Phylogenomic analyses of the genus Actinoplanes: description of four novel genera.

The genus Actinoplanes comprises 57 species (in February 2024) that are important components of ecosystems and are widely used in biotechnology, especially pharmaceuticals. Phylogenetic analysis of the family Micromonosporaceae (based on the 16S rRNA gene sequence) allowed us to group members of different genera into separate clades; however, the genus Actinoplanes was divided into three separate clades. Such phylogenetic heterogeneity could be due to the limitations of 16S rRNA gene analysis. In response to this heterogeneity, genomic phylogeny was performed. Phylogenomic reconstruction based on 324 single-copy orthologous genes allowed us to divide the genus Actinoplanes first into four clades and then, based on average nucleotide identity analysis, into five clades. Finally, chemotaxonomic analysis of each clade confirmed each clade's distinctiveness and the necessity to reclassify the genus Actinoplanes. The obtained data allowed us to divide the genus Actinoplanes into five genera: Actinoplanes, Paractinoplanes, Winogradskya, Symbioplanes and Amorphoplanes.

Microbe Profile: Salinispora tropica: natural products and the evolution of a unique marine bacterium.

Salinispora tropica was originally cultured from tropical marine sediments and described as the first obligate marine actinomycete genus. Soon after its discovery, it yielded the potent proteasome inhibitor salinosporamide A, a structurally novel natural product that is currently in phase III clinical trials for the treatment of cancer. If approved, it will be the first natural product derived from a cultured marine microbe to achieve clinical relevance. S. tropica produces many other biologically active natural products, including some linked to chemical defence, thus providing ecological context for their production. However, genomic analyses reveal that most natural product biosynthetic gene clusters remain orphan, suggesting that more compounds await discovery. The abundance of biosynthetic gene clusters in S. tropica supports the concept that the small molecules they encode serve important ecological functions, while their evolutionary histories suggest a potential role in promoting diversification. Better insights into the ecological functions of microbial natural products will help inform future discovery efforts.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Natural Products Produced in Culture by Biosynthetically Talented Salinispora arenicola Strains Isolated from Northeastern and South Pacific Marine Sediments.

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.

Abyssomicins-A 20-Year Retrospective View.

Abyssomicins represent a new family of polycyclic macrolactones. The first described compounds of the abyssomicin family were abyssomicin B, C, atrop-C, and D, produced by the marine actinomycete strain Verrucosispora maris AB-18-032, which was isolated from a sediment collected in the Sea of Japan. Among the described abyssomicins, only abyssomicin C and atrop-abyssomicin C show a high antibiotic activity against Gram-positive bacteria, including multi-resistant and vancomycin-resistant strains. The inhibitory activity is caused by a selective inhibition of the enzyme 4-amino-4-deoxychorismate synthase, which catalyzes the transformation of chorismate to para-aminobenzoic acid, an intermediate in the folic acid pathway.

Genomic analysis suggests Salinispora is a rich source of novel lanthipeptides.

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.

A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110.

Actinoplanes sp. SE50/110 is the industrially relevant producer of acarbose, which is used in the treatment of diabetes mellitus. Recent studies elucidated the expression dynamics in Actinoplanes sp. SE50/110 during growth. From these data, we obtained a large genomic region (ACSP50_3900 to ACSP50_3950) containing 51 genes, of which 39 are transcribed in the same manner. These co-regulated genes were found to be stronger transcribed on maltose compared with glucose as a carbon source. The transcriptional regulator MalT was identified as an activator of this maltose-regulated large genomic region (MRLGR). Since most of the genes are poorly annotated, the function of this region is farther unclear. However, comprehensive BLAST analyses indicate similarities to enzymes involved in amino acid metabolism. We determined a conserved binding motif of MalT overlapping the -35 promoter region of 17 transcription start sites inside the MRLGR. The corresponding sequence motif 5'-TCATCC-5nt-GGATGA-3' displays high similarities to reported MalT binding sites in Escherichia coli and Klebsiella pneumoniae, in which MalT is the activator of mal genes. A malT deletion and an overexpression mutant were constructed. Differential transcriptome analyses revealed an activating effect of MalT on 40 of the 51 genes. Surprisingly, no gene of the maltose metabolism is affected. In contrast to many other bacteria, MalT is not the activator of mal genes in Actinoplanes sp. SE50/110. Finally, the MRLGR was found partly in other closely related bacteria of the family Micromonosporaceae. Even the conserved MalT binding site was found upstream of several genes inside of the corresponding regions. KEY POINTS : • MalT is the maltose-dependent activator of a large genomic region in ACSP50_WT. • The consensus binding motif is similar to MalT binding sites in other bacteria. • MalT is not the regulator of genes involved in maltose metabolism in ACSP50_WT.

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production.

trans-4-Hydroxy- l-proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l-proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L -1 ·hr -1 in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110.

BACKGROUND: Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. RESULTS: The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5'-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC-MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. CONCLUSION: Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.

Regulation of teicoplanin biosynthesis: refining the roles of tei cluster-situated regulatory genes.

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.

Actinoplanes sediminis sp. nov., isolated from marine sediment.

An actinomycete strain M4I47T was isolated from sediment from Megas Gialos, Syros, Greece. The results of phylogenetic analysis of the 16S rRNA gene sequence of M4I47T indicated that the highest similarity was with Actinoplanes atraurantiacus Y16T (98.9 %), Actinoplanes deccanensis IFO 13994T (98.8 %), Actinoplanes digitatis IFO 12512T (98.1 %) and Actinoplanes abujensis A4029T (98.0 %). The cell wall of the novel isolate contained meso-diaminopimelic acid and the whole-cell sugars were xylose, arabinose and glucose. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2). The phospholipid profile comprised phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unknown phospholipid. The DNA G+C content was 71.5 mol%. Furthermore, a combination of DNA-DNA relatedness and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the most closely related species. On the basis of these phenotypic and genotypic data, M4I47T represents a novel species of the genus Actinoplanes, for which the name Actinoplanessediminis sp. nov. is proposed. The type strain is M4I47T (=CCTCC AA 2016022T=DSM 100965T).

Jishengella zingiberis sp. nov., isolated from root tissue of Zingiber montanum.

A novel actinomycete, strain PLAI 1-1T, which formed spiny single spore directly on substrate mycelium was isolated from root tissue of Zingiber montanum. The isolate contained meso-diaminopimelic acid and 3-hydroxydiaminopimelic acid in the cell-wall peptidoglycan. The acyl type of the cell-wall muramic acid was glycolyl. The whole-cell sugars of strain PLAI 1-1T were glucose, arabinose, xylose, ribose and a trace amount of mannose. The membrane phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. The major menaquinone was MK-9 (H4). The main cellular fatty acids were iso-C15 : 0 and C17 : 1ω8c. The G+C content of the genomic DNA was 70.6 mol%. 16S rRNA gene sequence analysis revealed that strain PLAI 1-1T was a member of the genus Jishengella and had the highest 16S rRNA gene sequence similarity to Jishengella endophytica DSM 45430T (99.2 %). Based on the data of physiological and biochemical tests, including the result of DNA-DNA hybridization, strain PLAI 1-1T represents a novel species of the genus Jishengella, for which the name Jishengellazingiberis sp. nov. is proposed. The type strain is PLAI 1-1T (=TBRC 7644T=NBRC 113144T).

Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters.

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.

Plantactinospora solaniradicis sp. nov., a novel actinomycete isolated from the root of a tomato plant (Solanum lycopersicum L.).

A Gram-positive, non-motile actinomycete, designated strain NEAU-FJL1T, was isolated from tomato root (Solanum lycopersicum L.) collected from Harbin, Heilongjiang province, north China. The strain formed single spores with smooth surfaces from substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-FJL1T should be affiliated with the genus Plantactinospora and forms a distinct branch with its close neighbour Plantactinospora soyae NEAU-gxj3T (99.2% sequence similarity). The cell wall was found to contain meso-diaminopimelic acid and the whole cell sugars were identified as xylose, glucose, arabinose and galactose. The predominant menaquinones were identified as MK-10(H6) and MK-10(H4). The phospholipid profile was found to consist of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acids were identified as C15:0, iso-C16:0, anteiso-C17:0, C17:0 and iso-C15:0. With reference to phenotypic characteristics, phylogenetic data and DNA-DNA hybridization results, strain NEAU-FJL1T can be distinguished from its most closely related strain and classified as a new species, for which the name Plantactinospora solaniradicis sp. nov. is proposed. The type strain is NEAU-FJL1T (= DSM 100596T = CGMCC 4.7284T).

Actinoplanes deserti sp. nov., isolated from a desert soil sample.

A novel actinomycete, designated strain YIM CF22T, was isolated from a desert soil sample collected from Turpan in Xinjiang Uyghur Autonomous Region, north-western China. The taxonomic position of the strain YIM CF22T is described based on a polyphasic approach. Strain YIM CF22T was found to form irregular sporangia on agar media. It contains meso-diaminopimelic acid in the cell wall peptidoglycan. The major menaquinone was identified as MK-9(H4); the polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, two unidentified phospholipids and two unidentified glycolipids. The whole cell sugars were found to be ribose, mannose, galactose, glucose and xylose. The major cellular fatty acids were found to be (> 5%) iso-C16:0 (43.5%), anteiso-C17:0 (10.2%), iso-C15:0 (7.1%), C17:1 ω8c (6.3%) and iso H-C16:1 (5.9%). The G+C content was determined to be 70.8%. 16S rRNA gene sequence analysis of strain YIM CF22T showed high similarity (97.0%) to Actinoplanes rishiriensis NBRC 108556T. The strain also showed high 16S rRNA gene sequence similarities to Verrucosispora sediminis CGMCC 4.3550T (96.9%) and Micromonospora tulbaghiae DSM 45142T (96.8%). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM CF22T clusters with A. rishiriensis NBRC 108556T, Actinoplanes globisporus JCM 3186T and Actinoplanes rhizophilus NEAU-A-2T. Based on the differential phenotypic characteristics and the results of DNA-DNA relatedness and phylogenetic analysis, it is proposed that strain YIM CF22T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes deserti sp. nov. is proposed. The type strain is YIM CF22T (= KCTC 39543T = CCTCC AB2018113T).

Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD 3',5'-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5'-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3' as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation.IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

Genomic insights into specialized metabolism in the marine actinomycete Salinispora.

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five-Membered Thiolactone Ring Formation.

Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered δ-thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.

Mangrovihabitans endophyticus gen. nov., sp. nov., a new member of the family Micromonosporaceae isolated from Bruguiera sexangula.

A novel endophytic actinobacterium, designated strain S3Cf-2T, was isolated from a surface-sterilized bark of Bruguiera sexangula collected from Dongzhaigang National Nature Reserve in Hainan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain S3Cf-2T fell within the family Micromonosporaceae and formed a distinct clade in the Micromonosporaceae phylogenetic tree. The 16S rRNA gene sequence similarity values between strain S3Cf-2T and the type species of 30 genera in the family Micromonosporaceae were 91.55-97.45 %. Strain S3Cf-2T formed extensively branched substrate mycelia without fragmentation. An oval or rod-like spore with a smooth surface was borne singly at the end of substrate mycelium. The novel isolate possessed meso-diaminopimelic acid as the diamino acid of the peptidoglycan, and glucose, galactose, mannose, arabinose, xylose and ribose as whole-cell sugars. The acyl type of the cell-wall peptidoglycan was glycolyl and mycolic acids were absent. The major polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown aminolipid, corresponding to phospholipid type PII. The major menaquinones were MK-9(H6) and MK-9(H8). The major cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0, anteiso-C17 : 0 and iso-C15 : 0. The G+C content of the genomic DNA was 71.4 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic analyses, strain S3Cf-2T represents a novel species of a new genus within the family Micromonosporaceae, for which the name Mangrovihabitans endophyticus gen. nov., sp. nov. is proposed. The type strain of the type species is S3Cf-2T (=DSM 100693T=CGMCC 4.7299T).

Verrucosispora endophytica sp. nov., isolated from the root of wild orchid (Grosourdya appendiculata (Blume) Rchb.f.).

A novel endophytic filamentous bacterium strain A-T 7972T, was isolated from wild orchid Grosourdya appendiculata (Blume) Rchb.f. collected in Thailand. The bacterium developed single non-motile spores with warty surface on substrate mycelia. The taxonomic position was described using a polyphasic approach. The 16S rRNA gene sequence and phylogenetic analysis indicated that strain A-T 7972T belonged to the genus Verrucosispora and shared the highest sequence similarity with V. lutea YIM 013T (98.71 %) and V. gifhornensis DSM 44337T (98.53 %). The values of DNA-DNA relatedness that distinguished this novel strain from its closest species were below 70 %. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars were mannose, ribose, glucose and xylose. The predominant menaquinone was MK-9(H4). The predominant fatty acids were branched fatty acids iso-C15 : 0 and iso-C16 : 0. The diagnostic phospholipids profile consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The G+C content of the genomic DNA was 70.5 mol%. Based on its phenotypic, chemotaxonomic and genotypic characteristics, the new isolate A-T 7972T (=BCC 50981T=TBRC 6031T=NBRC 112512T) is proposed to be the type strain of novel species, Verrucosispora endophytica sp. nov.