Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis.

Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N-acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis; sporangia of the gsmA null mutant (ΔgsmA) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N-acetylmuramoyl-l-alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (ΔasmA) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the ΔgsmAΔasmA double mutant released longer spore chains than those from the ΔgsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis.IMPORTANCEActinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2-20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis.

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Actinoplanes sandaracinus sp. nov., a novel ligninase- producing and cellulose-degrading actinobacterium isolated from soil.

A novel ligninase-producing and cellulose-degrading actinobacterium, designated strain NEAU-A12T, was isolated from a soil sample collected from Aohan banner, Chifeng City, Inner Mongolia Autonomous Region, PR China. A polyphasic taxonomic study was used to establish the status of strain NEAU-A12T. 16S rRNA gene sequence analysis revealed that strain NEAU-A12T belonged to the genus Actinoplanes and showed the highest similarity (98.3 %) to Actinoplanes palleronii DSM 43940T, while showing less than 98.3 % similarity to other members of the genus Actinoplanes. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and glycosylphosphatidylinositol. The diagnostic sugars in cell hydrolysates were determined to be arabinose, glucose and xylose. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2). The major fatty acids were C15 : 0, C16 : 0, C16 : 1 ω7c and C17 : 0. Meanwhile, genomic analysis revealed a genome size of 10 192 524 bp and a DNA G+C content of 70.6 mol%, and indicated that strain NEAU-A12T had the potential to degrade lignin and cellulose, as well as produce bioactive compounds. In addition, the average nucleotide identity values between strain NEAU-A12T and its reference strains A. palleronii DSM 43940T, Actinoplanes regularis DSM 43151T, Actinoplanes philippinensis DSM 43019T, Actinoplanes xinjiangensis DSM 45184T and Actinoplanes italicus DSM 43146T were 80.3, 80.3, 84.1, 84.3 and 84.0 %, respectively. The levels of digital DNA-DNA hybridization between them were found to be 23.6 % (21.3-26.1 %), 23.8 % (21.5-26.3 %), 28.3 % (25.9-30.8 %), 28.6 % (26.0-30.9 %) and 28.4 % (26.2-31.1 %), respectively. Based on phenotypic, chemotaxonomic and genotypic data, strain NEAU-A12T is considered to represent a novel species of the genus Actinoplanes, for which the name Actinoplanes sandaracinus sp. nov. is proposed, with NEAU-A12T (=CCTCC AA 2020039T=DSM 112043T) as the type strain.

Actinoplanes pyxinae sp. nov., a new lichen-derived rare actinobacterium exhibiting antimicrobial and anticancer activity.

A novel lichen-derived actinobacterium, designated Pm04-4T, was isolated from Pyxine cocoes (Sw.) Nyl. lichen collected from Chaiyaphum, Thailand. A polyphasic approach was used to describe the taxonomic position of the strain. The strain had morphological and chemotaxonomic properties similar to members of the genus Actinoplanes. It produced sporangia on the substrate mycelia. Meso-diaminopimelic acid, galactose, glucose and mannose were detected in the whole-cell hydrolysate of the strain. The major menaquinone was MK-9(H4). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Strain Pm04-4T showed the highest 16S rRNA gene sequence similarity to Actinoplanes akusuensis TRM 8003T (99.0 %). In the phylogenomic tree, strain Pm04-4T was positioned close to A. aksuensis TRM88003T, A. maris M416T, A. polyasparticus TRM66264T, A. hotanensis TRM88002T, A. abujensis DSM 45518T, A. bogorensis NBRC 110975T, A. brasiliensis DSM 43805T, A. lichenicola LDG1-01T and A. ovalisporus LDG1-06T. The average nucleotide identity and digital DNA-DNA hybridization values between strain Pm04-4T and its closely related neighbours were below the threshold values for describing new species. Moreover, the strain could be distinguished from its closely related type strains by phenotypic properties. Based on genotypic and phenotypic evidence, it can be concluded that strain Pm04-4T is a representative of a new Actinoplanes species for which the name Actinoplanes pyxinae sp. nov. is proposed. The type strain is Pm04-4T (=TBRC 16207T=NBRC 115836T). The type strain exhibited activity against Staphylococcus aureus ATCC 25923 as well as four yeast strains, namely Candida albicans TISTR 5554, Candida glabrata TISTR 5006, Candida krusei TISTR 5351 and Candida parapsilosis TISTR 5007. It also showed cytotoxicity against Caco-2, MNT-1 and MCF-7 cancer cells.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

Microscopic and molecular genetic analyses of morphological development of the actinomycete Actinoplanes missouriensis.

The survival strategy of members of the bacterial genus Actinoplanes is fascinating from morphological and evolutionary perspectives. A brief motile phase is incorporated in the filamentous and resting stages of the life cycle of Actinoplanes missouriensis. Spores either lie dormant or swim under different external conditions. This review presents microscopic observations and molecular genetic analyses of A. missouriensis morphological development. Selected examples of the characterization of developmental genes and their products are also introduced.

Enhancement of α-galactosidase production using novel Actinoplanes utahensis B1 strain: sequential optimization and purification of enzyme.

α-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzymes, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing α-1,6 linked terminal galacto-oligosaccharide residues such as melibiose, raffinose, and branched polysaccharides such as galacto-glucomannans and galactomannans. In this study, Actinoplanes utahensis B1 was explored for α-galactosidase production, yield improvement, and activity enhancement by purification. Initially, nine media components were screened using the Plackett-Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as the best-supporting nutrients for the highest enzyme secretion by A. Utahensis B1. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in α-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72 kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified α-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the isolation of Actinoplanes utahensis B1 and optimization of the process for the α-galactosidase production as well as single-step purification.

Three novel Actinoplanes species isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ.

Three novel actinomycete strains, designated TRM66264-DLMT, TRM88002T and TRM88003T, were isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ. The 16S rRNA gene sequence and phylogenetic analyses of three strains indicated that they belonged to the genus Actinoplanes. The phylogenetically closest strains of TRM66264-DLMT, TRM88002T and TRM88003T were Actinoplanes bogorensis LIPI11-2-Ac043T (98.4 %), Actinoplanes abujensis A4029T (98.0 %) and Actinoplanes ferrugineus IFO15555T (98.1 %), respectively. The major polar lipids of strains TRM66264-DLMT and TRM88002T were phosphatidylethanolamine and disphosphatidylglycerol, while strain TRM88003T only had phosphatidylethanolamine. The predominant menaquinones of strain TRM66264-DLMT were identified as MK-9(H4) and MK-9 (H6). Strains TRM88002T and TRM88003T had MK-9(H4). The cell-wall peptidoglycan of three strains contained meso-diaminopimelic acid. The whole-cell sugars of strain TRM66264-DLMT were identified as arabinose, glucose, galactose and xylose. Strains TRM88002T and TRM88003T mainly had arabinose and glucose. The DNA G+C content of strains TRM66264-DLMT, TRM88002T and TRM88003T were 70.48, 70.46 and 70.64 mol%, respectively. Genotypic and phenotypic analysis confirmed that all three strains sre new members of the genus Acinoplanes. Therefore, it is proposed that strains TRM66264-DLMT, TRM88002T and TRM88003T represent three novel species of the genus Actinoplanes, for which the names Actinoplanes polyasparticus sp. nov. (type strain TRM66264-DLMT=CCTCC AA 2021015T=LMG 32389T), Actinoplanes hotanensis sp. nov. (type strain TRM88002T=CCTCC AA 2021036T=LMG 32621T) and Actinoplanes aksuensis sp. nov. (type strain TRM88003T=CCTCC AA 2021037 T=LMG 32622T) are proposed.

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

A novel actinobacterium strain (M4I6T) was isolated from marine sediment collected in Megas Gialos, Syros, Greece. On the basis of 16S rRNA gene sequence analysis, strain M4I6T was indicated as belonging to the genus Actinoplanes, with high similarity to 'Actinoplanes solisilvae' LAM7112T (97.9 %), Actinoplanes ferrugineus IFO 15555T (97.6 %), Actinoplanes cibodasensis LIPI11-2-Ac042T (97.2 %) and Actinoplanes bogorensis LIPI11-2-Ac043T (97.2 %). Phylogenetic analysis of the 16S rRNA gene sequence of strain M4I6T showed that the strain formed a stable subclade with 'A. solisilvae' LAM7112T. The cell wall of the novel isolate contained meso-diaminopimelic acid and the whole-cell sugars were xylose, glucose and ribose. The predominant menaquinones were MK-9(H4), MK-9(H2) and MK-9(H8). The phospholipid profile comprised phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannosides and an unknown phospholipid. The major fatty acids (>5 %) were anteiso-C16 : 0, iso-C17 : 0, 10-methyl-C16 : 0, C15 : 0, iso-C16 : 0 and C17 : 0. Genome sequencing showed a DNA G+C content of 70.9 mol%. However, the low average nucleotide identity value, digital DNA-DNA hybridization and average amino acid identity values demonstrated that strain M4I6T could be readily distinguished from its closest related species. Based on data from this polyphasic study, strain M4I6T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes maris sp. nov. is proposed. The type strain is M4I6T (=DSM 101017T=CGMCC 4.7854T).

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

Enhancement of acarbose production by genetic engineering and fed-batch fermentation strategy in Actinoplanes sp. SIPI12-34.

BACKGROUND: Acarbose, as an alpha-glucosidase inhibitor, is widely used clinically to treat type II diabetes. In its industrial production, Actinoplanes sp. SE50/110 is used as the production strain. Lack of research on its regulatory mechanisms and unexplored gene targets are major obstacles to rational strain design. Here, transcriptome sequencing was applied to uncover more gene targets and rational genetic engineering was performed to increase acarbose production. RESULTS: In this study, with the help of transcriptome information, a TetR family regulator (TetR1) was identified and confirmed to have a positive effect on the synthesis of acarbose by promoting the expression of acbB and acbD. Some genes with low expression levels in the acarbose biosynthesis gene cluster were overexpressed and this resulted in a significant increase in acarbose yield. In addition, the regulation of metabolic pathways was performed to retain more glucose-1-phosphate for acarbose synthesis by weakening the glycogen synthesis pathway and strengthening the glycogen degradation pathway. Eventually, with a combination of multiple strategies and fed-batch fermentation, the yield of acarbose in the engineered strain increased 58% compared to the parent strain, reaching 8.04 g/L, which is the highest fermentation titer reported. CONCLUSIONS: In our research, acarbose production had been effectively and steadily improved through genetic engineering based on transcriptome analysis and fed-batch culture strategy.

Heterologous Expression Reveals Ancient Properties of Tei3­A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus­the producer of the last-resort-drug teicoplanin­has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.

Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.

Actinoplanes flavus sp. nov., a novel cellulase-producing actinobacterium isolated from coconut palm rhizosphere soil.

A novel cellulase-producing actinomycete, designated strain NEAU-H7T, was isolated from coconut palm rhizosphere soil collected from Wenchang City, Hainan Province, PR China. A polyphasic taxonomic study was carried out to establish the status of this strain. Results of 16S rRNA gene sequence analysis indicated that strain NEAU-H7T belonged to the genus Actinoplanes, with highest similarity to Actinoplanes hulinensis NEAU-M9T (99.2 % 16S rRNA gene sequence similarity). The diagnostic sugars in cell hydrolysates were determined to be ribose, galactose and mannose. The major fatty acids (>10%) were C16 : 0, C18 : 1 ω9c and C18 : 0. The predominant menaquinones were identified as MK-9(H4) and MK-9(H6). The major polar lipids were phosphatidylethanolamine, phosphatidylinositol and two phosphatidylinositol mannosides. The amino acid of the cell-wall peptidoglycan was determined to be meso-diaminopimelic acid. The DNA G+C content was 71.2 mol%. Phylogenetic analysis using 16S rRNA gene sequences showed that strain NEAU-H7T formed a stable phyletic line with A. hulinensis NEAU-M9T. However, whole-genome phylogeny showed strain NEAU-H7T formed a stable phyletic line with A. hulinensis NEAU-M9T (99.2%), Actinoplanes campanulatus DSM 43148T (98.6%), Actinoplanes capillaceus DSM 44859T (98.3%) and Actinoplanes lobatus DSM 43150T (97.6%). The digital DNA-DNA hybridization (dDDH) results between them were 53.6 (50.9-56.2), 54.1 (51.3-56.9), 53.1 (50.3-55.9) and 52.9 % (50.1-55.6 %), and whole-genome average nucleotide identity (ANI) values between them were 93.7, 93.6, 93.5 and 93.5 %. The low dDDH and ANI values demonstrated that strain NEAU-H7T could be distinguished from its reference strains. Moreover, genomic analysis indicated that the strain NEAU-H7T had the potential to decompose cellulose and produce bioactive compounds. On the basis of morphological, chemotaxonomic and phylogenetic characteristics, strain NEAU-H7T is proposed to represent a novel species of the genus Actinoplanes, with the name Actinoplanes flavus sp. nov. The type strain is NEAU-H7T (=CCTCC AA 2020034T=DSM 112042T).

Actinoplanes lichenicola sp. nov. and Actinoplanes ovalisporus sp. nov., isolated from lichen in Thailand.

Two actinobacteria, designated as strain LDG1-01T and LDG1-06T, were isolated from lichen samples collected in Thailand. Results of morphological characterization, chemotaxonomic studies and 16S rRNA gene analysis indicated that both strains were members of the genus Actinoplanes. MK-9(H4) was found as the major menaquinone. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were observed as the polar lipids, but differences in minor unidentified polar lipids were found between the strains. Results of comparative genome analysis based on average nucleotide identity and digital DNA-DNA hybridization calculations revealed that both strains showed values below 95 and 70 %, respectively, from each other and closely related Actinoplanes type strains. Based on data from this polyphasic study, strains LDG1-01T and LDG1-06T represent novel species of the genus Actinoplanes. The names proposed are Actinoplanes lichenicola sp. nov. (type strain, LDG1-01T (=JCM 33066T=TISTR 2982T) and Actinoplanes ovalisporus sp. nov. (type strain, LDG1-06T=JCM 33067T=TISTR 2983T).

Actinoplanes aureus sp. nov., a novel protease-producing actinobacterium isolated from soil.

A novel protease-producing actinobacterium, designated strain NEAU-A11T, was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China, and characterised using a polyphasic approach. The hydrolytic enzymes, such as proteases, played critical roles in destruction of fungi by degrading the protein linkages to disrupt integrity in the cell wall. This suggested that the isolate could be a good biocontrol candidate against pathogens to control fungal diseases. On the basis of 16S rRNA gene sequence analysis, strain NEAU-A11T was indicated to belong to the genus Actinoplanes and was most closely related to Actinoplanes rectilineatus JCM 3194 T (98.9%). Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were arabinose, xylose and glucose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major fatty acids were C18:0, C16:0, C18:1 ω9c, C17:0 and C15:0. Genome sequencing revealed a genome size of 10,742,096 bp, a G + C content of 70.5% and 9,514 protein-coding genes (CDS), including 102 genes coding for protease. Moreover, Genome analysis showed that strain NEAU-A11T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Genome mining analysis using antiSMASH 5.0 led to the identification of 20 putative gene clusters responsible for the production of diverse secondary metabolites. Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with A. rectilineatus JCM 3194 T in the genus Actinoplanes. Whole-genome phylogeny showed strain NEAU-A11T formed a stable phyletic line with Actinoplanes lutulentus DSM 45883 T (97.6%). However, whole-genome average nucleotide identity value between strain NEAU-A11T and its reference strains A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24.8% (22.5-27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95-96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G + C in genomic DNA was 70.5%, within the range of 67-76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11T (= CCTCC AA 2019063 T = JCM 33971 T) as the type strain.

Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase-like Sulfurtransferase.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensisIMPORTANCEActinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase.

BACKGROUND: Actinoplanes sp. SE50/110 is the natural producer of the diabetes mellitus drug acarbose, which is highly produced during the growth phase and ceases during the stationary phase. In previous works, the growth-dependency of acarbose formation was assumed to be caused by a decreasing transcription of the acarbose biosynthesis genes during transition and stationary growth phase. RESULTS: In this study, transcriptomic data using RNA-seq and state-of-the-art proteomic data from seven time points of controlled bioreactor cultivations were used to analyze expression dynamics during growth of Actinoplanes sp. SE50/110. A hierarchical cluster analysis revealed co-regulated genes, which display similar transcription dynamics over the cultivation time. Aside from an expected metabolic switch from primary to secondary metabolism during transition phase, we observed a continuously decreasing transcript abundance of all acarbose biosynthetic genes from the early growth phase until stationary phase, with the strongest decrease for the monocistronically transcribed genes acbA, acbB, acbD and acbE. Our data confirm a similar trend for acb gene transcription and acarbose formation rate. Surprisingly, the proteome dynamics does not follow the respective transcription for all acb genes. This suggests different protein stabilities or post-transcriptional regulation of the Acb proteins, which in turn could indicate bottlenecks in the acarbose biosynthesis. Furthermore, several genes are co-expressed with the acb gene cluster over the course of the cultivation, including eleven transcriptional regulators (e.g. ACSP50_0424), two sigma factors (ACSP50_0644, ACSP50_6006) and further genes, which have not previously been in focus of acarbose research in Actinoplanes sp. SE50/110. CONCLUSION: In conclusion, we have demonstrated, that a genome wide transcriptome and proteome analysis in a high temporal resolution is well suited to study the acarbose biosynthesis and the transcriptional and post-transcriptional regulation thereof.