Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.

A systematically biosynthetic investigation of lactic acid bacteria reveals diverse antagonistic bacteriocins that potentially shape the human microbiome.

BACKGROUND: Lactic acid bacteria (LAB) produce various bioactive secondary metabolites (SMs), which endow LAB with a protective role for the host. However, the biosynthetic potentials of LAB-derived SMs remain elusive, particularly in their diversity, abundance, and distribution in the human microbiome. Thus, it is still unknown to what extent LAB-derived SMs are involved in microbiome homeostasis. RESULTS: Here, we systematically investigate the biosynthetic potential of LAB from 31,977 LAB genomes, identifying 130,051 secondary metabolite biosynthetic gene clusters (BGCs) of 2,849 gene cluster families (GCFs). Most of these GCFs are species-specific or even strain-specific and uncharacterized yet. Analyzing 748 human-associated metagenomes, we gain an insight into the profile of LAB BGCs, which are highly diverse and niche-specific in the human microbiome. We discover that most LAB BGCs may encode bacteriocins with pervasive antagonistic activities predicted by machine learning models, potentially playing protective roles in the human microbiome. Class II bacteriocins, one of the most abundant and diverse LAB SMs, are particularly enriched and predominant in the vaginal microbiome. We utilized metagenomic and metatranscriptomic analyses to guide our discovery of functional class II bacteriocins. Our findings suggest that these antibacterial bacteriocins have the potential to regulate microbial communities in the vagina, thereby contributing to the maintenance of microbiome homeostasis. CONCLUSIONS: Our study systematically investigates LAB biosynthetic potential and their profiles in the human microbiome, linking them to the antagonistic contributions to microbiome homeostasis via omics analysis. These discoveries of the diverse and prevalent antagonistic SMs are expected to stimulate the mechanism study of LAB's protective roles for the microbiome and host, highlighting the potential of LAB and their bacteriocins as therapeutic alternatives. Video Abstract.

Genomic and metabolic analyses reveal antagonistic lanthipeptides in archaea.

BACKGROUND: Microbes produce diverse secondary metabolites (SMs) such as signaling molecules and antimicrobials that mediate microbe-microbe interaction. Archaea, the third domain of life, are a large and diverse group of microbes that not only exist in extreme environments but are abundantly distributed throughout nature. However, our understanding of archaeal SMs lags far behind our knowledge of those in bacteria and eukarya. RESULTS: Guided by genomic and metabolic analysis of archaeal SMs, we discovered two new lanthipeptides with distinct ring topologies from a halophilic archaeon of class Haloarchaea. Of these two lanthipeptides, archalan α exhibited anti-archaeal activities against halophilic archaea, potentially mediating the archaeal antagonistic interactions in the halophilic niche. To our best knowledge, archalan α represents the first lantibiotic and the first anti-archaeal SM from the archaea domain. CONCLUSIONS: Our study investigates the biosynthetic potential of lanthipeptides in archaea, linking lanthipeptides to antagonistic interaction via genomic and metabolic analyses and bioassay. The discovery of these archaeal lanthipeptides is expected to stimulate the experimental study of poorly characterized archaeal chemical biology and highlight the potential of archaea as a new source of bioactive SMs. Video Abstract.

Algibacter onchidii sp. nov., a symbiotic bacterium isolated from a marine invertebrate.

A novel symbiotic bacterium, designated strain XY-114T, was isolated from the cerata of an Onchidium marine invertebrate species collected in the South China Sea. Strain XY-114T was an aerobic, Gram-stain-negative, non-motile and short rod-shaped bacterium (0.5-0.8 µm wide and 1.0-1.5 µm long) without flagellum. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XY-114T belonged to the genus Algibacter with the highest similarity of 97.2 % to the closest phylogenetic relative Algibacter aestuarii KYW371T. Cells grew at 15-37 °C (optimum, 30 °C), at pH 5.5-9.0 (optimum 7.0-8.0) and at NaCl concentrations of 0.5-5.0 % (w/v; optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant respiratory quinone was MK-6. Flexirubin-type pigments were absent. The genome size of strain XY-114T was 3.4 Mbp, with 34.9 mol% of DNA G+C content. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain XY-114T and A. aestuarii KYW371T were 74.5 %, 17.0±1.8 % and 73.9 %. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain XY-114T represents a novel species of the genus Algibacter, for which the name Algibacter onchidii sp. nov. is proposed. The type strain is XY-114T (=KCTC 72217T=MCCC 1K03606T).

Anthraquinones as Potential Antibiofilm Agents Against Methicillin-Resistant Staphylococcus aureus.

Biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) are one of the contributing factors to recurrent nosocomial infection in humans. There is currently no specific treatment targeting on biofilms in clinical trials approved by FDA, and antibiotics remain the primary therapeutic strategy. In this study, two anthraquinone compounds isolated from a rare actinobacterial strain Kitasatospora albolonga R62, 3,8-dihydroxy-l-methylanthraquinon-2-carboxylic acid (1) and 3,6,8-trihydroxy-1-methylanthraquinone-2-carboxylic acid (2), together with their 10 commercial analogs 3-12 were evaluated for antibacterial and antibiofilm activities against MRSA, which led to the discovery of two potential antibiofilm anthraquinone compounds anthraquinone-2-carboxlic acid (6) and rhein (12). The structure-activity relationship analysis of these anthraquinones indicated that the hydroxyl group at the C-2 position of the anthraquinone skeleton played an important role in inhibiting biofilm formation at high concentrations, while the carboxyl group at the same C-2 position had a great influence on the antibacterial activity and biofilm eradication activity. The results of crystal violet and methyl thiazolyl tetrazolium staining assays, as well as scanning electron microscope and confocal scanning laser microscopy imaging of compounds 6 and 12 treatment groups showed that both compounds could disrupt preformed MRSA biofilms possibly by killing or dispersing biofilm cells. RNA-Seq was subsequently used for the preliminary elucidation of the mechanism of biofilm eradication, and the results showed upregulation of phosphate transport-related genes in the overlapping differentially expressed genes of both compound treatment groups. Herein, we propose that anthraquinone compounds 6 and 12 could be considered promising candidates for the development of antibiofilm agents.

Muricauda onchidii sp. nov., isolated from a marine invertebrate from South China Sea, and transfers of Flagellimonas algicola, Flagellimonas pacifica and Flagellimonas maritima to Muricauda algicola comb. nov., Muricauda parva nom. nov. and Muricauda aurantiaca nom. nov., respectively, and emended description of the genus Muricauda.

An aerobic, Gram-stain-negative, rod-shaped and non-motile strain (XY-359T) was isolated from the mouth of a marine invertebrate Onchidium species from the South China Sea. It grew at pH 6.0-8.5 (optimum, pH 7.5), at 15-37 °C (optimum, 30 °C) and in the presence of 0.5-4.5 % (w/v) NaCl (optimum, 2.5 %). It could not hydrolyse Tweens 20, 40, 60 or 80 and no flexirubin-type pigments were produced. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, six unidentified phospholipids and two unidentified polar lipids. The major fatty acids were iso-C17:0 3-OH, iso-C15:1 G and iso-C15:0 3-OH. The respiratory quinone was MK-6. Strain XY-359T showed the greatest degree of 16S rRNA sequence similarity to Flagellimonas algicola AsT0115T (96.54 %), followed by Muricauda flava DSM 22638T (96.27 %). Phylogenetic analysis based on 16S rRNA gene sequences and 31 core genes indicated that strain XY-359T belongs to the genus Muricauda. The genome size of strain XY-359T was 4 207 872 bp, with 39.1 mol% of DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between strain XY-359T and F. algicola AsT0115T were 74.58 % and 18.5 %, respectively, and those between strain XY-359T and M. flava DSM 22638T were 74.2 % and 18.3 %. The combined phenotypic, chemotaxonomic and phylogenetic data suggest that strain XY-359T represents a novel species of the genus Muricauda, for which the name Muricauda onchidii sp. nov. is proposed. The type strain is XY-359T (=MCCC 1K03658T =KCTC 72218T). Moreover, based on the proposal of nesting Spongiibacterium and Flagellimonas within Muricauda by García (Validation List No. 193) and the analyses of phylogenetic trees and average amino acid identities in this study, the transfers of F. algicola, F. pacifica and F. maritima to the genus Muricauda as Muricauda algicola comb. nov., Muricauda parva nom. nov. and M. aurantiaca nom. nov., respectively, are proposed, with an emended description of the genus Muricauda.

Algoriphagus kandeliae sp. nov., isolated from mangrove rhizosphere soil.

Strain XY-J91T, a Gram-stain-negative, reddish orange, non-spore-forming and short-rod-shaped marine bacterium, was isolated from rhizosphere soil of the mangrove plant Kandelia candel (L.) Druce in Mai Po Nature Reserve, Hong Kong. The strain showed growth at 15-50 °C (optimum 40 °C), at pH 5.5-9.5 (optimum 7.0-8.0) and with 0-8 % (w/v) NaCl (optimum 1-2 %). The only respiratory quinone was MK-7 and the major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and an unidentified phospholipid. The G+C content of strain XY-J91T was 40.4 mol%. Strain XY-J91T exhibited highest 16S rRNA gene sequence similarities to the type strains of Algoriphagus marincola SW-2T (96.66 %), Algoriphagus taiwanensis CC-PR-82T (96.21%), Algoriphagus ornithinivorans JC2052T (96.16%), Algoriphagus confluentis HJM-2T (95.73%) and Algoriphagus zhangzhouensis 12C11T (95.52 %). Based on the phylogenetic, phenotypic and chemotaxonomic evidence presented, strain XY-J91T represents a novel species of the genus Algoriphagus, for which the name Algoriphagus kandeliae sp. nov. is proposed. The type strain is XY-J91T (=MCCC 1K03612T=KCTC 72216T).

Pseudooceanicola onchidii sp. nov., isolated from a marine invertebrate from the South China Sea.

A novel bacterial strain, XY-99T, was isolated from the epidermis of a marine invertebrate of the genus Onchidium from seawater of the South China Sea. The cells of the strain were aerobic, Gram-stain-negative, non-motile, and oval-shaped (0.8-1.0 µm wide and 1.0-1.5 µm long) without a flagellum. The strain grew at temperatures of 15-37 °C (optimum, 35-37 °C), at pH 5.5-9.5 (optimum 7.5), and at NaCl concentrations of 0-12.0 % (w/v) (optimum 1.5-3.0 %). The major fatty acids (>10 %) were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and 11-methyl C18 : 1ω7c. The predominant polar lipid was diphosphatidylglycerol. The respiratory quinone was Q-10. The closet phylogenetic neighbours were Pseudooceanicola flagellatus DY470T and Pseudooceanicola nitratireducens JLT1210T, showing 97.5 and 97.3 % of 16 s rRNA gene sequence similarity. The genome size of XY-99T was 3 673 499 bp, with 64.5 % DNA G+C content. The average nucleotide identity and digital DNA-DNA hybridization values between XY-99T and Pseudooceanicola flagellatus DY470T were 72.8 and 14.0 %, respectively, while they were 79.3 and 22.3 % between XY-99T and Pseudooceanicola nitratireducens JLT1210T. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that XY-99T represents a novel species of the genus Pseudooceanicola, for which the name Pseudooceanicola onchidii sp. nov. is proposed. The type strain is XY-99T (=KCTC 72211T=MCCC 1K03607T).

Euzebya rosea sp. nov., a rare actinobacterium isolated from the East China Sea and analysis of two genome sequences in the genus Euzebya.

Rare Actinobacteria, known as non-Streptomyces, hold great potential to produce new bioactive compounds for drug development. A strain designated DSW09T, which belongs those rare Actinobacteria, was isolated from surface seawater of the East China Sea. The cells were aerobic, Gram-positive, non-motile, non-spore-forming and rod-shaped (0.4 µm wide and 1.5-4.0 µm long). The closest relative was Euzebya tangerina F10T (96.46 % of 16S rRNA gene similarity). Cell growth occurred at 15-45 °C (optimum, 25-30 °C), at pH 6.0-9.0 (pH 6.0-7.0) and at NaCl concentrations of 0.5-5.0 % (w/v; 1.0-4.0 %). The major cellular fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C15 : 0iso 2OH), C17 : 1ω8c and C16 : 0. The predominant polar lipid was diphosphatidylglycerol. The predominant menaquinone was MK-9(H4). The cell-wall peptidoglycan was A1 γ-type, containing meso-DPA. The major cell-wall sugars were rhamnose and ribose. The genome size was 5 509 297 bp with a 71.29 mol% G+C content for strain DSW09T, while 4 781 440 bp with a 68.87 mol% G+C content for E. tangerina F10T. The average nucleotide identity and digital DNA-DNA hybridization values between strain DSW09T and E. tangerina F10T were 73.44 % and 16.43 %, respectively. Based on phylogenetic, phenotypic, chemotaxonomic evidence and genomic analyses, strain DSW09T is a novel species of genus Euzebya, for which the name Euzebya rosea sp. nov. is proposed. The type strain is DSW09T (=DMS 104446T=MCCC 1K03290T).

Kandeliimicrobium roseum gen. nov., sp. nov., a new member of the family Rhodobacteraceae isolated from mangrove rhizosphere soil.

A Gram-stain-negative, non-flagellated, short rod-shaped bacterium, designated XY-R6T, was isolated from the rhizosphere soil of a mangrove plant, Kandelia candel (L.) Druce, in Mai Po Nature Reserve, Hong Kong. Growth of strain XY-R6T was observed at pH 5.0-9.5 (optimum 6.5-8.0), between 8 and 42 °C (optimum 28-34 °C), and in the presence of 0-9.5 % (w/v) NaCl (optimum 1-4 %). The predominant isoprenoid quinone was ubiquinone-10. The major fatty acids were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) (55.61 %), C19 : 0cycloω8c (21.59 %) and C16 : 0 (11.24 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, aminolipid, phosphatidylcholine and diphosphatidylglycerol. The genomic DNA G+C content of strain XY-R6T was 69.3 mol%. Phylogenetic analyses, based on 16S rRNA gene sequences, revealed that strain XY-R6T belonged to the family Rhodobacteraceae of the class Alphaproteobacteria and formed a distinct lineage, showing the highest gene sequence similarities to the members of genus Wenxinia(94.5-94.3 %), followed by the genera Profundibacterium (94.3 %), Defluviimonas(93.8-92.5 %), Oceanicola (93.8 %) and Cereibacter (93.7 %). Similarities to other genera within the family Rhodobacteraceae were below 94.0 %. Based on comprehensive phenotypic, phylogenetic and chemotaxonomic characterization, it is indicated that strain XY-R6T represents a novel species of a new genus in the family Rhodobacteraceae, for which the name Kandeliimicrobium roseum gen. nov., sp. nov. is proposed, with XY-R6T (=MCCC 1K01498T=KCTC 52266T=DSM 104294T) as the type strain.