Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism

Recent developments in whole-cell spectroscopic methods allow rapid characterization of microorganisms of interest to human health, but have yet to be widely applied to marine microbiological studies. In this study of bacteria associated with the kelp Laminaria digitata, we have isolated 18 epiphytic bacterial strains from several thalli, sequenced their 16S rDNA, built corresponding phylogenetic trees, and characterized them using spectroscopic methods. Molecular taxonomy revealed Gram⁺Actinobacteria and Gram⁻Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. Twelve marine reference strains (Gram⁺Firmicutes, and Gram⁻Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes) were treated accordingly. Whole-cell MALDI-TOF MS spectral profiles of 29 of the 30 strains were built into a database against which 16 replicate spectra of each strain were compared and categorized into groups. The proton HR-MAS NMR stack plots allowed visual delineation into taxonomic groups according to their most common peaks, in agreement with identifiable compounds from corresponding D₂O solution spectra. With both methods, these groups corresponded to taxa identified by 16S rDNA sequences, MALDI-TOF MS being more discriminative than HR-MAS NMR. Culture age did not influence the spectral signatures in both approaches. Most cells grown under minimal conditions (VNSS medium) afforded HR-MAS NMR profiles markedly different to those grown in enriched conditions (ZoBell medium), indicating different adaptive metabolic responses between the two media. Spectral signatures obtained under strictly controlled conditions can be used as rapid and reliable tools for taxonomic purposes and as markers of physiological status.     

Effect of Processing Treatment and Modified Atmosphere Packing on Carrot’s Microbial Community Structure by Illumina MiSeq Sequencing

The aim of this study was to analyze the microbiome of carrot (Daucus carota subsp. sativus) subjected to minimal pre-treatment (rinsing in organic acid solution) and packaging in a high-oxygen modified atmosphere, and then stored for 17 days under refrigeration conditions (4 °C). The highest levels of bacteria in the carrot microbiome were characterized, at almost 78%, by bacteria belonging to the Enterobacteriaceae and Pseudomonadaceae families. Rinsing in a solution of ascorbic and citric acids resulted in the improvement of microbiological quality in the first day of storage. However, the use of a high-oxygen modified atmosphere extended the shelf life of the minimally processed product. Compared to carrots stored in air, those stored in high oxygen concentration were characterized by a greater ratio of bacteria belonging to the Serratia and Enterobacter genera, and a lower ratio belonging to the Pseudomonas and Pantoea genera. Moreover, the β-biodiversity analysis confirmed that the oxygen concentration was the main factor influencing the differentiation of the metabiomes of the stored carrots. The bacterial strains isolated from carrots identified by molecular methods were mostly pathogenic or potentially pathogenic microorganisms. Neither the minimal pre-treatment nor packaging in high-oxygen atmosphere was able to eliminate the threat of pathogenic bacteria emerging in the product.     

Discovery and biosynthesis of bosamycins from Streptomyces sp. 120454

Nonribosomal peptides (NRPs) that are synthesized by modular megaenzymes known as nonribosomal peptide synthetases (NRPSs) are a rich source for drug discovery. By targeting an unusual NRPS architecture, we discovered an unusual biosynthetic gene cluster (bsm) from Streptomyces sp. 120454 and identified that it was responsible for the biosynthesis of a series of novel linear peptides, bosamycins. The bsm gene cluster contains a unique monomodular NRPS, BsmF, that contains a cytochrome P450 domain at the N-terminal. BsmF (P450 + A + T) can selectively activate tyrosine with its adenylation (A) domain, load it onto the thiolation (T) domain, and then hydroxylate tyrosine to form 5-OH tyrosine with the P450 domain. We demonstrated a NRPS assembly line for the formation of bosamycins by genetic and biochemical analysis and heterologous expression. Our work reveals a genome mining strategy targeting a unique NRPS domain for the discovery of novel NRPs.

Genome mining targeting a unique NRPS domain led to the identification of a novel class of peptides named bosamycins.     

Profils d'activités enzymatiques des genres Agrobacterium,alcaligenes, alteromonas,flavobacterium et pseudomonas

Profiles of enzymatic activities were studied using 19 chromogenic substrates for 22 species (211 strains) belonging to the genera Agrobacterium, Alcaligenes, Alteromonas, Flavobacterium and Pseudomonas. The observed patterns of reactions may be useful as an aid in identification of these species and for epidemiological studies.     

Applying Fourier-transform infrared spectroscopy and chemometrics to the characterization and identification of lactic acid bacteria

The infrared absorption spectra of 55 lactic acid bacteria belonging to the genera Lactobacillus, Weissella and Carnobacterium were obtained and mathematically analyzed. Sixteen reference strains and 39 food strains isolated from meat and meat products and belonging to the genera Lactobacillus (6 species), Weissella (3 species) and Carnobacterium (2 species) were processed under standardized conditions and their medium infrared spectra obtained using Fourier-transform infrared (FT-IR) spectroscopy. Reproducibility indexes and similarities between FT-IR spectra were calculated using modified correlation coefficients to detect the ranges with the best reproducibility and discrimination properties. Hierarchical cluster analysis and stepwise discriminant analysis (SDA) were subsequently carried out to detect classes and create library groups. Reference strains could be distinguished on the basis of their spectral data and their clustering was in agreement with differences in chemical composition of the cell wall. For the 39 food isolates, the capability of two identification systems was compared. Unknown strains were identified (a) using the linear functions obtained from SDA (canonical variables) of the variables that provide the best discrimination of spectra, and (b) by calculating a differentiation index when a range of the unknown's transformed IR spectrum was compared to all spectra included in a reference library. The system based on the differentiation index obtained a higher rate of identification, allowing for detection of outliers. FT-IR spectroscopy is shown to afford additional information to phenotypic and genotypic data which may help to establish a more robust taxonomic classification.     

Postdiagenetic Changes in Kerogen Properties and Type by Bacterial Oxidation and Dehydrogenation

A significant part of organic carbon found on the earth is deposited as fossil organic matter in the lithosphere. The most important reservoir of carbon is shale rocks enriched with organic matter in the form of kerogen created during diagenesis. The purpose of this study was to analyze whether the bacterial communities currently inhabiting the shale rocks have had any impact on the properties and type of kerogen. We used the shale rock located on the Fore-Sudetic Monocline, which is characterized by oil-prone kerogen type II. We were able to show that shale rock inhabited by bacterial communities are characterized by oxidized and dehydrated kerogen type III (gas-prone) and type IV (nonproductive, residual, and hydrogen-free). Bacterial communities inhabiting shale rock were dominated by heterotrophs of the Proteobacteria, Firmicutes, and Actinobacteria phyla. Additionally, we detected a number of protein sequences in the metaproteomes of bacterial communities matched with enzymes involved in the oxidative metabolism of aliphatic and aromatic hydrocarbons, which may potentially contribute to the postdiagenetic oxidation and dehydrogenation of kerogen. The kerogen transformation contributes to the mobilization of fossil carbon in the form of extractable bitumen dominated by oxidized organic compounds.     

Preventive Effects of Anthocyanins from Lycium ruthenicum Murray in High-Fat Diet-Induced Obese Mice Are Related to the Regulation of Intestinal Microbiota and Inhibition of Pancreatic Lipase Activity

Lyciumruthenicum Murray (L. ruthenicum) has been used both as traditional Chinese medicine and food. Recent studies indicated that anthocyanins are the most abundant bioactive compounds in the L. ruthenicum fruits. The purpose of this study was to investigate the preventive effects and the mechanism of the anthocycanins from the fruit of L. ruthenicum (ACN) in high-fat diet-induced obese mice. In total, 24 male C57BL/6J mice were divided into three groups: control group (fed a normal diet), high-fat diet group (fed a high-fat diet, HFD), and HFD +ACN group (fed a high-fat diet and drinking distilled water that contained 0.8% crude extract of ACN). The results showed that ACN could significantly reduce the body weight, inhibit lipid accumulation in liver and white adipose tissue, and lower the serum total cholesterol and low-density lipoprotein cholesterol levels compared to that of mice fed a high-fat diet. 16S rRNA gene sequencing of bacterial DNA demonstrated that ACN prevent obesity by enhancing the diversity of cecal bacterial communities, lowering the Firmicutes-to-Bacteroidota ratio, increasing the genera Akkermansia, and decreasing the genera Faecalibaculum. We also studied the inhibitory effect of ACN on pancreatic lipase. The results showed that ACN has a high affinity for pancreatic lipase and inhibits the activity of pancreatic lipase, with IC50 values of 1.80 (main compound anthocyanin) and 3.03 mg/mL (crude extract), in a competitive way. Furthermore, fluorescence spectroscopy studies showed that ACN can quench the intrinsic fluorescence of pancreatic lipase via a static mechanism. Taken together, these findings suggest that the anthocyanins from L. ruthenicum fruits could have preventive effects in high-fat-diet induced obese mice by regulating the intestinal microbiota and inhibiting the pancreatic lipase activity.     

Molecular characterization,modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein’s structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme’s possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D₃, 25-hydroxyvitamin D₃ and 1α-hydroxyvitamin D₃, in which all substrates docked proximally to the enzyme’s heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D₃ from 1α-hydroxyvitamin D₃. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D₃ to calcitriol, or related metabolic products.     

Identification of potential sources of thymoquinone and related compounds in Asteraceae, Cupressaceae, Lamiaceae, and Ranunculaceae families

In this study, forty-seven plant species belonging to seven families were analysed by GC and GC-MS for the contents of pharmacologically effective quinones: dithymoquinone (DTQ), thymohydroquinone (THQ), and thymoquinone (TQ). The results showed that detectable amounts (??1 mg kg^?1) of at least one of these compounds have been found in three species of both Monarda (M. didyma, M. media, and M. menthifolia) and Thymus (T. pulegioides, T. serpyllum, and T. vulgaris) genera, two Satureja (S. hortensis and S. montana) species, and in single representatives of Eupatorium (E. cannabinum), Juniperus (J. communis), and Nigella (N. sativa) genera. The maximum contents of THQ and TQ were found in M. media aerial parts and M. didyma inflorescences (2674 and 3564 mg kg^?1 of dried weight, respectively) in amounts significantly exceeding their maximum contents in N. sativa seeds (THQ = 530 mg kg^?1 and TQ = 1881 mg kg^?1), which are generally considered as the main natural source of both of these compounds. As a conclusion, M. didyma (bergamot) and M. media (purple bergamot) can be recommended as new prospective natural sources of THQ and TQ for pharmaceutical or food industries.      

Analysis of CRISPR-Cas systems in Gardnerella suggests its potential role in the mechanisms of bacterial vaginosis

Bacterial vaginosis (BV) is the principal cause of vaginal discharge among women, and it can lead to many comorbidities with a negative impact in women’s daily activities. Despite the fact that the pathophysiological process of BV remains unclear, great advances had been achieved in determining consequences of the shift in the vaginal community, and it was defined that Gardnerella spp., plays a key role in the pathogenesis of BV. Interactions of vaginal phage communities and bacterial hosts may be relevant in eubiosis/dysbiosis states, so defense mechanisms in Gardnerella spp., against phage infections could be relevant in BV development. In this study, we analyzed CRISPR-Cas systems among the 13 Gardnerella species recently classified, considering that these systems act as prokaryotic immune systems against phages, plasmids, and other mobile genetic elements. In silico analyses for CRISPR-Cas systems mining over the 81 Gardnerella spp., strains genomes analyzed led to the identification of subtypes I-E and II-C. Spacers analyses showed a hypervariable region across species, providing a high resolution level in order to distinguish clonality in strains, which was supported with phylogenomic analyses based on Virtual Genomic Fingerprinting. Moreover, most of the spacers revealed interactions between Gardnerella spp., strains and prophages over the genus. Furthermore, virulence traits of the 13 species showed insights of potential niche specificity in the vaginal microbiome. Overall, our results suggest that the CRISPR-Cas systems in the genus Gardnerella may play an important role in the mechanisms of the development and maintenance of BV, considering that the Gardnerella species occupies different niches in the vaginal community; in addition, spacer sequences can be used for genotyping studies.     

Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in Nas_F5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.     

Discovery of daspyromycins A and B, 2-aminovinyl-cysteine containing lanthipeptides,through a genomics-based approach

Lanthipeptides are one of the largest groups of ribosomally synthesized and post-translationally modified peptides(RiPPs) and are characterized by the presence of lanthionine(Lan) or methyllanthionine residues(MeLan). Only very few lanthipeptides contain a C-terminal 2-aminovinyl-cysteine(AviCys) motif, but all of them show potent antibacterial activities. Recent advances of genome sequencing led to the rapid accumulation of new biosynthetic gene clusters(BGCs) for lanthipeptides. In this study,...     

Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450-Catalysed Oxidations

P450_cin and P450_cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein–substrate H bonding has been previously proposed as crucial in the selectivity of P450_cin oxidations, but not as essential for P450_cam. To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein–substrate H bond to selectivity in both P450_cin and P450_cam. The H bonding in P450_cam was re-examined using camphane, another substrate for which the protein–substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein–substrate H bond was not a factor in substrate affinity for the enzyme.     

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway

Nonribosomal peptide synthetases (NRPSs) are modular enzymes that use a thiotemplate mechanism to assemble the peptide backbones of structurally diverse and biologically active natural products in bacteria and fungi. Unlike these canonical multi-modular NRPSs, single-module NRPS-like enzymes, which lack the key condensation (C) domain, are rare in bacteria, and have been largely unexplored to date. Here, we report the discovery of a gene cluster (gup) encoding a NRPS-like megasynthetase through genome mining. Heterologous expression of the gup cluster led to the production of two unprecedented alkaloids, guanipiperazines A and B. The NRPS-like enzyme activates two l-tyrosine molecules, reduces them to the corresponding amino aldehydes, and forms an unstable imine product. The subsequent enzymatic reduction affords piperazine, which can be morphed by a P450 monooxygenase into a highly strained compound through C–O bond formation. Further intermolecular oxidative coupling forming the C–C or C–O bond is catalyzed by another P450 enzyme. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.

Genome mining of a NRPS-like gene cluster led to the identification of two novel alkaloids with antimicrobial activity. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.     

Rapid isolation of DNA from Actinomyces

DNA could not be quickly extracted from members of the genus Actinomyces by the usual methods of lysis. Treatment of 7 different actinomyces cells with lysozyme and achromopeptidase, both 5 mg/g wet cells, for 2 h, followed by SDS (0.2 %), proteinase K (5 mg/g wet cells) and EDTA (lmM) for 1 h, lysed the cells. The yield obtained in one day was 337 μg per 200 mg of bacterial cells. The treatment was also found to work effectively on strains belonging to Veillonella, Staphylococcus, Fusobacterium and Bifidobacterium genera.     

Progress in the Study of Bioluminescent Earthworms

Even though bioluminescent oligochaetes rarely catch people's eyes due to their secretive lifestyle, glowing earthworms sighting reports have come from different areas on all continents except Antarctica. A major breakthrough in the research of earthworm bioluminescence occurred in the 1960s with the studies of the North American Diplocardia longa. Comparative studies conducted on 13 earthworm species belonging to six genera showed that N‐isovaleryl‐3‐aminopropanal (Diplocardia luciferin) is the common substrate for bioluminescence in all examined species, while luciferases appeared to be responsible for the color of bioluminescence. The second momentous change in the situation has occurred with the discovery in Siberia (Russia) of two unknown luminous enchytraeids. The two bioluminescent systems belong to different types, have different spectral characteristics and localization, and different temperature and pH optima. They are unique, and this fact is confirmed by the negative results of all possible cross‐reactions. The bioluminescent system of Henlea sp. comprises four essential components: luciferase, luciferin, oxygen and calcium ion. For Friderica heliota, the luminescent reaction requires five components: luciferase, luciferin, ATP, magnesium ion and oxygen. Along with luciferin, more than a dozen analogues were isolated from worm biomass. These novel peptide‐like natural compounds represent an unprecedented chemistry found in terrestrial organisms.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C?H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C?H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Analysis of Rhizonin Biosynthesis Reveals Origin of Pharmacophoric Furylalanine Moieties in Diverse Cyclopeptides

Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3-furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non-ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l -DOPA as Fua precursors and provided the first mechanistic insight. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme-dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.