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

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide natural products characterized by the presence of lanthionine and methyllanthionine cross-linked amino acids formed by dehydration of Ser/Thr residues followed by conjugate addition of Cys to the resulting dehydroamino acids. Class I lanthipeptide dehydratases utilize glutamyl-tRNAGlu as a cosubstrate to glutamylate Ser/Thr followed by glutamate elimination. A vast majority of lanthipeptides identified from class I synthase systems have been from Gram-positive bacteria. Herein, we report the heterologous expression and modification in Escherichia coli of two lanthipeptides from the Gram-negative Bacteroidetes Pedobacter lusitanus NL19. These peptides are representative of a group of compounds frequently encoded in Pedobacter genomes. Structural characterization of the lanthipeptides revealed a novel ring pattern as well as an unusual ll-lanthionine stereochemical configuration and a cyclase that lacks the canonical zinc ligands found in most LanC enzymes.

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

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

Feeding on fungi: genomic and proteomic analysis of the enzymatic machinery of bacteria decomposing fungal biomass.

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community.

Pedobacter schmidteae sp. nov., a new bacterium isolated from the microbiota of the planarian Schmidtea mediterranea.

Pedobacter schmidteae sp. nov. strain EGT (Collection de Souches de l'Unité des Rickettsie CSUR P6417 = Colección Española de Cultivos Tipo CECT 9771) is a new Pedobacter species isolated from the planarian Schmidtea mediterranea. Schmidtea mediterranea are flatworms living in freshwater and exhibiting an unusual ability to regenerate amputated parts. To date, the gut microbiota of Schmidtea mediterranea remains poorly studied. Here, via the culturomics strategy that consists in using diversified culture conditions, we isolated a new bacterium, strain EG, that we characterized using the taxono-genomics approach that combines phenotypic assays and genome sequencing and analysis. Strain EG exhibits a 16S rRNA sequence similarity of 98.29% with Pedobacter nyackensis strain NWG-II14T, its closest neighbour with standing in nomenclature. It is an aerobic bacterium belonging to the family Sphingobacteriaceae. Colonies are small, round, smooth and transparent. Bacterial cells are Gram-negative, rod-shaped, motile and non-spore-forming bacilli with positive catalase and oxidase activities. The genome sequence is 6,198,518 bp-long with a G + C content of 41.13%, and the Ortho-ANI and dDDH values when compared to P. nyackensis are 77.34% and 21.50%, respectively. Strain EGT exhibits unique characteristics that classify it as the type strain of new bacterial species for which we propose the name Pedobacter schmidteae sp. nov.

Characterization and antifungal activity of chitosanase produced by Pedobacter sp. PR-M6.

To investigate the temperature requirements of chitosanase activity, as well as the degradation patterns generated by enzyme-induced chitosan oligomer hydrolysis, Pedobacter sp. PR-M6 was inoculated onto 0.5% colloidal chitosan medium agar plates. Cell growth was higher at 30 °C than at 20 °C during the initial 2 days of incubation. The protein content rapidly increased on day 1 at both temperatures and then it slowly increased at 20 °C and slowly decreased at 30 °C during the following 5 days of incubation. In order to characterize the electrophoretic pattern, Pedobacter sp. PR-M6 was cultured in 1% powder chitosan medium at 20 °C and 30 °C for 5 days after incubation and analyzed by SDS-PAGE. Four bands were visible, corresponding to ct1 (25 kDa), ct2 (17 kDa), ct3 (15 kDa), and ct4 (14 kDa), at both 20 °C and 30 °C. The optimal conditions for the activity of chitosanase produced from Pedobacter sp. PR-M6 were 60 °C and 1.81 enzyme units/mg protein. Two major isozyme bands (ct3 and ct4) exhibited their strongest chitosanase activity at 50 °C in SDS-PAGE gel. The reaction products generated from (GlcN)2-(GlcN)5 substrates at 60 °C after a 1 h incubation were investigated by thin-layer chromatography. Low-molecular weight chitosan and oligochitosan (LCOC) and soluble chitosan showed antifungal activity against A. brassicicola, B. cinerea, F. solani, and R. solani. LCOC exhibited higher antifungal activity than soluble chitosan. Moreover, LCOC treatments (500 ppm and 1000 ppm) inhibited conidia germination in A. brassicicola.

High-level expression and characterization of a new κ-carrageenase from marine bacterium Pedobacter hainanensis NJ-02.

A novel κ-carrageenase gene (CgkB) has been cloned from Pedobacter hainanensis NJ-02 and expressed heterologously in Escherichia coli BL21 (DE3). It consisted of 1935 bp and encoded 644 amino acid residues with a molecular weight of 71·61 kDa. The recombinant enzyme showed maximal activity of 2458 U mg-1 at 40°C and pH 8·0. Additionally, it could retain more than 70% of its maximal activity after being incubated at pH of 5·5-10·0 below 40°C. K+ and a broad range of NaCl can activate the enzyme. The Km and Vmax of CgkB was 2·4 mg ml-1 and 126 mmol mg-1  min-1 . The ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the carrageenan into tetrasaccharides and hexasaccharides. The results indicated that the enzyme with high activity could be a valuable enzyme tool to produce carrageenan oligosaccharides with various activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymatic preparation of carrageenan oligosaccharides has drawn increased attention due to their various physiological activities. It is urgent to explore enzyme tools with higher activity and better stability. In this work, a novel κ-carrageenase was identified and characterized from marine bacterium Pedobacter hainanensis NJ-02. The enzyme with high activity could be a valuable tool to produce carrageenan oligosaccharides with various activities.

Pedobacter panacis sp. nov., isolated from Panax ginseng soil.

A novel strain, DCY108T was isolated from soil of a Panax ginseng field, Yeoncheon province (38°04'N 126°57'E), Republic of Korea. Strain DCY108T is Gram-negative, non-motile, non-flagellate, rod-shaped, and aerobic. The bacterium grows optimally at 25-30 °C, pH 6.5-7.0 and 1 % NaCl. Phylogenetically, strain DCY108T is closely related to Pedobacter jejuensis JCM 18824T, Pedobacter aquatilis JCM 13454T, Pedobacter kyungheensis LMG 26577T and the type strain of the genus Pedobacter heparinus DSM 2366T. The DNA-DNA relatedness values between strain DCY108T and its close phylogenetic neighbors were below 30.0 %. The DNA G+C content of strain DCY108T was determined to be 45.1 mol%. The predominant quinone was menaquinone 7 (MK-7). The major polar lipids were identified as phosphatidylethanolamine and three unidentified aminolipids AL1, AL13 and AL17. Iso-C15:00, iso-C17:03OH and summed feature 3 (C16:1 ω7c/C16:1 ω6c) were identified as the major fatty acids present in strain DCY108T. The results of physiological and biochemical tests allowed strain DCY108T to be differentiated phenotypically from other recognized species belonging to the genus Pedobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Pedobacter panacis sp. nov is proposed with the type strain designated as DCY108T (=CCTCCAB 2015196T = KCTC 42748T).

Production of a low molecular weight heparin using recombinant glycuronidase [corrected].

The Δ4,5 unsaturated uronate (4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid) residue is produced through the depolymerization of heparin, heparosan, and heparan sulfate with heparin lyases. The recovery of unsaturated uronate containing products is necessary to prepare low molecular weight heparin (LMWH) from heparin or heparosan. In this study, the gene of Δ4,5 and Δ4,5(Δ20) unsaturated glycuronidase (EC# 3.2.1.56) from Pedobacter heparinus (formerly Flavobacterium heparinum) was cloned into pMAL-c2x plasmid. Its fusion protein with MBP was expressed in Escherichia coli TB1. After purification, Δ4,5 unsaturated glycuronidase was evaluated. The Δ4,5(Δ20) glycuronidase showed excellent activity on the unsaturated bonds of the different depolymerized products from Hep I, Hep II, and Hep III on heparin, heparosan, and heparan sulfate.

Pedobacter kyungheensis sp. nov., with ginsenoside converting activity.

The Gram-negative, aerobic, non-motile, non-spore forming, and rod-shaped bacterium designated as THG-T17(T) was isolated from the soil of a ginseng field of Pocheon in Korea, and its taxonomic position was investigated by using a polyphasic approach. The growth of strain THG-T17(T) occurred at 4-40°C and pH 4.0-9.0 with 1-2% (w/v) NaCl on nutrient agar. Strain THG-T17(T) displayed ß-glucosidase activity that was responsible for its ability to transform ginsenoside Rb(1) (one of the dominant ginsenosides of ginseng) to compound F2 via gypenoside XVII. On the basis of 16S rRNA gene sequence similarity, strain THG-T17(T) was shown to belong to the genus Pedobacter and was related to Pedobacter soli 15-51(T) (98.8%), Pedobacter sandarakinus DS-27(T) (98.0%) and Pedobacter terrae DS-57(T) (98.1%). The G+C content of the genomic DNA was 42.4 mol%. The DNA-DNA relatedness values between strain THG-T17(T) and its phylogenetically closest neighbors were below 14%. Phenotypic and chemotaxonomic data, especially analysis of cellular fatty acid, supported the affiliation of strain THG-T17(T) to the genus Pedobacter. The results of genotyping and biochemical tests showed strain THG-T17(T) to be differentiated genotypically and phenotypically from the recognized species of the genus Pedobacter. Therefore, the novel isolate represents a novel species, for which the name Pedobacter kyungheensis sp. nov. is proposed, with the type strain THG-T17(T) (=KACC 16221(T) = LMG 26577(T)).

Antioxidant capacity of novel pigments from an Antarctic bacterium.

In Antarctica microorganisms are exposed to several conditions that trigger the generation of reactive oxygen species, such as high UV radiation. Under these conditions they must have an important antioxidant defense system in order to prevent oxidative damage. One of these defenses are pigments which are part of the non-enzymatic antioxidant mechanisms. In this work we focused on the antioxidant capacity of pigments from an Antarctic microorganism belonging to Pedobacter genus. This microorganism produces different types of pigments which belong to the carotenoids group. The antioxidant capacity of a mix of pigments was analyzed by three different methods: 1,1-diphenyl-2-picrylhydrazyl, ROS detection and oxygen electrode. The results obtained from these approaches indicate that the mix of pigments has a strong antioxidant capacity. The oxidative damage induced by UVB exposure to liposomes was also analyzed. Intercalated pigments within the liposomes improved its resistance to lipid peroxidation. Based on the analysis carried out along this research we conclude that the antioxidant properties of the mix of pigments protect this bacterium against oxidative damage. These properties make this mix of pigments a powerful antioxidant mixture with potential biotechnological applications.

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.

Pedobacter rhizosphaerae sp. nov. and Pedobacter soli sp. nov., isolated from rhizosphere soil of Chinese cabbage (Brassica campestris).

Two bacterial strains, 01-96(T) and 15-51(T), isolated from rhizosphere soil of Chinese cabbage (Brassica campestris) were characterized by using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain 01-96(T) was phylogenetically related most closely to Pedobacter suwonensis 15-52(T) and Pedobacter roseus CL-GP80(T) (96.7 and 96.7% similarity, respectively), and strain 15-51(T) to Pedobacter borealis G-1(T) and P. suwonensis 15-52(T) (97.2 and 97.1%, respectively). However, levels of DNA-DNA relatedness between strain 15-51(T) and P. borealis KACC 14287(T) and P. suwonensis KACC 11317(T) were low (<50%). The G+C content of the genomic DNA was 37.8 mol% for strain 01-96(T) and 38.6 mol% for strain 15-51(T). The major fatty acids of the two strains were iso-C(17:0) 3-OH, iso-C(15:0) and summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)ω7c). The results of DNA-DNA hybridization and phenotypic data showed that strains 01-96(T) and 15-51(T) could be distinguished from their closest phylogenetic relatives, and that these strains represented two novel species of the genus Pedobacter, for which the names Pedobacter rhizosphaerae sp. nov. (type strain 01-96(T) =KACC 14938(T) =NBRC 107690(T)) and Pedobacter soli sp. nov. (type strain 15-51(T) =KACC 14939(T) =NBRC 107691(T)) are proposed.