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.

Production of cadmium sulfide quantum dots by the lithobiontic Antarctic strain Pedobacter sp. UYP1 and their application as photosensitizer in solar cells.

BACKGROUND: Microbes are present in almost every environment on Earth, even in those with extreme environmental conditions such as Antarctica, where rocks may represent the main refuge for life. Lithobiontic communities are composed of microorganisms capable of colonizing rocks and, as it is a not so well studied bacterial community, they may represent a very interesting source of diversity and functional traits with potential for biotechnological applications. In this work we analyzed the ability of Antarctic lithobiontic bacterium to synthesize cadmium sulfide quantum dots (CdS QDs) and their potential application in solar cells. RESULTS: A basaltic andesite rock sample was collected from Fildes Peninsula, King George Island, Antarctica, and processed in order to isolate lithobiontic bacterial strains. Out of the 11 selected isolates, strain UYP1, identified as Pedobacter, was chosen for further characterization and analysis due to its high cadmium tolerance. A protocol for the biosynthesis of CdS QDs was developed and optimized for this strain. After 20 and 80 min of synthesis, yellow-green and orange-red fluorescent emissions were observed under UV light, respectively. QDs were characterized through spectroscopic techniques, dynamic light scattering analysis, high-resolution transmission electron microscopy and energy dispersive x-ray spectroscopy. Nanostructures of 3.07 nm, composed of 51.1% cadmium and 48.9% sulfide were obtained and further used as photosensitizer material in solar cells. These solar cells were able to conduct electrons and displayed an open circuit voltage of 162 mV, a short circuit current density of 0.0110 mA cm-2, and had an efficiency of conversion up to 0.0016%, which is comparable with data previously reported for solar cells sensitized with biologically produced quantum dots. CONCLUSIONS: We report a cheap, rapid and eco-friendly protocol for the production of CdS QDs by an Antarctic lithobiontic bacterium, Pedobacter, a genus that was not previously reported as a quantum dot producer. The application of the biosynthesized QDs as sensitizer material in solar cells was validated.

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.

Isopedopeptins A-H: Cationic Cyclic Lipodepsipeptides from Pedobacter cryoconitis UP508 Targeting WHO Top-Priority Carbapenem-Resistant Bacteria.

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 µg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 µg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

Bacteroidetes can be a rich source of novel lanthipeptides: The case study of Pedobacter lusitanus.

Lanthipeptides are intriguing peptides known since 1928, the year of penicillin's discovery. At that time, they were known as lantibiotics due to their (methyl)lanthionine amino acids and antibacterial activity. Their body of knowledge expanded tremendously over the last few years. Our analysis reveals that Bacteroidetes has a high state of clusters encoding the biosynthesis of class I lanthipeptides. We show that some strains of Pedobacter have a number of LanBs/genome comparable to that of some Actinobacteria. The case study selected was Pedobacter lusitanus NL19. Its clusters identified encode LanBs associated with LanCs as well as orphan LanBs. The first are concomitant with LanT transporters typical of class II lanthipeptides (and not class I), making their clusters into a hybrid class I and class II type. So far, this kind of operon was described only once and is involved in the production of pinensins, the first lanthipeptide with antifungal activity. A particular feature of pinensins is their splitted LanBs and we found that these enzymes are also widely encoded in Bacteroides. The function of a high percentage of proteins predicted to play a role in the production of Pedobacter lanthipeptides is unknown. Other major fraction of these proteins is expected to be enrolled in signal-transduction pathways. We demonstrate that the occurrence of lanthipeptides clusters in the genomes of Gram-negative bacteria is higher than previously reported. More importantly, we show that their genetic background is highly diverse, which is an undeniable foreshadowing of novel peptide structures, biochemistry and biological function.

Pedobacter pollutisoli sp. nov., Isolated from Tetrabromobisphenol A-Contaminated Soil.

A Gram-stain negative, strictly aerobic, non-spore forming, non-motile, rod-shaped bacterium, designated TBBPA-24T, was isolated from tetrabromobisphenol A-contaminated soil in China. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain TBBPA-24T was most closely related to Pedobacter nanyangensis Q4T (96.5%) and Pedobacter 'zeaxanthinifaciens' TDMA-5T (96.1%). Chemotaxonomic analysis revealed that strain TBBPA-24T possessed MK-7 as the major respiratory quinone and lipid, aminolipid, phospholipid, phosphatidylethanolamine, and phosphoaminolipid as the major polar lipid. The major fatty acids were iso-C15:0 (40.2%), summed feature 3 (C16:1ω6c and/or C16:1ω7c, 25.6%) and iso-C17:0 3-OH (16.4%). The genomic DNA G+C content of strain TBBPA-24T was 43.9 mol%. Based on the phylogenetic, phenotypic characteristics, and chemotaxonomic data, strain TBBPA-24T is considered a novel species of the genus Pedobacter, for which the name Pedobacter pollutisoli sp. nov. is proposed. The type strain TBBPA-24T (= KCTC 62314T = CCTCC AB 2017244T) is proposed.

Pedobacter aquicola sp. nov., isolated from freshwater.

A non-motile, pink-pigmented bacterial strain designated IMCC25679T, was isolated from freshwater Lake Chungju of Korea. Phylogenetic trees based on 16S rRNA gene sequences showed that the strain IMCC25679T formed a lineage within the genus Pedobacter. The strain IMCC25679T was closely related to Pedobacter daechungensis Dae 13T (96.4% sequence similarity), Pedobacter rivuli HME8457T (95.3%) and Pedobacter lentus DS-40T (94.3%). The major fatty acids of IMCC- 25679T were iso-C15:0, iso-C16:0 and summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c). The major respiratory quinone was MK-7. The major polar lipids were phosphatidylethanolamine (PE), an unidentified sphingolipid (SL), an unidentified aminolipid (AL) and three unidentified polar lipids (PL). The DNA G + C content of IMCC25679T was 32.2 mol%. Based on the evidence presented in this study, the strain IMCC25679T represents a novel species within the genus Pedobacter, with the proposed name Pedobacter aquicola, sp. nov. The type strain is IMCC25679T (= KACC 19486T = NBRC113131T).

Chemical characterization and surface properties of a new bioemulsifier produced by Pedobacter sp. strain MCC-Z.

A novel biopolymer was described in the form of an extracellular polysaccharide (EPS) by Pedobacter sp. strain MCC-Z, a member of a bacterial genus not previously described as an emulsifier producer. The new biomolecule was extracted, purified and characterized, and its surface and emulsifying properties were evaluated. The purified bioemulsifier, named Pdb-Z, showed high emulsifying activity (E24% = 64%) and reduced the surface tension of water up to 41 mN/m with a critical micelle concentration value of 2.6 mg/mL. The chemical characterization of Pdb-Z was performed using (1)H NMR, FT-IR, HPLC/MS/MS and GC/MS. Pdb-Z was found to contain 67% of carbohydrates, consisting mainly of galactose and minor quantities of talose, 30% of lipids, being pentadecanoic acid the major lipidic constituent, and 3% of proteins. The bioemulsifier was a glycolipids-protein complex with an estimated molecular mass of 10(6)Da. Furthermore, Pdb-Z emulsified pure aliphatic and aromatic hydrocarbons as well as diesel more efficiently than commercial synthetic surfactants, used for comparison. Our results suggest Pdb-Z has interesting properties for applications in remediation of hydrocarbon-contaminated environments and bioremediation processes.

Electrospray ionization mass spectrometric analysis of κ-carrageenan oligosaccharides obtained by degradation with κ-carrageenase from Pedobacter hainanensis.

κ-Carrageenan was degraded with a novel κ-carrageenase isolated from Pedobacter hainanensis, which was first isolated from seaside soil under the stacks of red algae in Hainan province of China. The κ-carrageenase was detected with a molecular weight of ∼55 kDa estimated from SDS-PAGE and yielded enzymatic activity of 700.53 units/mg of protein under the conditions of pH 7.0 and 40 °C. Analysis of the degradation products by TLC and HPLC indicated that the enzyme degraded κ-carrageenan to sulfated oligosaccharides with even-numbered degree of polymerization, of which the tetrasaccharide was the major product. All the degradation components during different time courses were analyzed by ESI-MS, and their structures were assigned. Structural analysis by CID MS/MS revealed that each carrageenan oligosaccharide was composed of An-G4S-type neocarrabiose units, which consisted of a 3,6-anhydro-α-d-galactose (An) residue in the nonreducing end and a ß-d-galactose-4-sulfate (G4S) residue in the reducing end. These results demonstrated that the κ-carrageenase cleaved κ-carrageenan at the internal ß-1,4 linkage of κ-carrageenan. This enzymatic degradation offers an alternative approach to prepare κ-carrageenan oligosaccharides, which could be used as a powerful tool for further study on biological activity-structure relationship and thorough industrial exploitation of κ-carrageenan.

Screening and biological activities of pedopeptins, novel inhibitors of LPS produced by soil bacteria.

Lipopolysaccharide (LPS) is a strong endotoxin and is delivered to the cell surface signaling receptor, Toll-like receptor 4 and MD-2 complex, via soluble cluster of differentiation (CD) 14 or membranous CD14, resulting in the induction of the inflammatory response. To obtain new compounds that block LPS binding to CD14, we designed a high-throughput screening based on time-resolved intermolecular fluorescence resonance energy transfer. This cell-free screening system successfully led to the discovery of novel inhibitors of LPS-CD14 interaction from the library of the secondary metabolites of microorganisms. We identified the novel compounds pedopeptin A, B and C from a culture broth of Pedobacter sp. SANK 72003. Pedopeptins blocked LPS binding to CD14 in vitro with IC50 values of 20, 11 and 47 nM, respectively, and also inhibited LPS binding to the cells expressing CD14, leading to the suppression of cytokine production. Moreover, they showed antimicrobial activities against Escherichia coli with minimum inhibitory concentration ranging from 2 to 4 µg ml(-1).

Pedopeptins, novel inhibitors of LPS: taxonomy of producing organism, fermentation, isolation, physicochemical properties and structural elucidation.

In the course of our screening for inhibitors of lipopolysaccharide (LPS) binding to cellular receptor CD14, potent inhibitory activity was detected in the cultured broth of Pedobacter sp. SANK 72003. Three active compounds, pedopeptin A, B and C, were isolated from the broth and their structures were elucidated by physicochemical and spectral data to be new cyclic depsipeptides.

Pedobacter hainanensis sp. nov., isolated from seaside soil.

A Gram-negative, aerobic, rod-shaped, motile, non-spore-forming bacterial strain, designated 13-Q(T), was isolated from seaside soil under the stacks of the red algae in Hainan province in China. Identification was carried out on the basis of polyphasic taxonomy. Phylogenetic analysis of 16S rRNA gene sequences showed that strain 13-Q(T) belonged to the genus Pedobacter, and the highest similarity was 94.4 % with Pedobacter terricola KCTC 12876(T). Strain 13-Q(T) was able to grow at 10-40 °C, in pH 5.0-10.0, in the presence of 0-2.0 % NaCl. The major fatty acids were iso-C(15:0) (40.4 %), summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) ω7c) (18.9 %) and iso-C(17:0) 3-OH (18.4 %). The predominant menaquinone was MK-7. The G+C content of the genomic DNA was 42.7 mol%. Strain 13-Q(T) could be distinguished from the nearest phylogenetic neighbors by various chemotaxonomic and phenotypic properties. The results of the polyphasic analyses suggested that strain 13-Q(T) should be considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter hainanensis sp. nov. is proposed. The type strain is 13-Q(T) (=CCTCC AB 2012076(T) = NRRL B-59850(T)).

Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow.

Organisms without a sequenced genome and lacking a complete protein database encounter an added level of complexity to protein identification and quantitation. De novo sequencing, new bioinformatics tools, and mass spectrometry (MS) techniques allow for advances in this area. Here, the proteomic characterization of an unsequenced psychrophilic bacterium, Pedobacter cryoconitis, is presented employing a novel workflow based on (15) N metabolic labelling, 2DE, MS/MS, and bioinformatics tools. Two bioinformatics pipelines, based on nitrogen constraint (N-constraint), ortholog searching, and de novo peptide sequencing with N-constraint similarity database search, are compared based on proteome coverage and throughput. Results demonstrate the effect of different growth temperatures (1°C, 20°C) and different carbon sources (glucose, maltose) on the proteome. Seventy-six and 69 proteins were identified and validated from the glucose- and maltose-grown bacterium, respectively, from which 21 and 22 were differentially expressed at different growth temperatures. Differentially expressed proteins are involved in stress response and carbohydrate metabolism, with higher expression at 20°C than at 1°C, while antioxidants were upregulated at 1°C. This study provides an alternative workflow to identify, validate, and quantify proteins from unsequenced organisms distantly related to other species in the protein database. Furthermore, it provides further understanding on bacterial adaptation mechanisms to cold environments, and a comparative proteomic analyses with other psychrophilic microorganisms.