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.

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.

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.

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.

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.

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.

Evolving trends in next-generation probiotics: a 5W1H perspective.

In recent years, scientific community has been gathering increasingly more insight on the dynamics that are at play in metabolic and inflammatory disorders. These rapidly growing conditions are reaching epidemic proportions, bringing clinicians and researcher's new challenges. The specific roles and modulating properties that beneficial/probiotic bacteria hold in the context of the gut ecosystem seem to be key to avert these inflammatory and diet-related disorders. Faecalibacterium prausnitzii, Akkermansia muciniphila and Eubacterium hallii have been identified as candidates for next generation probiotics (NGPs) with exciting potential for the prevention and treatment of such of dysbiosis-associated diseases. The challenges of these non-conventional native gut bacteria lie mainly on their extreme sensitivity to O2 traces. If these strains are to be used successfully in food, supplements or drugs they need to be stable and active in humans. In the present review, we present an overall perspective of the most updated scientific literature on the newly called NGPs through the 5W1H (What, Why, Who, Where, When, and How) method, an innovative and attractive problem-solving approach that provides the reader an effective understanding of the issue at hand.

Lipidomic signature of Bacillus licheniformis I89 during the different growth phases unravelled by high-resolution liquid chromatography-mass spectrometry.

Bacillus licheniformis I89 is a non-pathogenic, Gram-positive bacterium, frequently found in soil. It has several biotechnological applications as producer of valuable compounds such as proteases, amylases, surfactants, and lantibiotics. Herein, it is reported the identification of the polar lipidome of B. licheniformis I89 during the different growth phases (lag, exponential and stationary) at 37 °C. The analytical approach relied on hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry (HILIC-ESI-MS), accurate mass measurements and tandem mass spectrometry (MS/MS). In the lipidome of B. licheniformis I89 were identified four phospholipid classes: phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin; two glycolipid classes: monoglycosyldiacylglycerol and diglycosyldiacylglycerol; and two phosphoglyceroglycolipid classes: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. The same lipid species were identified at the different growth phases, but there were significant differences on the relative abundance of some molecular species. There was a significant increase in the 30:0 lipid species and a significant decrease in the 32:0 lipid species, between exponential and stationary phases, when compared to lag phase. No differences were observed between exponential and stationary phases. The lipidomic-based approach used herein is a very promising tool to be employed in the study of bacterial lipid composition, which is a requirement to understand its metabolism and response to growth conditions.

Lichenicidin rational site-directed mutagenesis library: A tool to generate bioengineered lantibiotics.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure-activity relationship studies carried out for other lantibiotics.

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine.

Strain NL19T is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19T is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacterhartonius DSM 19033T. It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included C16 : 1ω7c, C14 : 1ω5c, C4 : 0, iso-C17 : 0, iso-C17 : 0 3-OH, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0 3-OH. The main lipids present were phospholipids (60 %) and sphingolipids (35 %). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA-DNA hybridization similarities between strain NL19T and Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacter hartonius DSM 19033T were 15.3 , 16.2 , 11.5 and 16.0 %, respectively. Strain NL19T can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19Trepresents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19T (=LMG 29220T=CECT 9028T). An amended description of Pedobacter himalayensis is also included.

Oxidative stress and genotoxicity of an organic and an inorganic nanomaterial to Eisenia andrei: SDS/DDAB nano-vesicles and titanium silicon oxide.

In the past few years the number of studies on the toxic effects of nanomaterials (NMs) in the environment increased significantly. Nonetheless, the data is still scarce, since there is a large number of NMs and new ones are being developed each day. Soils are extremely important for life, and are easily exposed to the released NMs, thus enhanced efforts are needed to study the impacts on soil biota. The objective of the present work was to determine if different concentrations of two NMs, one inorganic (TiSiO4) and other organic (nano-vesicles of sodium sodecyl sulfate/ didodecyl dimethylammonium bromide - SDS/DDAB), are genotoxic to soil invertebrates. Additionally, it was intended to understand whether, in the event of occurring, genotoxicity was caused by the incapability of the cells to deal with the oxidative stress caused by these NMs. With that purpose, Eisenia andrei were exposed for 30 days to the artificial OECD soil contaminated with different concentrations of the NMs being tested. After the exposure, coelomocytes were extracted from earthworms and DNA damage was measured by the comet assay. The activity of antioxidant enzymes (e.g. glutathione peroxidase, glutathione reductase and glutathione-S-Transferase) and lipid peroxidation were also assessed. The results showed that both NMs were genotoxic, particularly TiSiO4 for which significant DNA damages were recorded for concentrations above 444mg of TiSiO4-NM/kg of soildw. Since no statistically significant differences were found in the tested antioxidant enzymes and in lipid peroxidation, the mechanism of genotoxicity of these NMs seemed to be unrelated with oxidative stress.

Class I and Class II Lanthipeptides Produced by Bacillus spp.

The increasing number of multidrug-resistant pathogens, along with the small number of new antimicrobials under development, leads to an increased need for novel alternatives. Class I and class II lanthipeptides (also known as lantibiotics) have been considered promising alternatives to classical antibiotics. In addition to their relevant medical applications, they are used as probiotics, prophylactics, preservatives, and additives in cosmetics and personal-care products. The genus Bacillus is a prolific source of bioactive compounds including ribosomally and nonribosomally synthesized antibacterial peptides. Accordingly, there is significant interest in the biotechnological potential of members of the genus Bacillus as producers of antimicrobial lanthipeptides. The present review focuses on aspects of the biosynthesis, gene cluster organization, structure, antibacterial spectrum, and bioengineering approaches of lanthipeptides produced by Bacillus strains. Their efficacy and potency against some clinically relevant strains, including MRSA and VRE, are also discussed. Although no lanthipeptides are currently in clinical use, the information herein highlights the potential of these compounds.

Impact of domestication in the production of the class II lanthipeptide lichenicidin by Bacillus licheniformis I89.

Investigation on lantibiotics biosynthesis constitutes an emergent field, since these molecules have demonstrated a great potential to replace the so-called "traditional antibiotics". The adaptation of bacteria to laboratory conditions (domestication) is an unpredictable phenomenon, which sometimes is associated with the loss of important biotechnological properties. In this study, the domestication of Bacillus licheniformis was associated with the production of the lantibiotic lichenicidin, a two-peptide lantibiotic with activity against several Gram-positive bacteria.

Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna.

In the environment, chemical substances appear as complex mixtures and consequently organisms are exposed to a variety of chemicals from different sources (e.g. wastewater treatment plants, agriculture runoffs). When studying chemical mixtures, there are two conceptual models usually used to predict toxicity: the Independent Action (IA) and Concentration Addition (CA) models. However, deviations from these reference models can occur as synergism or antagonism, dose ratio or dose level dependency. The aim of the present study was to investigate the effects of triclosan and carbendazim, and their binary mixture to Daphnia magna. With this purpose, immobilisation, feeding inhibition, and reproduction were assessed as main ecotoxicity endpoints. In addition, in vivo genotoxicity of both chemicals was investigated using the comet assay. In the single exposure, carbendazim was more toxic to D. magna than triclosan. When daphnids were exposed to both single compounds, DNA damage was observed. Concerning mixture exposures, different endpoints followed different patterns of response, from additivity: IA model (feeding inhibition and reproduction data), to deviations that indicate interaction between chemicals inside the organism: dose level dependency (immobilisation data) and dose ratio dependency (DNA damage). This study showed that additivity does not rule the dose-effect relation in chemical mixtures of carbendazim and triclosan and interactions between both chemicals might induce generally higher toxicity than predicted based on single chemical exposures.

A microcosm approach to evaluate the degradation of tributyltin (TBT) by Aeromonas molluscorum Av27 in estuarine sediments.

Tributyltin (TBT) is a biocide extremely toxic to a wide range of organisms, which has been used for decades in antifouling paints. Despite its global ban in 2008, TBT is still a problem of great concern due to the high levels trapped in sediments. Aeromonas molluscorum Av27 is a TBT degrading bacterium that was isolated from an estuarine system. We investigated the ability and the role of this bacterium on TBT degradation in this estuarine system, using a microcosm approach in order to mimic environmental conditions. The experiment was established and followed for 150 days. Simultaneously, changes in the indigenous bacterial community structure were also investigated. The results revealed a maximum TBT degradation rate of 28% accompanied by the detection of the degradation products over time. Additionally, it was observed that TBT degradation was significantly enhanced by the presence of Av27. In addition a significantly higher TBT degradation occurred when the concentration of Av27 was higher. TBT degradation affected the bacterial community composition as revealed by the changes in the prevalence of Proteobacteria subdivisions, namely the increase of Deltaproteobacteria and the onset of Epsilonproteobacteria. However, the addition of Av27 strain did not affect the dominant phylotypes. Total bacterial number, bacterial biomass productivity, 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analyses also indicated alterations on the bacterial community structure over time, with bacteria non-tolerant to pollutants increasing their representativeness, as, for instance, the increase of the number of Alphaproteobacteria clones from 6% in the beginning to 12% at the end of the experiment. The work herein presented confirms the potential of Av27 strain to be used in the decontamination of TBT-polluted environments.

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus.

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this 'nonmodel' species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator-activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application.

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 µg ml(-1)), amoxicillin/clavulanic acid (30 µg ml(-1)) and cephalothin (30 µg ml(-1)) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.

SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine.

The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.

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.

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.