Lasso Peptides Research Articles

Unexpected Methyllanthionine Stereochemistry in the Morphogenetic Lanthipeptide SapT.

Lanthipeptides are polycyclic peptides characterized by the presence of lanthionine (Lan) and/or methyllanthionine (MeLan). They are members of the ribosomally synthesized and post-translationally modified peptides (RiPPs). The stereochemical configuration of (Me)Lan cross-links is important for the bioactivity of lanthipeptides. To date, MeLan residues in characterized lanthipeptides have either the 2S,3S or 2R,3R stereochemistry. Herein, we reconstituted in Escherichia coli the biosynthetic pathway toward SapT, a class I lanthipeptide that exhibits morphogenetic activity. Through the synthesis of standards, the heterologously produced peptide was shown to possess three MeLan residues with the 2S,3R stereochemistry (d-allo-l-MeLan), the first time such stereochemistry has been observed in a lanthipeptide. Bioinformatic analysis of the biosynthetic enzymes suggests this stereochemistry may also be present in other lanthipeptides. Analysis of another gene cluster in Streptomyces coelicolor that is widespread in actinobacteria confirmed another example of d-allo-l-MeLan and verified the bioinformatic prediction. We propose a mechanism for the origin of the unexpected stereochemistry and provide support using site-directed mutagenesis.

Tandem repeats in precursor protein stabilize transcript levels and production levels of the fungal ribosomally synthesized and post-translationally modified peptide ustiloxin B

Ustiloxin B is a ribosomally synthesized and post-translationally modified peptide (RiPP) first reported in Ascomycetes. Its biosynthetic pathway was recently identified in the filamentous fungus Aspergillus flavus. The precursor protein of ustiloxin B, UstA, has a signal peptide to the endoplasmic reticulum at its N-terminal and a subsequent tandemly highly repeated segment cleaved at Lys-Arg dipeptides by Kex2 protease; such proteins are called Kex2-processed repeat proteins (KEPs). RiPP biosynthetic pathways using KEPs as precursor proteins are widely distributed in the Fungi kingdom, with high diversity of precursor protein sequences. UstA in A. flavus has a 16-fold tandemly repeated segment containing the core peptide Tyr-Ala-Ile-Gly, which forms the ustiloxin B backbone structure, but it is unknown why such a costly-to-maintain highly repeated sequence is retained. Here, we replaced ustA, the gene encoding the ustiloxin B precursor protein, with synthetic genes encoding 1-, 3-, 5-, 7-, and 11-fold tandem-repeat segments in A. flavus, to investigate the relationship between the repeat number and ustiloxin B production. Ustiloxin B production increased quadratically with increasing repeat number in ustA variants, although it dropped in a previously constructed ustA variant that had a substituted synthetic gene encoding a 16-fold repeat segment probably because of the presence of the many rare codons in the sequence. We also examined the transcript levels of substituted synthetic genes in ustA variants, and surprisingly we found that the transcript levels of the synthetic genes increased linearly with increasing repeat number. This result implies that an unknown mechanism stabilizes ustA transcripts via the highly repeated structure in a feedback manner. We also constructed a transformant without the intron in native ustA, but no effect of intron removal was observed on either ustiloxin B production or the precursor gene transcript level. The costly-to-maintain highly repeated sequence in KEPs probably serves the purpose of maintaining stable transcripts and thus increasing the amount of substrate.

Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin.

Antibiotic resistance is a growing threat to public health, making the development of antibiotics of critical importance. One promising class of potential new antibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs), which include klebsidin, a lasso peptide from Klebsiella pneumoniae that inhibits certain bacterial RNA polymerases. We develop a high-throughput assay based on growth inhibition of Escherichia coli to analyze the mutational tolerance of klebsidin. We transform a library of klebsidin variants into E. coli and use next-generation DNA sequencing to count the frequency of each variant before and after its expression, thereby generating functional scores for 320 of 361 single amino acid changes. We identify multiple positions in the macrocyclic ring and the C-terminal tail region of klebsidin that are intolerant to mutation, as well as positions in the loop region that are highly tolerant to mutation. Characterization of selected peptide variants scored as active reveals that each adopts a threaded lasso conformation; active loop variants applied extracellularly as peptides slow the growth of E. coli and K. pneumoniae. We generate an E. coli strain with a mutation in RNA polymerase that confers resistance to klebsidin and similarly carry out a selection with the klebsidin library. We identify a single variant, klebsidin F9Y, that maintains activity against the resistant E. coli when expressed intracellularly. This finding supports the utility of this method and suggests that comprehensive mutational analysis of lasso peptides can identify unique and potentially improved variants.

Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products with potent antibiotic, antiviral, and anticancer properties. RiPP enzymes known as cyclodehydratases and dehydrogenases work together to catalyze intramolecular, inter-residue condensation and dehydrogenation reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here, we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers—including aminobenzoic acid derivatives and beta-amino acids—within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse non-α-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond—both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

RaS-RiPPs in Streptococci and the Human Microbiome.

Radical S-adenosylmethionine (RaS) enzymes have quickly advanced to one of the most abundant and versatile enzyme superfamilies known. Their chemistry is predicated upon reductive homolytic cleavage of a carbon–sulfur bond in cofactor S-adenosylmethionine forming an oxidizing carbon-based radical, which can initiate myriad radical transformations. An emerging role for RaS enzymes is their involvement in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a natural product family that has become known as RaS-RiPPs. These metabolites are especially prevalent in human and mammalian microbiomes because the complex chemistry of RaS enzymes gives rise to correspondingly complex natural products with minimal cellular energy and genomic fingerprint, a feature that is advantageous in microbes with small, host-adapted genomes in competitive environments. Herein, we review the discovery and characterization of RaS-RiPPs from the human microbiome with a focus on streptococcal bacteria. We discuss the varied chemical modifications that RaS enzymes introduce onto their peptide substrates and the diverse natural products that they give rise to. The majority of RaS-RiPPs remain to be discovered, providing an intriguing avenue for future investigations at the intersection of metalloenzymology, chemical ecology, and the human microbiome.

Macrocyclization and Backbone Modification in RiPP Biosynthesis.

The past decade has seen impressive advances in understanding the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the most common modifications found in these natural products is macrocyclization, a strategy also used by medicinal chemists to improve metabolic stability and target affinity and specificity. Another tool of the peptide chemist, modification of the amides in a peptide backbone, has also been observed in RiPPs. This review discusses the molecular mechanisms of biosynthesis of a subset of macrocyclic RiPP families, chosen because of the unusual biochemistry involved: the five classes of lanthipeptides (thioether cyclization by Michael-type addition), sactipeptides and ranthipeptides (thioether cyclization by radical chemistry), thiopeptides (cyclization by [4+2] cycloaddition), and streptide (cyclization by radical C-C bond formation). In addition, the mechanisms of backbone amide methylation, backbone epimerization, and backbone thioamide formation are discussed, as well as an unusual route to small molecules by posttranslational modification.

Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

Diverse Protein Architectures and α-N-Methylation Patterns Define Split Borosin RiPP Biosynthetic Gene Clusters.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.

Disrupting Transcription and Folate Biosynthesis Leads to Synergistic Suppression of Escherichia coli Growth

The use of synergistic antibiotic combinations has emerged as a viable approach to contain the rapid spread of antibiotic‐resistant pathogens. Here we report the discovery of a new strongly synergistic pair – microcin J25 and sulfamonomethoxine. The former is a lasso peptide that inhibits the function of RNA polymerase and the latter is a sulfonamide antibacterial agent that disrupts the folate pathway. Key to our discovery was a screening strategy that focuses on an antibiotic (microcin J25) that targets a hub (transcription) in the densely interconnected network of cellular pathways. The rationale was that disrupting such a hub likely weakens the entire network, generating weak links that potentiate the growth inhibitory effect of other antibiotics. We found that MccJ25 potentiates five other antibiotics as well. These results showcase the merit of taking a more targeted approach in the search and study of synergistic antibiotic pairs.

Microbial metabolites: cause or consequence in gastrointestinal disease?

Systems biology studies have established that changes in gastrointestinal microbiome composition and function can adversely impact host physiology. Notable diseases synonymously associated with dysbiosis include inflammatory bowel diseases, cancer, metabolic disorders, and opportunistic and recurrent pathogen infections. However, there is a scarcity of mechanistic data that advances our understanding of taxonomic correlations with pathophysiological host-microbiome interactions. Generally, to survive a hostile gut environment, microbes are highly metabolically active and produce trans-kingdom signaling molecules to interact with competing microorganisms and the host. These specialized metabolites likely play important homeostatic roles, and identifying disease-specific taxa and their effector pathways can provide better strategies for diagnosis, treatment, and prevention, as well as the discovery of innovative therapeutics. The signaling role of microbial biotransformation products such as bile acids, short-chain fatty acids, polysaccharides, and dietary tryptophan is increasingly recognized, but little is known about the identity and function of metabolites that are synthesized by microbial biosynthetic gene clusters, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), nonribosomal peptides (NRPs), polyketides (PKs), PK-NRP hybrids, and terpenes. Here we consider how bioactive natural products directly encoded by the human microbiome can contribute to the pathophysiology of gastrointestinal disease, cancer, autoimmune, antimicrobial-resistant bacterial and viral infections (including COVID-19). We also present strategies used to discover these compounds and the biological activities they exhibit, with consideration of therapeutic interventions that could emerge from understanding molecular causation in gut microbiome research.

Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.

Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.

Characterization of Histidine Functionalization and Its Timing in the Biosynthesis of Ribosomally Synthesized and Posttranslationally Modified Thioamitides.

Thioamitides are ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products that hold great potential in anticancer drug development. Members in this RiPP family feature a thioamidated peptidyl chain conjugated with a macrocyclic ring system that contains two nonproteinogenic residues, 2-aminovinyl-cysteine (AviCys) and β-hydroxy-N,N-dimethyl-l-histidine (hdmHis). Focusing on the hdmHis residue that is unique to thioamitides, we report the enzymatic process for His functionalization and, more importantly, the timing of its related reactions with the other posttranslational modifications (PTMs) involved in thioamitide biosynthesis. His functionalization involves the activities of an S-adenosyl-l-methionine-dependent protein and a 2-oxoglutarate-Fe(II) monooxygenase for His bis-N-dimethylation and subsequent β-hydroxylation in a highly ordered manner. This process relies on the leader peptide sequence of the precursor peptide and on the establishment of the AviCys-containing, C-terminal macrocyclic ring system in particular. In contrast, prior peptide thioamidation is not required. Knowledge gained from the catalytic logic, specificity, and compatibility of His functionalization greatly furthers our understanding of the process through which nature develops thioamitides from a ribosomally synthesized peptide precursor.

Biochemical Reconstitution Reveals the Biosynthetic Timing and Substrate Specificity for Thioamitides.

Thioamitides are apoptosis-inducing ribosomally synthesized and post-translationally modified peptides (RiPPs) with substantial post-translational modifications (PTMs), whose biosynthetic details remain elusive. We reconstituted their key PTMs through in vitro enzymatic reactions and gene coexpressions in E. coli and rigorously demonstrated the order of those modifications. Notably, thioamitide biosynthesis involves N- to C-terminal thioamidations and employs both leader-dependent and leader-independent reactions followed by leader removal by successive degradation. Our study provides a comprehensive overview of thioamitide biosynthesis and lays the foundation for thioamitide engineering in E. coli.

High-throughput discovery of novel lanthipeptides and producers by metagenomic mining of isolates population (MMIP) from Chinese spicy cabbage

Lanthipeptides are known antimicrobial agents having great potential for application in food preservation. Many lanthipeptide biosynthetic gene clusters (BGCs) were mined in fermented food microbiota, however, it is difficult to obtain the bioactive lanthipeptides and their producing strains. Here, we established a high-throughput strategy designated Metagenomic Mining of Isolates Population (MMIP) to efficiently excavate and obtain novel lanthipeptides, especially their potential producing strains. MMIP procedure involves gathering bacteria isolates using culturable strategy, metagenomic mining for lanthipeptides and screening their producers, and characterization of specific lanthipeptides. 928 biosynthetic gene clusters including 139 ribosomally synthesized and post-translationally modified peptides (RiPPs) gene clusters were discovered in the metagenomic data of the isolates by antiSMASH. Entianin, lactocin S, lichenicidin, and 17 novel lanthipeptides gene clusters corresponding to 29 possible producers were further found from the harvested isolates population. Entianin and a novel two-component lanthipeptide paralicin were purified from Bacillus subtilis C5B1 and Bacillus paralicheniformis BaC1-8, respectively. They showed strong inhibitory activity to food spoilage bacteria Bacillus cereus and Listeria monocytogenes, and have great potential for application in food preservation. A novel lanthipeptide polysacin was also obtained using semi-in vitro biosynthesis. MMIP affords a novel strategy for effectively excavating lanthipeptides, especially their producers from diverse environmental niches.

Complex peptide natural products: Biosynthetic principles, challenges and opportunities for pathway engineering

Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.

Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Protein Engineering in Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) make up a rapidly growing superfamily of natural products. RiPPs exhibit an extraordinary range of structures, but they all begin as gene-encoded precursor peptides that are linear chains of amino acids produced by ribosomes. Given the gene-encoded nature of RiPP precursor peptides, the toolbox of protein engineering can be directly applied to these precursors. This Perspective will discuss examples of site-directed mutagenesis, noncanonical amino acid mutagenesis, and the construction and screening of combinatorial libraries as applied to RiPPs. These studies have led to important insights into the biosynthesis and bioactivity of RiPPs and the reengineering of RiPPs for entirely new functions.

Combined thermal and carboxypeptidase Y stability assays for probing the threaded fold of lasso peptides.

Lasso peptides are natural products belonging to the superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). The defining characteristic of lasso peptides is their threaded structure, which is reminiscent of a lariat knot. When working with lasso peptides, it is therefore of major importance to understand and evidence their threaded folds. While the full elucidation of their three-dimensional structures via NMR spectroscopy or crystallization remains the gold standard, these methods are time-consuming, require large quantities of highly pure lasso peptides, and therefore might not always be applicable. Instead, the unique properties of lasso peptides in context of their behavior at elevated temperatures and toward carboxypeptidase Y treatment can be leveraged as a tool to investigate and evidence the threaded lasso fold using only minute amounts of compound that does not need to be purified first. This chapter will provide insights into the thermal stability properties of lasso peptides and their behavior when treated with carboxypeptidase Y in comparison to a branched-cyclic peptide with the same amino acid sequence. Furthermore, it will be described in detail how to set up a combined thermal and carboxypeptidase Y stability assay and how to analyze its outcomes.

Generation of Lasso Peptide-Based ClpP Binders.

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.

Pseudaestuariivita rosea sp. nov., isolated from Acmaea sp., a marine mollusk.

A Gram-stain-negative, pink-pigmented, aerobic, non-motile and rod-shaped bacterium, designated as strain H15T, was isolated from Acmaea sp., collected from Weihai, Shandong Province, China. The novel isolate was able to grow at 4-37°C (optimum 33°C), pH 5.5-9.0 (optimum 7.0) and with 0.0-7.0% NaCl (optimum 4%, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the strain belonged to the family Rhodobacteraceae and was associated to the type strain of Pseudaestuariivita atlantica (96.7%). Genome analysis showed that the genome size was 3,893,398bp and the DNA G + C content obtained from the draft genome sequence was 56.7%. The secondary metabolites predicated that the strain H15Tcontained one cluster of lasso peptide, one cluster of bacteriocin, two clusters of terpene production, two clusters of homoserine lactone and one cluster of beta lactone. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between genome sequences of strain H15Tand all the related strains compared were lower than 63.1, 72.0 and 19.7%, respectively. Based on the analysis of chemical components, the predominant cellular fatty acids were summed featured 8 (C18:1ω7c/ω6c, 46.1%), C20:1 ω7c (17.1%), the major polar lipids contained phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid and the predominant menaquinone was Q10. Therefore, the combined chemotaxonomic, phenotypic and phylogenetic data indicated thatthe strain was considered to represent a novel species of the genus Pseudaestuariivitaand the name Pseudaestuariivita rosea sp. nov. was proposed for strain H15T (MCCC 1K04420T = KCTC 82505T).