The Pangenome of Gram-Negative Environmental Bacteria Hides a Promising Biotechnological Potential.

Secondary metabolites (SMs) from environmental bacteria offer viable solutions for various health and environmental challenges. Researchers are employing advanced bioinformatic tools to investigate less-explored microorganisms and unearth novel bioactive compounds. In this research area, our understanding of SMs from environmental Gram-negative bacteria lags behind that of its Gram-positive counterparts. In this regard, Pedobacter spp. have recently gained attention, not only for their role as plant growth promoters but also for their potential in producing antimicrobials. This study focuses on the genomic analysis of Pedobacter spp. to unveil the diversity of the SMs encoded in their genomes. Among the 41 genomes analyzed, a total of 233 biosynthetic gene clusters (BGCs) were identified, revealing the potential for the production of diverse SMs, including RiPPs (27%), terpenes (22%), hybrid SMs (17%), PKs (12%), NRPs (9%) and siderophores (6%). Overall, BGC distribution did not correlate with phylogenetic lineage and most of the BGCs showed no significant hits in the MIBiG database, emphasizing the uniqueness of the compounds that Pedobacter spp. can produce. Of all the species examined, P. cryoconitis and P. lusitanus stood out for having the highest number and diversity of BGCs. Focusing on their applicability and ecological functions, we investigated in greater detail the BGCs responsible for siderophore and terpenoid production in these species and their relatives. Our findings suggest that P. cryoconitis and P. lusitanus have the potential to produce novel mixtures of siderophores, involving bifunctional IucAC/AcD NIS synthetases, as well as carotenoids and squalene. This study highlights the biotechnological potential of Pedobacter spp. in medicine, agriculture and other industries, emphasizing the need for a continued exploration of its SMs and their applications.

Class II two-peptide lanthipeptide proteases: exploring LicTP for biotechnological applications.

The enzymatic machinery involved in the biosynthesis of lantibiotic is an untapped source of proteases with different specificities. Lanthipeptide biosynthesis requires proteolysis of specific target sequences by known proteases, which are encoded by contiguous genes. Herein, the activity of lichenicidin A2 (LicA2) trimming proteases (LicP and LicT) was investigated in vivo. Firstly, the impact of some residues and the size of the peptide were evaluated. Then followed trials in which LicA2 leader was evaluated as a tag to direct production and secretion of other relevant peptides. Our results show that a negatively charged residue (preferably Glu) at cleavage site is important for LicP efficacy. Some mutations of the lichenicidin hexapeptide such as Val-4Ala, Asp-5Ala, Asn-6Ser, and the alteration of GG-motif to GA resulted in higher processing rates, indicating the possibility of improved lichenicidin production in Escherichia coli. More importantly, insulin A, amylin (non-lanthipeptides), and epidermin were produced and secreted to E. coli supernatant, when fused to the LicA2 leader peptide. This work aids in clarifying the activity of lantibiotic-related transporters and proteases and to evaluate their possible application in industrial processes of relevant compounds, taking advantage of the potential of microorganisms as biofactories. KEY POINTS: • LicM2 correct activity implies a negatively charged residue at position -1. • Hexapeptide mutations can increase the amount of fully processed Bliß. • LicA2 leader peptide directs LicTP cleavage and secretion of other peptides.

Towards the Understanding of the Function of Lanthipeptide and TOMM-Related Genes in Haloferax mediterranei.

Research on secondary metabolites produced by Archaea such as ribosomally synthesized and post-translationally modified peptides (RiPPs) is limited. The genome of Haloferax mediterranei ATCC 33500 encodes lanthipeptide synthetases (medM1, medM2, and medM3) and a thiazole-forming cyclodehydratase (ycaO), possibly involved in the biosynthesis of lanthipeptides and the TOMMs haloazolisins, respectively. Lanthipeptides and TOMMs often have antimicrobial activity, and H. mediterranei has antagonistic activity towards haloarchaea shown to be independent of medM genes. This study investigated (i) the transcription of ycaO and medM genes, (ii) the involvement of YcaO in bioactivity, and (iii) the impact of YcaO and MedM-encoding genes' absence in the biomolecular profile of H. mediterranei. The assays were performed with biomass grown in agar and included RT-qPCR, the generation of knockout mutants, bioassays, and FTIR analysis. Results suggest that ycaO and medM genes are transcriptionally active, with the highest number of transcripts observed for medM2. The deletion of ycaO gene had no effect on H. mediterranei antihaloarchaea activity. FTIR analysis of medM and ycaO knockout mutants suggest that MedMs and YcaO activity might be directly or indirectly related t lipids, a novel perspective that deserves further investigation.

Linking Pedobacter lusitanus NL19 volatile exometabolome with growth medium composition: what can we learn using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry?

Microbial metabolomics allows understanding and to comprehensively analyse metabolites, and their related cellular and metabolic processes, that are produced and released to the extracellular environment under specific conditions. In that regard, the main objective of this research is to understand the impact of culture media changes in the metabolic profile of Pedobacter lusitanus NL19 (NL19) and Pedobacter himalayensis MTCC 6384 (MTCC6384) and respective influence on the production of biotechnologically relevant compounds. Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry with time-of-flight analyser (GC × GC-ToFMS) was applied to comprehensively study the metabolites produced by NL19 and MTCC6384 both in tryptic soy broth 100% (TSB100) and tryptic soy broth with 25% casein peptone (PC25). A total of 320 metabolites were putatively identified, which belong to different chemical families: alcohols, aldehydes, esters, ethers, hydrocarbons, ketones, nitrogen compounds, sulphur compounds, monoterpenes, and sesquiterpenes. Metabolites that were statistically different from the control (sterile medium) were selected allowing for the construction of the metabolic profile of both strains. A set of 80 metabolites was tentatively associated to the metabolic pathways such as the metabolism of fatty acids, branched-chain aminoacids, phenylalanine, methionine, aromatic compounds, and monoterpene and sesquiterpene biosynthesis. This study allowed to better understand how slight changes of the culture media and thus the composition of nutrients impair the metabolic profile of bacteria, which may be further explored for metabolomics pipeline construction or biotechnological applications.

Unravelling the Diversity and Abundance of the Red Fox (Vulpes vulpes) Faecal Resistome and the Phenotypic Antibiotic Susceptibility of Indicator Bacteria.

The WHO considers that antimicrobial resistance (AMR) is among the ten greatest global public health risks of the 21st century. The expansion of human populations and anthropogenically related activities, accompanied by the fragmentation of natural habitats, has resulted in increased human-wildlife interaction. Natural ecosystems are therefore subjected to anthropogenic inputs, which affect the resistome of wild animals. Thus, urgent multisectoral action is needed to achieve the Sustainable Development Goals following the One Health approach. The present work falls within the scope of this approach and aims to characterize the AMR of the faecal microbiome of the red fox (Vulpes vulpes), an opportunistic and generalist synanthropic species whose abundance has been increasing in urban and peri-urban areas. A high number of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) were screened and quantified using a high-throughput qPCR approach, and the antimicrobial susceptibility of cultivable E. coli and Enterococcus spp. were assessed interpreted with both ECOFFs and clinical breakpoints. The most abundant ARGs detected confer resistance to trimethoprim and tetracyclines, although the first were absent in one of the locations studied. Several ARGs considered to be threats to human health were identified in high relative abundances (blaTEM, ermB, aadA, tetM, tetW, tetL, drfA1 and drfA17), especially in the geographical area with greater anthropogenic influence. Although at a low percentage, resistant and multidrug-resistant (MDR) E. coli and Enterococcus spp. were isolated, including one MDR E. coli showing resistance to 12 antimicrobials from 6 different classes.

First characterization of the faecal resistome of eurasian otter (Lutra lutra), a sentinel species for aquatic environments.

Antimicrobial resistance (AMR) is a global health concern. Nowadays, antibiotic resistance genes (ARGs) are considered emerging pollutants. This study followed the One Health framework, in which AMR surveillance in the environment, including in wild animals, is advisable to mitigate this problem. Here we investigated AMR associated with Eurasian otter, a semi-aquatic mammal considered an indicator of freshwater health. To do so, otter's faecal resistome was characterized by a high-throughput qPCR array. This technique has a high-capacity of ARGs profiling. Additionally, we have assessed the antimicrobial susceptibility of two indicator bacteria, E. coli and Enterococcus spp, isolated from otter spraints and interpreted the results according to clinical and epidemiological cut-offs (ECOFFs).

Electrically Conductive and Antimicrobial Agro-Food Waste Biochar Functionalized with Zinc Oxide Particles.

Carbonaceous materials derived from biomass have been used as sustainable platforms for the growth of ZnO particles aiming the production of functional composite fillers. Kidney-bean pods were pyrolyzed by applying an experimental design that demonstrates that the specific surface area (SBET) of biochar is improved with increasing pyrolysis temperature combined with a short air-oxidation time. Meanwhile, the graphitization degree and the electrical conductivity (EC) of biochars were negatively affected by increasing the air-oxidation time. The biochar sample with the higher EC and the one with the higher SBET were selected to be functionalized with ZnO particles by a solvothermal methodology, obtaining composites with an EC and SBET properties superior to the ZnO-rGO composite, in addition to a similar antibacterial activity. The developed ZnO-biochar composite structures, which are more ecological and biocompatible than the ZnO composites derived from graphene sheets, can be applied as electrically conductive and active fillers.

Draft Genome Sequence of Vibrio mediterranei Strain CyArs1.

Here, we report on the draft genome sequence of Vibrio mediterranei strain CyArs1, isolated from the marine sponge Cinachyrella sp. Genome annotation revealed multiple genomic features, including eukaryotic-like repeat protein- and multidrug resistance-encoding genes, potentially involved in symbiotic relationships with the sponge host.

Oh, deer! How worried should we be about the diversity and abundance of the faecal resistome of red deer?

The emergence of antimicrobial resistance (AMR) is a global threat to public health. Antimicrobials are used in animal production and human medicine, which contribute to the circulation of antibiotic resistance genes (ARGs) in the environment. Wildlife can be reservoirs of pathogens and resistant bacteria. Furthermore, anthropogenic pressure can influence their resistome. This work aimed to study the AMR of the faecal microbiome of red deer, one of the most important game species in Europe. To this end, a high-throughput qPCR approach was employed to screen a high number of ARGs and the antimicrobial susceptibility of indicator bacteria was determined. Several genes that confer resistance to different classes of antibiotics were identified, with the most abundant being tetracycline ARGs. Other genes were also present that are considered current and future threats to human health, and some of these were relatively abundant. Multidrug-resistant E. coli and Enterococcus spp. were isolated, although the overall level of antibiotic resistance was low. These results highlight the pressing need to know the origin and transmission of AMR in wildlife. Thus, and considering the One Health concept, studies such as this one shows the need for surveillance programs to prevent the spread of drug-resistant strains and ARGs.

Antibiotic resistance and potential bacterial pathogens identified in red deer's faecal DNA.

In the last decades, the wildlife-human interface has been increasing due to several anthropogenic factors. Therefore, it is crucial to be aware of the impact of these new dynamics on the health of wild animals and their associated zoonotic disease risks. This study aimed to characterize the faecal microbiota of two populations of red deer (Cervus elaphus) by metabarcoding, with a particular focus on potential human and veterinary pathogens, and to perform an assessment of antibiotic resistance genes (ARGs) occurrence. The faecal microbiota of red deer was assessed by metabarcoding using the 16S rRNA marker, and OTUs of the genera Treponema, Yersinia, Clostridium, Mycobacterium, and Rickettsia were identified. Two of them affiliated with species more commonly regarded as pathogens (Clostridium piliforme and Yersinia enterocolitica). The quantification of ARGs was performed by quantitative real-time PCR, using a metagenomic approach, and the most abundant genes were found to be blaTEM , sul1, tetracycline resistance genes (tetW, tetO, and tetQ) and ermF. From these, tetO and tetW are rank II ARGs, which were recently considered future threats for human health. Our results suggest the need for screening programs for the occurrence of pathogens and ARGs in wildlife and particularly in-game species.

Assessing the potential of the two-peptide lantibiotic lichenicidin as a new generation antimicrobial.

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliß. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliß. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative.

Insights into the mode of action of the two-peptide lantibiotic lichenicidin.

Lantibiotics are promising candidates to address the worldwide problem of antibiotic resistance. They belong to a class of natural compounds exhibiting strong activity against clinically relevant Gram-positive bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Lichenicidin is a class II two-peptide lantibiotic. The presence of the two mature peptides, Bliα and Bliß, is necessary for full activity against target bacteria. This work aims at clarifying the synergistic activity of both peptides in their interaction with the target membranes. The effect of lichenicidin was tested against S. aureus cells and large unilamellar vesicles. Lichenicidin increases the net surface charge of S. aureus, as shown by zeta-potential measurements, without reaching electroneutralization. In addition, lichenicidin causes cell surface perturbations that culminate in the leakage of its internal contents, as observed by atomic force microscopy. Bliα seems to have low affinity for S. aureus, however, it contributes to increase the affinity of Bliß, because together they present higher affinity than separately. In contrast, Bliα seems to provide an anchoring site for lichenicidin in lipid II-containing membranes. Interestingly, Bliß alone can induce high levels of membrane leakage, but this effect appears to be faster in the presence of Bliα. Based on this information, we propose a mechanism of action of lichenicidin.

A closer look on the variety and abundance of the faecal resistome of wild boar.

Antimicrobial resistance (AMR) is a serious problem for public and animal health, and also for the environment. Monitoring and reporting the occurrence of AMR determinants and bacteria with the potential to disseminate is a priority for health surveillance programs around the world and critical to the One Health concept. Wildlife is a reservoir of AMR, and human activities can strongly influence their resistome. The main goal of this work was to study the resistome of wild boar faecal microbiome, one of the most important game species in Europe using metagenomic and culturing approaches. The most abundant genes identified by the high-throughput qPCR array encode mobile genetic elements, including integrons, which can promote the dissemination of AMR determinants. A diverse set of genes (n = 62) conferring resistance to several classes of antibiotics (ARGs), some of them included in the WHO list of critically important antimicrobials were also detected. The most abundant ARGs confer resistance to tetracyclines and aminoglycosides. The phenotypic resistance of E. coli and Enterococcus spp. were also investigated, and together supported the metagenomic results. As the wild boar is an omnivorous animal, it can be a disseminator of AMR bacteria and ARGs to livestock, humans, and the environment. This study supports that wild boar can be a key sentinel species in ecosystems surveillance and should be included in National Action Plans to fight AMR, adopting a One Health approach.

The Unexplored Wealth of Microbial Secondary Metabolites: the Sphingobacteriaceae Case Study.

Research on secondary metabolites (SMs) has been mostly focused on Gram-positive bacteria, especially Actinobacteria. The association of genomics with robust bioinformatics tools revealed the neglected potential of Gram-negative bacteria as promising sources of new SMs. The family Sphingobacteriaceae belongs to the phylum Bacteroidetes having representatives in practically all environments including humans, rhizosphere, soils, wastewaters, among others. Some genera of this family have demonstrated great potential as plant growth promoters, bioremediators and producers of some value-added compounds such as carotenoids and antimicrobials. However, to date, Sphingobacteriaceae's SMs are still poorly characterized, and likewise, little is known about their chemistry. This study revealed that Sphingobacteriaceae pangenome encodes a total of 446 biosynthetic gene clusters (BGCs), which are distributed across 85 strains, highlighting the great potential of this bacterial family to produce SMs. Pedobacter, Mucilaginibacter and Sphingobacterium were the genera with the highest number of BGCs, especially those encoding the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), terpenes, polyketides and nonribosomal peptides (NRPs). In Mucilaginibacter and Sphingobacterium genera, M. lappiensis ATCC BAA-1855, Mucilaginibacter sp. OK098 (both with 11 BGCs) and Sphingobacterium sp. 21 (6 BGCs) are the strains with the highest number of BGCs. Most of the BGCs found in these two genera did not have significant hits with the MIBiG database. These results strongly suggest that the bioactivities and environmental functions of these compounds, especially RiPPs, PKs and NRPs, are still unknown. Among RiPPs, two genera encoded the production of class I and class III lanthipeptides. The last are associated with LanKC proteins bearing uncommon lyase domains, whose dehydration mechanism deserves further investigation. This study translated genomics into functional information that unveils the enormous potential of environmental Gram-negative bacteria to produce metabolites with unknown chemistries, bioactivities and, more importantly, unknown ecological roles.

Pathogenicity of Shiga toxin-producing Escherichia coli (STEC) from wildlife: Should we care?

Shiga toxin-producing Escherichia coli (STEC) is one of the most frequent bacterial agents associated with food-borne outbreaks in Europe. In humans, the infection can lead to life-threatening diseases. Domestic and wild animals can harbor STEC, and ruminants are the main STEC reservoirs, although asymptomatic. In the present study we have characterized STEC from wildlife (wild boar (n = 56), red deer (n = 101), red fox (n = 37) and otter (n = 92)). Cultivable STEC (n = 52) were isolated from 17% (n = 49) of the faecal samples. All the isolates were non-O157 STEC encoding stx1 (n = 2; 4%) and/or stx2 genes (n = 51; 98%). Only one strain (2%) isolated from red fox had an antibiotic resistant phenotype. However, when the normalized resistance interpretation of epidemiological cutoffs (NRI ECOFFs) were used, 23% (n = 12) of the strains were non-wildtype to at least one of the antibiotics tested. After analysis by pulsed-field gel electrophoresis (PFGE), 20 strains were selected for whole genome sequencing and belonged to the following serotypes: O27:H30 (n = 15), O146:H28 (n = 2), O146:H21 (n = 1), O178:H19 (n = 1), and O103:H2 (n = 1). In addition to stx, all strains encode several virulence factors such as toxins, adhesins, fimbriae and secretion systems, among others. All sequenced genomes carried several mobile genetic elements (MGEs), such as prophages and/or plasmids. The core genome and the phylogenetic analysis showed close evolutionary relationships between some of the STEC recovered from wildlife and strains of clinical origin, highlighting their pathogenic potential. Overall, our results show the zoonotic potential of STEC strains originating from wildlife, highlighting the importance of monitoring their genomic characteristics following a One Health perspective, in which the health of humans is related to the health of animals, and the environment.

Haloarchaea have a high genomic diversity for the biosynthesis of carotenoids of biotechnological interest.

Haloarchaea are mostly components of the microbial biomass of saline aquatic environments, where they can be a dietary source of heterotrophic metazoans or contribute to flamingo's plumage coloration. The diversity of secondary metabolites (SMs) produced by haloarchaea, which might play multiple ecological roles and have diverse biotechnological applications has been largely understudied. Herein, 67 haloarchaeal complete genomes were analyzed and 182 SMs biosynthetic gene clusters (BGCs) identified that encode the production of terpenes (including carotenoids), RiPPs and siderophores. Terpene BGCs were further analysed and it was concluded that all haloarchaea might produce squalene and bacterioruberin, which one a strong antioxidant. Most of them have other carotenoid BGCs that include a putative ß-carotene ketolase that was not characterized so far in haloarchaea, but may be involved with canthaxanthin's biosynthesis. The production of bacterioruberin by Haloferax mediterranei ATCC 33500 was found to be not related to its antimicrobial activity.

The lanthipeptide biosynthetic clusters of the domain Archaea.

Research on Archaea's secondary metabolites is still lagging behind that of Bacteria and Eukarya. Our goal was to contribute to this knowledge gap by analyzing the lanthipeptide's clusters in Archaea. As previously proposed, Archaea encodes only class II synthetases (LanMs), which we found to be confined to the class Halobacteria (also known as haloarchaea). In total, we analyzed the phylogeny and the domains of 42 LanMs. Four types were identified, and the majority of them belong to the CCG group due to their cyclization domain, which includes LanMs of Cyanobacteria. Putative cognate peptides were predicted for most of LanMs and are a very diverse group of molecules that share a Kx(Y/F)(D/E)xx(F/Y) motif in their leader peptides. According to their homology, some of them were categorized into subfamilies, including Halolancins, Haladacins, Haloferaxcins and Halobiforcins. Many LanM genes were associated with mobile genetic elements, and their vicinities mainly encode ABC and MFS transporters, tailoring enzymes and uncharacterized proteins. Our results suggest that the biosynthesis of lanthipeptides in haloarchaea can entail distinct enzymology that must lead to the production of peptides with novel structures and unpredicted biological and ecological roles. Finally, an Haloferax mediterranei knockout, lacking its three lanM genes, was generated, and it was concluded that its antimicrobial activity is not primarily related to the production of lanthipeptides.

Design of Alginate-Based Bionanocomposites with Electrical Conductivity for Active Food Packaging.

Bionanocomposite materials have been designed as a promising route to enhance biopolymer properties, especially for food packaging application. The present study reports the preparation of bionanocomposite films of alginate with different loadings of pure reduced graphene oxide (rGO) or of mixed zinc oxide-rGO (ZnO-rGO) fillers by solvent casting. Sepiolite is used to make compatible rGO with the hydrophilic matrix. The addition of fillers to alginate matrix maintains the low water solubility promoted by the calcium chloride treatment, and, additionally, they demonstrate a weaker mechanical properties, and a slight increase in water vapor permeability and wettability. Due to the properties of ZnO-rGO, the alginate bionanocomposites show an increase of electrical conductivity with the increase of filler content. While the highest electrical conductivity (0.1 S/m) is achieved by the in-plane measurement, it is in the through-plane measurement the remarkable enhancement of almost 30 times greater than the alginate film. With 50% of ZnO-rGO filler, the bionanocomposites present the highest antioxidant and antibacterial activities. The combination of electrical conductivity with bioactive properties makes these films promising not only to extend food shelf-life but also to allow packaged food sterilization at low temperature.

Structural Analysis of Class I Lanthipeptides from Pedobacter lusitanus NL19 Reveals an Unusual Ring Pattern.

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-tRNAGlu 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.

Peptone from casein, an antagonist of nonribosomal peptide synthesis: a case study of pedopeptins produced by Pedobacter lusitanus NL19.

Novel natural products are urgently needed to address the worldwide incidence of bacterial resistance to antibiotics. Extreme environments are a major source of novel compounds with unusual chemical structures. Pedobacter lusitanus NL19 is a new bacterial species that was isolated from one such environment and which produces compounds with potent activity against relevant microorganisms in the clinical, food, veterinary and aquaculture areas. The production of antimicrobials by P. lusitanus NL19 was identified in tryptic soy agar (TSA), but not in its equivalent broth (TSB). It was observed that in TSB medium a high concentration of casein peptone (PC) repressed the production of antibacterial compounds. HPLC, MS and MS/MS spectra with de novo sequencing revealed that the bioactivity of P. lusitanus NL19 was due to the production of pedopeptins. Hence, biosynthesis of pedopeptins is inhibited by high concentrations of PC in the broth medium. Furthermore, a nonribosomal peptide synthetase (NRPS) gene cluster was identified in the genome of NL19 encoding the biosynthesis of the peptides. qPCR analysis confirmed that the transcription of these genes is repressed in cells cultivated in high concentrations of PC. It is shown that pedopeptins are nonribosomal peptides with a broad-spectrum activity, including against Gram-positive and Gram-negative bacteria and yeasts.