow for the design and production of a large variety of new-to-nature peptides with enhanced or new bioactivities, using known peptides as templates. Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs). The modularity of post-translational modification (PTM) enzymes and ribosomal biosynthesis inherent to lanthipeptides enables their engineering and screening in a high-throughput manner. The field of RiPPs research is rapidly evolving, with many novel PTMs and their associated modification enzymes being identified and characterized. The modularity presented by these diverse and promiscuous modification enzymes has made them promising tools for further in vivo engineering of lanthipeptides, allowing for the diversification of their structures and activities. In this review, we explore the diverse modifications occurring in RiPPs and discuss the potential applications and feasibility of combining various modification enzymes for lanthipeptide engineering. We highlight the prospect of lanthipeptide- and RiPP-engineering to produce and screen novel peptides, including mimics of potent non-ribosomally produced antimicrobial peptides (NRPs) such as daptomycin, vancomycin, and teixobactin, which offer high therapeutic potential., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Due to the accelerated appearance of antimicrobial-resistant (AMR) pathogens in clinical infections, new first-in-class antibiotics, operating via novel modes of action, are desperately needed. Brevicidine, a bacterial nonribosomally produced cyclic lipopeptide, has shown potent and selective antimicrobial activity against Gram-negative pathogens. However, before our investigations, little was known about how brevicidine exerts its potent bactericidal effect against Gram-negative pathogens. In this study, we find that brevicidine has potent antimicrobial activity against AMR Enterobacteriaceae pathogens, with MIC values ranging between 0.5 μM (0.8 mg/L) and 2 μM (3.0 mg/L). In addition, brevicidine showed potent antibiofilm activity against the Enterobacteriaceae pathogens, with the same 100% inhibition and 100% eradication concentration of 4 μM (6.1 mg/L). Further mechanistic studies showed that brevicidine exerts its potent bactericidal activity by interacting with lipopolysaccharide in the outer membrane, targeting phosphatidylglycerol and cardiolipin in the inner membrane, and dissipating the proton motive force of bacteria. This results in metabolic perturbation, including the inhibition of ATP synthesis; the inhibition of the dehydrogenation of NADH; the accumulation of reactive oxygen species in bacteria; and the inhibition of protein synthesis. Finally, brevicidine showed a good therapeutic effect in a mouse peritonitis-sepsis model. Our findings pave the way for further research on the clinical applications of brevicidine to combat prevalent infections caused by AMR Gram-negative pathogens worldwide., Competing Interests: The authors declare no conflict of interest.

Bacillus subtilis is a soil-dwelling bacterium that can interact with a plethora of other microorganisms in its natural habitat. Due to the versatile interactions and its ability to form nanotubes, i.e., recently described membrane structures that trade cytoplasmic content between neighbouring cells, we investigated the potential of HGT from B. subtilis to industrially-relevant members of lactic acid bacteria (LAB). To explore the interspecies HGT events, we developed a co-culturing protocol and provided proof of transfer of a small high copy non-conjugative plasmid from B. subtilis to LABs. Interestingly, the plasmid transfer did not involve conjugation nor activation of the competent state by B. subtilis. Moreover, our study shows for the first time non-conjugative cell-to-cell intraspecies plasmid transfer for non-competent Lactococcus lactis sp. cremoris strains. Our study indicates that cell-to-cell transformation is a ubiquitous form of HGT and can be potentially utilized as an alternative tool for natural (non-GMO) strain improvement., (© 2022 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader- and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helix-forming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the C-terminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.

Antimicrobial resistance is a natural and inevitable phenomenon that constitutes a severe threat to global public health and economy. Innovative products, active against new targets and with no cross- or co-resistance with existing antibiotic classes, novel mechanisms of action, or multiple therapeutic targets are urgently required. For these reasons, antimicrobial peptides such as bacteriocins constitute a promising class of new antimicrobial drugs under investigation for clinical development. Here, we review the potential therapeutic use of AS-48, a head-to-tail cyclized cationic bacteriocin produced by Enterococcus faecalis . In the last few years, its potential against a wide range of human pathogens, including relevant bacterial pathogens and trypanosomatids, has been reported using in vitro tests and the mechanism of action has been investigated. AS-48 can create pores in the membrane of bacterial cells without the mediation of any specific receptor. However, this mechanism of action is different when susceptible parasites are studied and involves intracellular targets. Due to these novel mechanisms of action, AS-48 remains active against the antibiotic resistant strains tested. Remarkably, the effect of AS-48 against eukaryotic cell lines and in several animal models show little effect at the doses needed to inhibit susceptible species. The characteristics of this molecule such as low toxicity, microbicide activity, blood stability and activity, high stability at a wide range of temperatures or pH, resistance to proteases, and the receptor-independent effect make AS-48 unique to fight a broad range of microbial infections, including bacteria and some important parasites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cebrián, Martínez-García, Fernández, García, Martínez-Bueno, Valdivia, Kuipers, Montalbán-López and Maqueda.)

ProcM-like enzymes are class II promiscuous lanthipeptide synthetases that are an attractive tool in synthetic biology for producing lanthipeptides with biotechnological or clinically desired properties. SyncM is a recently described modification enzyme from this family used to develop a versatile expression platform for engineering lanthipeptides. Most remarkably, SyncM can modify up to 79 SyncA substrates in a single strain. Six SyncAs were previously characterized from this pool of substrates. They showed particular characteristics, such as the presence of one or two lanthionine rings, different flanking residues influencing ring formation, and different ring directions, demonstrating the relaxed specificity of SyncM toward its precursor peptides. To gain a deeper understanding of the potential of SyncM as a biosynthetic tool, we further explored the enzyme's capabilities and limits in dehydration and ring formation. We used different SyncA scaffolds for peptide engineering, including changes in the ring's directionality (relative position of Ser/Thr to Cys in the peptide) and size. We further aimed to rationally design mimetics of cyclic antimicrobials and introduce macrocycles in prochlorosin-related and nonrelated substrates. This study highlights the largest lanthionine ring with 15 amino acids (ring-forming residues included) described to date. Taking advantage of the amino acid substrate tolerance of SyncM, we designed the first single-SyncA-based antimicrobial. The insights gained from this work will aid future bioengineering studies. Additionally, it broadens SyncM's application scope for introducing macrocycles in other bioactive molecules.

Peptide natural products are important lead structures for human drugs and many nonribosomal peptides possess antibiotic activity. This makes them interesting targets for engineering approaches to generate peptide analogues with, for example, increased bioactivities. Nonribosomal peptides are produced by huge mega-enzyme complexes in an assembly-line like manner, and hence, these biosynthetic pathways are challenging to engineer. In the past decade, more and more structural features thought to be unique to nonribosomal peptides were found in ribosomally synthesised and posttranslationally modified peptides as well. These streamlined ribosomal pathways with modifying enzymes that are often promiscuous and with gene-encoded precursor proteins that can be modified easily, offer several advantages to produce designer peptides. This review aims to provide an overview of recent progress in this emerging research area by comparing structural features common to both nonribosomal and ribosomally synthesised and posttranslationally modified peptides in the first part and highlighting synthetic biology strategies for emulating nonribosomal peptides by ribosomal pathway engineering in the second part., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Circular bacteriocins, also known as bacterial head-to-tail cyclized peptides, are a subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs). Compared with their conventional linear counterparts, circular bacteriocins are highly stable over a broad temperature and pH range, and circularization decreases proteolytic degradation by exopeptidases. These features render them great potential as scaffold candidates to withstand strident conditions in food- and pharmaceutical applications. However, the biosynthesis and bioactivity of circular bacteriocins still remain largely unknown. To investigate and gain more insights into the biosynthesis of circular bacteriocins and to achieve efficient production and characterization of bacteriocin variants, we developed an efficient cloning and heterologous expression system for clostridial circularin A and successfully produced this circular peptide in Lactococcus lactis NZ9000. We report three system formats with single plasmid or plasmid combinations to achieve successful cloning and functional production of circularin A in L. lactis . These systematic varieties enabled us to choose the appropriate method to efficiently obtain various constructs with desired properties. With the established heterologous systems in L. lactis , we performed several mutagenesis studies in the precursor peptide to study its structure/function relationships. The overlay activity assay revealed that these mutant variants had variable effects on different indicator strains: lysine substitution for certain glutamine residue(s) greatly decreased its bioactivity against Clostridium perfringens and L. lactis NZ9000, and alanine replacement for the cationic residues significantly reduced the activity against Lactobacillus sake ATCC 15521, whereas alanine substitution for the aromatic residues decreased its bioactivity against all three testing strains dramatically. Moreover, the conditions for bacteriocin production were optimized. Results show that supplementing the minimal medium with extra glucose (or sucrose) and immediate nisin-induction improved the peptide yield significantly. Briefly, we developed an excellent system for the production of circularin A and a wide range of variant peptides in a convenient host, as well as a method for fast detection of peptide production and activity. This system facilitated our mutagenesis studies which provided valuable insights into the effects of mutating specific residues on its biosynthesis and bioactivity, and will eventually enable more complex research into the biosynthesis of circularin A., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, van Heel, Chen and Kuipers.)

Many bacteria adapt their physiology and enter the viable but non-culturable state to survive prolonged exposure to adverse environmental conditions. The VBNC cells maintain active metabolism, membrane integrity and gene transcription. However, they lose the ability to form colonies on a conventional culture media. Thus, standard colony counting methods cannot detect these alive but dormant cells. The Gram-positive bacterium Bacillus subtilis was found to enter the VBNC state when pre-exposed to osmotic stress and treated with a lethal dose of kanamycin. These cells reduced their metabolic activity, ceased growth and division and became kanamycin-tolerant. Interestingly, despite active metabolism, the majority of the kanamycin tolerant cells could not be revived on LB agar. In this study, we use a robust RNA-Seq technique to elucidate the differences in transcriptional profiles of B. subtilis VBNC cells. A comparative analysis of differently expressed genes and operons performed in this study indicates high similarities in transcriptional responses of VBNC and kanamycin-sensitive cells to antibiotic treatment. Moreover, this work reveals that VBNC cells strongly upregulate genes involved in proline uptake and catabolism, suggesting a putative role of proline as nutrient in VBNC cells., (© 2022. The Author(s).)

Five environmental Bacillus strains were sequenced, of which three were isolated from the rhizosphere of agricultural soil and one each from Attock Oil Refinery and Khewra Salt Mine in Pakistan. The strains can be used for plant growth promotion and biosurfactant activity brought about by secondary metabolites.

Mersacidin is an antimicrobial class II lanthipeptide. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by intramolecular lanthionine rings. These rings give lanthipeptides their bioactive structure and stability. RiPPs are produced from a gene cluster that encodes a precursor peptide and its dedicated unique modification enzymes. The field of RiPP engineering aims to recombine modification enzymes from different RiPPs to modify new substrates, resulting in new-to-nature molecules with novel or improved functionality. The enzyme MrsM from the mersacidin gene cluster installs the four lanthionine rings of mersacidin, including the uniquely small ring A. By applying MrsM in RiPP engineering, this ring could be installed in linear peptides to achieve stabilization by a very small lanthionine or to create small lanthionine-stabilized modules for chemical modification. However, the formation of unique intramolecular structures like that of mersacidin's ring A can be very stringent. Here, the formation of ring A of mersacidin is characterized by mutagenesis. A range of truncated mersacidin variants was made to identify the smallest possible construct in which this ring could still be formed. Additionally, mutants were created to study the flexibility of ring A formation. It was found that although the formation of ring A is stringent, it can be formed in a core peptide as small as five amino acids. The truncated mersacidin core peptide CTFAL is the smallest ribosomally produced lanthipeptide reported to date, and it has exciting prospects as a new module for application in RiPP engineering.

Bacillus subtilis, in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically-induced biosynthesis requires activation of a SigA-type promoter that drives the expression of the proHJ operon. Population-wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single-cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically-controlled proline biosynthesis pathway is also involved in the antibiotic-mediated stress response., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Recent advances in the field of high throughput (meta-)transcriptomics and proteomics call for easy and rapid methods enabling to explore not only single genes or proteins but also extended biological systems. Gene set enrichment analysis is commonly used to find relations in a set of genes and helps to uncover the biological meaning in results derived from high-throughput data. The basis for gene set enrichment analysis is a solid functional classification of genes. Here, we describe a comprehensive database containing multiple functional classifications of genes of all (>55 000) publicly available complete bacterial genomes. In addition to the most common functional classes such as COG and GO, also KEGG, InterPro, PFAM, eggnog and operon classes are supported. As classification data for features is often not available, we offer fast annotation and classification of proteins in any newly sequenced bacterial genome. The web server FUNAGE-Pro enables fast functional analysis on single gene sets, multiple experiments, time series data, clusters, and gene network modules for any prokaryote species or strain. FUNAGE-Pro is freely available at http://funagepro.molgenrug.nl., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial communities exposed to rapid changes in their habitat encounter different forms of stress. Fluctuating conditions of the microenvironment drive microorganisms to develop several stress responses to sustain growth and division, like altering gene expression and changing the cell's physiology. It is commonly known that these protection systems may give rise to differently adapted subpopulations and indirectly impact bacterial susceptibility to antimicrobials. This study focuses on the adaptation of a soil-dwelling bacterium, Bacillus subtilis , to sudden osmotic changes, including transient and sustained osmotic upshift. Here, we demonstrate that physiological changes caused by pre-exposure to osmotic stress facilitate B. subtilis ' entry into a quiescent state, helping them survive when exposed to a lethal antibiotic concentration. We show that the adaptation to transient osmotic upshift with 0.6 M NaCl causes decreased metabolic rates and lowered antibiotic-mediated ROS production when cells were exposed to the aminoglycoside antibiotic kanamycin. Using a microfluidic platform combined with time-lapse microscopy, we followed the uptake of fluorescently labelled kanamycin and examined the metabolic activity of differently preadapted populations at a single-cell level. The microfluidics data revealed that under the conditions tested, B. subtilis escapes from the bactericidal activity of kanamycin by entering into a nongrowing dormant state. Combining single-cell studies and population-wide analysis of differently preadapted cultures, we demonstrate that kanamycin-tolerant B. subtilis cells are entrapped in a viable but nonculturable (VBNC) state., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)