Reproducible mass spectrometry data processing and compound annotation in MZmine 3.

Untargeted mass spectrometry (MS) experiments produce complex, multidimensional data that are practically impossible to investigate manually. For this reason, computational pipelines are needed to extract relevant information from raw spectral data and convert it into a more comprehensible format. Depending on the sample type and/or goal of the study, a variety of MS platforms can be used for such analysis. MZmine is an open-source software for the processing of raw spectral data generated by different MS platforms. Examples include liquid chromatography-MS, gas chromatography-MS and MS-imaging. These data might typically be associated with various applications including metabolomics and lipidomics. Moreover, the third version of the software, described herein, supports the processing of ion mobility spectrometry (IMS) data. The present protocol provides three distinct procedures to perform feature detection and annotation of untargeted MS data produced by different instrumental setups: liquid chromatography-(IMS-)MS, gas chromatography-MS and (IMS-)MS imaging. For training purposes, example datasets are provided together with configuration batch files (i.e., list of processing steps and parameters) to allow new users to easily replicate the described workflows. Depending on the number of data files and available computing resources, we anticipate this to take between 2 and 24 h for new MZmine users and nonexperts. Within each procedure, we provide a detailed description for all processing parameters together with instructions/recommendations for their optimization. The main generated outputs are represented by aligned feature tables and fragmentation spectra lists that can be used by other third-party tools for further downstream analysis.

Isolation and Characterization of an Antioxidant Aryl Polyene Pigment from Antarctic Bacterium Lysobacter sp. A03.

Lysobacter is known as a bacterial genus with biotechnological potential, producing an array of enzymes, antimicrobial metabolites, and bioactive antioxidant compounds, including aryl polyene (APE) pigments that have been described as protecting substances against photooxidative damage and lipid peroxidation. In this study, the pigment extracted from keratinolytic Lysobacter sp. A03 isolated from Antarctic environment was characterized. The results of KOH test, UV-vis spectroscopy, CIELAB color system, 1H-NMR, and FTIR-ATR spectroscopy suggest the pigment is a yellow xanthomonadin-like pigment. The in vitro antioxidant activity of the pigment was confirmed by the scavenging of ABTS and DPPH radicals. In silico analysis of the genome through antiSMASH software was also performed and the secondary metabolite gene clusters for APE and resorcinol synthesis were identified, suggesting that proteins responsible for the pigment biosynthesis are encoded in Lysobacter A03 genome.

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.

Decoding the molecular interplay in the central dogma: An overview of mass spectrometry-based methods to investigate protein-metabolite interactions.

With the emergence of next-generation nucleotide sequencing and mass spectrometry-based proteomics and metabolomics tools, we have comprehensive and scalable methods to analyze the genes, transcripts, proteins, and metabolites of a multitude of biological systems. Despite the fascinating new molecular insights at the genome, transcriptome, proteome and metabolome scale, we are still far from fully understanding cellular organization, cell cycles and biology at the molecular level. Significant advances in sensitivity and depth for both sequencing as well as mass spectrometry-based methods allow the analysis at the single cell and single molecule level. At the same time, new tools are emerging that enable the investigation of molecular interactions throughout the central dogma of molecular biology. In this review, we provide an overview of established and recently developed mass spectrometry-based tools to probe metabolite-protein interactions-from individual interaction pairs to interactions at the proteome-metabolome scale.

Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

Proteomic dataset of Listeria monocytogenes exposed to sublethal concentrations of free and nanoencapsulated nisin.

The cellular proteins of L. monocytogenes exposed to free and liposome-encapsulated nisin at sublethal concentration were hydrolyzed by trypsin and examined by tandem mass spectrometry (MS/MS) to obtain proteomic data. In the present study, we use the STRING v11.05 database analyze the interactions among the 78 upregulated proteins from L. monocytogenes obtained after treatment with sublethal concentrations of free and nanoliposome-encapsulated nisin. As result, from the upregulated proteins by free nisin was determined a network with 140 edges with two relevant nodes, containing ribosomal proteins and transmembrane transport proteins (SecD and ABC transport system). These two sets of proteins present biological connection as a group, with strong interactions and are related to detoxification and other Listeria response mechanisms. In addition, a high amount of membrane proteins was identified in the free nisin treatment. On the other hand, in the interaction analysis of upregulated proteins by liposome-loaded nisin, was found 156 edges with a single protein network, the same observed in free nisin, related to ribosomal proteins. Therefore, according with this analysis, the encapsulation of nisin into liposomes cause upregulation of ribosomal and decrease of L. monocytogenes response proteins as compared with free nisin.

Listeria monocytogenes exposed to antimicrobial peptides displays differential regulation of lipids and proteins associated to stress response.

With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides.

Genomic characterization and production of antimicrobial lipopeptides by Bacillus velezensis P45 growing on feather by-products.

AIMS: To investigate the potential of novel Bacillus velezensis P45 as an eco-friendly alternative for bioprocessing poultry by-products into valuable antimicrobial products. METHODS AND RESULTS: The complete genome of B. velezensis P45 was sequenced using the Illumina MiSeq platform, showing 4455 protein and 98 RNA coding sequences according to the annotation on the RAST server. Moreover, the genome contains eight gene clusters for the production of antimicrobial secondary metabolites and 25 putative protease-related genes, which can be related to feather-degrading activity. Then, in vitro tests were performed to determine the production of antimicrobial compounds using feather, feather meal and brain-heart infusion (BHI) cultures. Antimicrobial activity was observed in feather meal and BHI media, reaching 800 and 3200 AU ml-1 against Listeria monocytogenes respectively. Mass spectrometry analysis indicates the production of antimicrobial lipopeptides surfactin, fengycin and iturin. CONCLUSIONS: The biotechnological potential of B. velezensis P45 was deciphered through genome analysis and in vitro studies. This strain produced antimicrobial lipopeptides growing on feather meal, a low-cost substrate. SIGNIFICANCE AND IMPACT OF STUDY: The production of antimicrobial peptides by this keratinolytic strain may represent a sustainable alternative for recycling by-products from poultry industry. Furthermore, whole B. velezensis P45 genome sequence was obtained and deposited.

Antimicrobial activity of Baccharis dracunculifolia DC and its synergistic interaction with nisin against food-related bacteria.

The antimicrobial activities of Baccharis dracunculifolia DC essential oil (EO) and hydroalcoholic extract (HE) were evaluated. The EO showed broad antimicrobial activity and its synergistic combination with nisin was tested. Major components of EO were nerolidol, beta-pinene and D-limonene, while artepillin C, rutin and cafeic acid were major phenolics of HE. EO and HE were tested by agar diffusion assay against several strains of bacteria and yeasts, and mixed cultures of bacterial strains. The EO presented the largest spectrum of antimicrobial activity inhibiting all Gram-positive bacteria tested. Yeasts were not inhibited. The effect of EO against mixtures of sensitive and non-sensitive bacteria was tested on milk agar, being the inhibitory effect only observed on mixtures containing susceptible strains. The combination of EO and nisin at ½ MIC was evaluated on the growth curve of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Salmonella Enteritidis during 24 h at 37 °C. The combination EO-nisin was effective and no viable counts of B. cereus, L. monocytogenes and S. Enteritidis was observed, while the individual antimicrobials caused no inhibition. The counts of S. aureus were about 4 log CFU/mL lower in comparison with EO or nisin alone. B. dracunculifolia DC may be a potential source of natural antimicrobials, and its synergistic effect with nisin would reduce the working concentration, minimizing the organoleptic effects associated with this plant antimicrobial.

Proteomic analysis reveals differential responses of Listeria monocytogenes to free and nanoencapsulated nisin.

The ability of Listeria monocytogenes grow on ready-to-eat food is a major concern in food safety. Natural antimicrobials, such as nisin, can be used to control this pathogen, but the increasing reports of nisin tolerance and resistance make necessary novel approaches to increase its effectiveness, such as encapsulation. The goal of this study was to investigate how L. monocytogenes ATCC7644 regulates and shapes its proteome in response to sublethal doses of nisin and nisin-loaded phosphatidylcholine liposomes (lipo-nisin), compared to untreated cells growing under optimal conditions. Total proteins were extracted from L. monocytogenes cells treated for 1 h with free and lipo-nisin. As result, of 803 proteins that were initially identified, 64 and 53 proteins were differentially upregulated and downregulated respectively, in the treatments with nisin and lipo-nisin. Changes of Listeria proteome in response to treatments containing nisin were mainly related to ATP-binding cassette (ABC) transporter systems, transmembrane proteins, RNA-binding proteins and diverse stress response proteins. Some of the proteins uniquely detected in samples treated with free nisin were the membrane proteins SecD, Lmo1539 and the YfhO enzyme, which are related to translocation of L. monocytogenes virulence factors, activation of the LiaR-mediated stress defense and glycosylation of wall teichoic acid, respectively. The L. monocytogenes treated with liposome encapsulated nisin showed no expression of some stress response factors as compared with the free nisin, suggesting a reduction of stress mediated response and production of nisin-resistance factors by exposure to encapsulated nisin.

Proteomic analysis of Listeria monocytogenes exposed to free and nanostructured antimicrobial lipopeptides.

In this work, the effect of antimicrobial lipopeptide P34 on Listeria monocytogenes was evaluated for the first time through a proteomics approach. Bacteria were treated with sub-lethal doses of peptide P34 (F-P34) and P34 encapsulated into nanoliposomes (N-P34), while empty nanoliposomes (NE) and fresh buffer were used as controls. The proteomic analysis allowed the detection of one group of proteins commonly differentially represented in response to free and encapsulated P34 exposure. A second group of proteins was found to be exclusively differentially represented after exposure with encapsulated P34 only. The antimicrobial peptide P34 caused a significant downregulation of proteins associated with the transport of manganese and the over-representation of proteins related with iron transport in L. monocytogenes. In addition, reduction of stress tolerance proteins related to the σB and VirR regulons, together with the modulation of phosphoenolpyruvate phosphotransferase systems (PTS) for sugar transport were observed. The sugar and oligopeptide transporters regulated by antimicrobial action may influence the key virulence factor PrfA, reducing the pathogenicity of this microorganism.

Nisin influence on the expression of Listeria monocytogenes surface proteins.

In this work, a comparative analysis of the peripheral cell component (PCC) proteins of Listeria monocytogenes was carried out. The study was conducted on two set of samples consisting of bacteria treated with sub-lethal concentration of nisin and untreated bacteria as control. PCC proteins were extracted by Tris-Urea-EDTA treatment and then subjected to trypsin digestion and mass spectrometry analysis. The whole cell proteome was analyzed through label-free quantitative proteomics approach. Proteomic analysis was carried out using OrbiTrap Mass Spectrometer coupled to nanoflow liquid chromatography. The treatment with sub-lethal nisin concentration resulted in 62 up regulated and 97 down regulated proteins compared to untreated samples. Using PSORTb 3.0, 19 and 18 surface proteins were detected among the up regulated and down regulated proteins, respectively. Proteins related with increased biofilm formation by L.monocytogenes, such as moonlight proteins of the pyruvate dehydrogenase complex and flagellin-related proteins, were identified as up regulated surface proteins. Proteins associated with virulence of L.monocytogenes, including listeriolysin O, internalin B and actin assembly-inducing protein, were detected among the down regulated proteins. To confirm proteomics data, increased production of biofilm was experimentally confirmed in nisin-treated cells through crystal violet method. BIOLOGICAL SIGNIFICANCE: Proteosurfaceomics can be defined as the "omics" science applied to the proteins of the peripheral cell component (PCC). The surface proteins of Listeria monocytogenes, an important foodborne pathogen were investigated after treatment with nisin, a bacteriocin approved as a natural food preservative by regulatory agencies. Recent cases of nisin tolerance by Listeria spp. were documented, and deeper studies on the molecular process behind the bacterial survival may help in both understanding the development of tolerance process and comparing nisin effect with other antimicrobial compounds.

Diversity of cyclic antimicrobial lipopeptides from Bacillus P34 revealed by functional annotation and comparative genome analysis.

Cyclic lipopeptides (CLPs) from Bacillus strains have demonstrated a wide range of bioactivities making them interesting candidates for different applications in the pharmaceutical, food and biotechnological industries. Genome sequencing, together with phylogenetic analysis of the Bacillus sp. P34, isolated from a freshwater fish gut, showed that the bacterial strain belongs to the Bacillus velezensis group. In silico investigation of metabolic gene clusters of nonribosomal peptide synthetases (NRPS) revealed the genetic elements associated with the synthesis of surfactin, fengycin and iturin family component bacillomycin. Further, an assay was conducted to investigate the production of CLPs in the presence of heat inactivated bacterial cultures or fungal spores. Maximum fengycin concentration was observed at 24 h (2300-2700 mg/mL), while maximum iturin amounts were detected at 48 h (250 mg/mL) in the presence of heat-inactivated spores of Aspergillus niger. Heat-inactivated cells of Listeria monocytogenes caused a reduction of both fengycin and iturin amounts. The production of fengycins A and B and the iturin family component bacillomycin L was confirmed by mass spectrometry analyses. This study reinforces the potential of B. velezensis P34 as a valuable strain for biotechnological production of CLPs recognized as important antimicrobial substances.

Marine bacteria as source of antimicrobial compounds.

The marine environment encompasses a huge biological diversity and can be considered as an underexplored location for prospecting bioactive molecules. In this review, the current state of art about antimicrobial molecules from marine bacteria has been summarized considering the main phylum and sources evolved in a marine environment. Considering the last two decades, we have found as most studied group of bacteria producers of substances with antimicrobial activity is the Firmicutes phylum, in particular strains of the Bacillus genus. The reason for that can be attributed to the difficult cultivation of typical Actinobacteria from a marine sediment, whose members are the major producers of antimicrobial substances in land environments. However, a reversed trend has been observed in recent years with an increasing number of reports settling on Actinobacteria. Great diversity of chemical structures have been identified, such as fijimicyns and lynamicyns from Actinomycetes and macrolactins produced by Bacillus.