Direct 16S/ITS rRNA Gene PCR Followed by Sanger Sequencing for Detection of Mycetoma Causative Agents in Dakar, Senegal: A Pilot Study Among Patients with Mycetoma Attending Aristide Le Dantec University Hospital.

Mycetoma can be caused either by fungi or aerobic Actinomycetes. A precise identification of the causal agents is critical for the therapeutic outcome. Thus, this study aimed to identify the pathogens of mycetoma using 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains. In sum, 32 samples including 15 black grains, 12 red grains, and five white/yellow grains collected from patients with mycetoma at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020 were submitted to PCR/sequencing. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. The age of the patients ranged from 14 to 72 years with a mean age of 36 ± 14 years. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced with only one established eumycetoma pathogen, Madurella mycetomatis identified in 11 (73%). Cladosporium sphaerospermum was identified in one sample. For the 16S rRNA sequencing of red grains, a 58.3% (7/12) success rate was obtained with Actinomadura pelletieri identified in six samples. Among the five samples sequenced twice, the 16S rRNA allowed us to identify the causative agent in 2 cases, A. madurae in one, and A. geliboluensis in the other. The ITS rRNA identified 3 fungi, of which none was a mycetoma agent. Overall, direct 16S/ITS rRNA sequencing of the grains for detecting and identifying mycetoma pathogens was successful in 59.4% of cases. Fungi, led by M. mycetomatis, were the predominant pathogens identified. Two probable new mycetoma agents, C. sphaerospermum, and A. geliboluensis were identified and both deserve to be confirmed in further studies.

Variations of soil metal content, soil enzyme activity and soil bacterial community in Rhododendron delavayi natural shrub forest at different elevations.

BACKGROUND: Rhododendron delavayi is a natural shrub that is distributed at different elevations in the karst region of Bijie, China, and that has an important role in preventing land degradation in this region. In this study, we determined the soil mineral element contents and soil enzyme activities. The composition of the soil bacterial community of R. delavayi at three elevations (1448 m, 1643 m, and 1821 m) was analyzed by high-throughput sequencing, and the interrelationships among the soil bacterial communities, mineral elements, and enzyme activities were determined. RESULTS: The Shannon index of the soil bacterial community increased and then decreased with increasing elevation and was highest at 1643 m. Elevations increased the number of total nodes and edges of the soil bacterial community network, and more positive correlations at 1821 m suggested stronger intraspecific cooperation. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla at all three elevations. The Mantel test and correlation analysis showed that Fe and soil urease significantly affected bacterial communities at 1448 m; interestingly, Chloroflexi was positively related to soil urease at 1448 m, and Actinobacteria was positively correlated with Ni and Zn at 1821 m. Fe and soil urease significantly influenced the bacterial communities at lower elevations, and high elevation (1821 m) enhanced the positive interactions of the soil bacteria, which might be a strategy for R. delavayi to adapt to high elevation environments. CONCLUSION: Elevation significantly influenced the composition of soil bacterial communities by affecting the content of soil mineral elements and soil enzyme activity.

Actinobacteria Isolated from Soils of Arid Saharan Regions Display Simultaneous Antifungal and Plant Growth Promoting Activities.

Application of actinobacteria has grown exponentially in recent years in sustainable agricultural. Most actinobacterial inoculants are tailored to function as either biocontrol agents or biofertilizers. Hence, there is the need to obtain and include multifunctional actinobacterial strains in inocula formulations. In this research, 90 actinobacterial isolates were isolated from rhizospheric and non-rhizospheric soils of Algerian Saharan arid regions and were screened for their activity against the phytopathogenic fungi Alternaria alternata, Aspergillus flavus, Botrytis cinerea, Fusarium oxysporum, and Fusarium solani. Five isolates that inhibited at least three of these fungi were characterized according to morphological, environmental and biochemical parameters, and were preliminarily identified as Streptomyces enissocaesilis A1, Streptomyces olivoverticillatus A5, Streptomyces erumpens A6, Streptomyces cavourensis A8, and Streptomyces microflavus A20. These strains were then screened for plant growth promoting activities. All strains produced siderophores, hydrocyanic acid, ammonia and the auxin indole-3-acetic acid (IAA) and were capable of solubilizing phosphate. The highest producer of siderophores (69.19 percent siderophore units), ammonia (70.56 µg mL-1) and IAA (148.76 µg mL-1) was strain A8, A20, and A5, respectively. These findings showed that the five actinobacteria are multipurpose strains with simultaneous antifungal and plant growth promoting activities and have the potential to be used for sustainable agricultural practices, particularly in arid regions.

Alternative protocol leading to rapid identification of Actinomycetes isolated from Algerian desertic soil by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is the first-line method for the rapid identification of most cultured microorganisms. As for Streptomyces strains, MALDI-TOF MS identification is complicated by the characteristic incrustation of colonies in agar and the strong cell wall of Actinomycetes cells requiring the use of alternative protein extraction protocols. In this study, we developed a specific protocol to overcome these difficulties for the MALDI-TOF MS identification of Actinomycetes made on solid medium. This protocol includes incubation of colony removed from agar plate with the beta-agarase enzyme, followed by a mechanical lysis and two washes by phosphate buffer and ethanol. Twenty-four Streptomyces and two Lentzea strains isolated from Algerian desertic soils were first identified by 16S rRNA sequencing as gold standard method, rpoB gene was used as a secondary gene target when 16S rRNA did not allow species identification. In parallel the isolates were identified by using the MALDI-TOF MS protocol as reported. After the expansion of the database with the inclusion of this MSPS, the strains were analyzed again in MALDI Biotyper, and all were identified. This work demonstrates that the rapid identification of Actinomycetes can be obtained without protein extraction step frequently used in MALDI-TOF mass spectrometry with this type of microorganisms.

Atacama desert actinomycetes: taxonomic analysis, drought tolerance and plant growth promoting potential.

This study was designed to recover representative culturable actinomycetes from the Atacama Desert, and to detect their ability to promote plant growth under drought conditions. Environmental samples were taken from three Atacama Desert habitats, namely, from the Aguas Calientes, Lomas Bayas and Yungay core regions. With one exception higher actinomycete counts were obtained when isolation media were inoculated with mineral particles than with corresponding aliquots of serial dilution. Comparative 16S rRNA gene sequencing showed that representative isolates belonged to thirteen genera including putative novel Blastococcus, Kocuria, Micromonospora, Pseudonocardia, Rhodococcus and Streptomyces species. Representative isolates produced indole-3-acetic acid, siderophore and solubilized phosphate as well as displaying an ability to grow under drought conditions. In conclusion, the current findings open up exciting prospects for the promising potential of actinomycetes from the Atacama Desert to be used as bioinoculants to promote plant growth in arid and semi-arid biomes.

Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.

Demequina capsici sp. nov., a novel plant growth-promoting actinomycete isolated from the rhizosphere of bell pepper (Capsicum annuum).

Demequina, commonly found in coastal and marine environments, represents a genus of Actinomycetes. In this study, strains Demequina PMTSA13T and OYTSA14 were isolated from the rhizosphere of Capsicum annuum, leading to the discovery of a novel species, Demequina capsici. Bacteria play a significant role in plant growth, yet there have been no reports of the genus Demequina acting as plant growth-promoting bacteria (PGPB). Comparative genomics analysis revealed ANI similarity values of 74.05-80.63% for PMTSA13T and 74.02-80.54% for OYTSA14, in comparison to various Demequina species. The digital DNA-DNA hybridization (dDDH) values for PMTSA13T ranged from 19 to 39%, and 19.1-38.6% for OYTSA14. Genome annotation revealed the presence of genes associated with carbohydrate metabolism and transport, suggesting a potential role in nutrient cycling and availability for plants. These strains were notably rich in genes related to 'carbohydrate metabolism and transport (G)', according to their Cluster of Orthologous Groups (COG) classification. Additionally, both strains were capable of producing auxin (IAA) and exhibited enzymatic activities for cellulose degradation and catalase. Furthermore, PMTSA13T and OYTSA14 significantly induced the growth of Arabidopsis thaliana seedlings primarily attributed to their capacity to produce IAA, which plays a crucial role in stimulating plant growth and development. These findings shed light on the potential roles of Demequina strains in plant-microbe interactions and agricultural applications. The type strain is Demequina capsici PMTSA13T (= KCTC 59028T = GDMCC 1.4451T), meanwhile OYTSA14 is identified as different strains of Demequina capsici.

Existence of rare actinobacterial forms in the Indian sector of Southern Ocean: 16 S rRNA based metabarcoding study.

The significance of the Southern Ocean (SO) as a sink of atmospheric CO2 and other greenhouse gases is well established. Earlier studies have highlighted the role of microbes in various SO ecosystem processes. However, the diversity and role of actinobacteria in the Indian sector of SO (ISO) water and sediments are unknown. This study aimed to analyze the diversity of actinobacteria in water and sediment samples of SO based on amplicon microbiome analyses. The taxonomic analysis identified a total number of 27 phyla of which Proteobacteria (40.2%), Actinobacteria (13.6%), and Firmicutes (8.7%) were found to be dominant. The comparative study of water and sediment samples revealed the dominance of different actinobacteria in water and sediments. While the order Streptomycetales was dominant in the water samples, Micrococcales was found to be dominant in the sediment samples. The genus level analysis found the presence of eight and seventeen genera in the sediment and water samples, respectively. The genus Streptomyces, Saccharopolyspora, Nocardioides, Sva0996 marine group, and Mycobacterium were seen both in sediment and water samples. Marmoricola, Ilumatobacter, and Glaciihabitans were observed only in sediment samples whereas Rhodococcus, Corynebacterium, Micrococcus, Turicella, Pseudonocardia, Bifidobacterium, Nesterenkonia, Collinsella, Knoellia, Cadidatus, Actinomarina, Libanicoccus and Cutibacterium were noticed exclusively in water samples. Our study also emphasizes the need for further detailed study to understand the links between actinobacterial diversity and their ecological functions in the ISO. The available metabarcoding data paves the way for future research in cultivable forms of novel and rare Actinobacteria for their bioprospecting applications.

Isoptericola haloaureus sp. nov., a dimorphic actinobacterium isolated from mangrove sediments of southeast India, implicating biosaline agricultural significance through nitrogen fixation and salt tolerance genes.

Strain MP-1014T, an obligate halophilic actinobacterium, was isolated from the mangrove soil of Thandavarayancholanganpettai, Tamil Nadu, India. A polyphasic approach was utilized to explore its phylogenetic position completely. The isolate was Gram-positive, filamentous, non-motile, and coccoid in older cultures. Ideal growth conditions were seen at 30 °C and pH 7.0, with 5% NaCl (W/V), and the DNA G + C content was 73.3%. The phylogenic analysis of this strain based upon 16S rRNA gene sequence revealed 97-99.8% similarity to the recognized species of the genus Isoptericola. Strain MP-1014T exhibits the highest similarity to I. sediminis JC619T (99.7%), I. chiayiensis KCTC19740T (98.9%), and subsequently to I. halotolerans KCTC19646T (98.6%), when compared with other members within the Isoptericola genus (< 98%). ANI scores of strain MP-1014T are 86.4%, 84.2%, and 81.5% and dDDH values are 59.7%, 53.6%, and 34.8% with I. sediminis JC619T, I. chiayiensis KCTC19740T and I. halotolerans KCTC19646T respectively. The major polar lipids of the strain MP-1014T were phosphatidylinositol, phosphatidylglycerol, diphosphotidylglycerol, two unknown phospholipids, and glycolipids. The predominant respiratory menaquinones were MK9 (H4) and MK9 (H2). The major fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C14:0, C15:0, and C16:0. Also, initial genome analysis of the organism suggests it as a biostimulant for enhancing agriculture in saline environments. Based on phenotypic and genetic distinctiveness, the strain MP-1014 T represents the novel species of the genus Isoptericola assigned Isoptericola haloaureus sp. nov., is addressed by the strain MP-1014 T, given its phenotypic, phylogenetic, and hereditary uniqueness. The type strain is MP-1014T [(NCBI = OP672482.1 = GCA_036689775.1) ATCC = BAA 2646T; DSMZ = 29325T; MTCC = 13246T].

Tessaracoccus lacteus sp. nov., Isolated from the Sludge of a Wastewater Treatment Plant.

A bacterium, designated strain T21T, that is non-motile, rod-shaped, and formed pale white colonies, was isolated from the sludge of a wastewater treatment plant's secondary sedimentation tank in China. Strain T21T could grow at 20-40 °C (optimum growth at 30 °C), pH 3.0-10.0 (optimum growth at pH 5.0) and in the presence of 0-8.0% (w/v) NaCl (optimum growth at 2.0%). Based on phylogenetic analysis of 16S rRNA gene sequences and genome sequences, the isolate belongs to the genus Tessaracoccus in the phylum Actinomycetota. It exhibited a close relationship with Tessaracoccus palaemonis J1M15T, Tessaracoccus defluvii LNB-140T, Tessaracoccus flavescens SST-39T, and Tessaracoccus coleopterorum HDW20T. The 16S rRNA gene sequence similarities are 99.8%, 97.9%, 97.9%, and 97.8%, respectively. The major cellular fatty acids were anteiso-C15:0 and C16:0. The main respiratory quinone was MK-9(H4). The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, glycolipid, and phospholipid. Genome annotation of strain T21T predicted the presence of 2829 genes, of which 2754 are coding proteins and 59 are RNA genes. The genomic DNA G+C content was 69.2%. Based on the results of phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, we propose the name Tessaracoccus lacteus sp. nov. for this novel species within the genus Tessaracoccus. The type strain is T21T (=CCTCC AB 2023031T = KCTC 49936T).

Actinotalea lenta sp. nov., a novel actinomycete isolated from tidal flat sediment.

Two Gram-stain-positive, aerobic, oxidase- and catalase-negative, non-motile, and short rod-shaped actinomycetes, named SYSU T00b441T and SYSU T00b490, were isolated from tidal flat sediment located in Guangdong province, PR China. The 16S rRNA gene sequence similarity, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SYSU T00b441T and SYSU T00b490 were 99.3, 99.5 and 97.1 %, respectively. Strains SYSU T00b441T and SYSU T00b490 exhibited the highest 16S rRNA gene sequence similarities to Actinotalea ferrariae CF 5-4T (97.1 %/98.2 %), with ANI values of 74.01/73.88 % and dDDH values of 20.5/20.4 %. In the phylogenomic tree, the two isolates were affiliated with the genus Actinotalea. The genomes of strains SYSU T00b441T and SYSU T00b490 were 3.31 and 3.34 Mb, and both had DNA G+C contents of 72.8 mol%, coding 3077 and 3085 CDSs, three and three rRNA genes, and 53 and 51 tRNAs, respectively. Growth occurred at 15-40 °C (optimum, 28-30 °C), pH 4.0-10.0 (optimum, 7.0) and in the presence of 0-7 % (w/v) NaCl (optimum, 3 %). The major fatty acids (>10  %) of strains SYSU T00b441T and SYSU T00b490 were anteiso-C15 : 0 and C16 : 0. The major respiratory quinone was identified as MK-10(H4). The polar lipids of strains SYSU T00b441T and SYSU T00b490 were diphosphatidyl glycerol, phosphatidylglycerol, phosphoglycolipid, phosphatidyl ethanolamine, two phosphatidylinositol mannosides, two glycolipids and two phospholipids. Based on these data, the two strains (SYSU T00b441T and SYSU T00b490) represent a novel species of the genus Actinotalea, for which the name Actinotalea lenta sp. nov is proposed. The type strain is SYSU T00b441T (=GDMCC 1.3827T=KCTC 49943T).

A treasure trove of 1034 actinomycete genomes.

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.

Biological activities and metabolomic profiles of extracts from the marine sediment bacterium Nocardiopsis alba DP1B cultivated in different media.

The soil bacterium DP1B was isolated from a marine sediment collected off the coast of Randayan Island, Kalimantan Barat, Indonesia and identified based on 16S rDNA as Nocardiopsis alba. The bacterium was cultivated in seven different media (A1, ISP1, ISP2, ISP4, PDB, PC-1, and SCB) with three different solvents [distilled water, 5 % NaCl solution, artificial seawater (ASW)] combinations, shaken at 200 rpm, 30 °C, for 7 days. The culture broths were extracted with ethyl acetate and each extract was tested for its antimicrobial activity and brine shrimp lethality, and the chemical diversity was assessed using thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The result showed that almost all extracts showed antibacterial but not antifungal activity, whereas their brine shrimp toxicity levels vary from high to low. The best medium/solvent combinations for antibacterial activity and toxicity were PC-1 (in either distilled water, 5% NaCl solution, or ASW) and SCB in ASW. Different chemical diversity profiles were observed on TLC, GC-MS, and LC-MS/MS. Extracts from the PC-1 cultures seem to contain a significant number of cyclic dipeptides, whereas those from the SCB cultures contain sesquiterpenes, indicating that media and solvent compositions can affect the secondary metabolite profiles of DP1B. In addition, untargeted metabolomic analyses using LC-MS/MS showed many molecular ions that did not match with those in the Global Natural Products Social Molecular Networking (GNPS) database, suggesting that DP1B has great potential as a source of new natural products.

Pradimicin U, a promising antimicrobial agent isolated from a newly found Nonomuraea composti sp. nov.

Pradimicin U is a new dihydrobenzo[a]naphthacenequinone compound found to be active on a screen designed to investigate compounds with antimicrobial activity, produced by the actinomycete designated strain FMUSA5-5T. The strain was isolated from a bio-fertilizer of Musa spp. collected from Suphanburi province, Thailand. The chemotaxonomic characteristics and 16S rRNA gene analysis revealed that strain FMUSA5-5T is a member of the genus Nonomuraea. Low genome-based taxonomic criteria, average nucleotide identity (ANI) (82.8-88.3%), average amino-acid identity (AAI) (79.4-87.3%), and digital DNA-DNA hybridization (dDDH) (29.5-38.5%) values and several phenotypic differences between strain FMUSA5-5T and its closest type strains of the genus Nonomuraea indicated that strain FMUSA5-5T represents a novel species of the genus Nonomuraea and the name Nonomuraea composti sp. nov. is proposed for the strain. The crude extract from the culture broth of strain FMUSA5-5T displayed promising antimicrobial activity against several pathogens and led to the isolation of a novel secondary metabolite, pradimicin U. Interestingly, this compound displayed a broad spectrum of biological activities such as antimalarial activity against Plasmodium falciparum K1 (IC50 value = 3.65 µg/mL), anti-Mycobacterium tuberculosis H37Ra (MIC value = 25.0 µg/mL), anti-Alternaria brassicicola BCC 42724 (MIC value = 25.0 µg/mL), anti-Bacillus cereus ATCC 11778 and anti-Staphylococcus aureus ATCC 29213 (MIC values = 6.25 and 1.56 µg/mL, respectively). Moreover, the compound possessed strong anti-human small cell lung cancer (NCI-H187) activity with IC50 value of 5.69 µg/mL, while cytotoxicity against human breast cancer (MCF-7) and Vero cells was very weak (IC50 values of 52.49 and 21.84 µg/mL, respectively).

Biodegradation and antimicrobial capability-induced heavy metal resistance of the marine-derived actinomycetes Nocardia harenae JJB5 and Amycolatopsis marina JJB11.

Currently, heavy metal-resistant (HMR) marine actinomycetes have attracted much attention worldwide due to their unique capabilities. In this study, 27 marine-derived actinomycetes were isolated from coastal beaches in the Arabian Gulf of Al-Jubail in Saudi Arabia and screened for resistance to 100 mg/L of the heavy metals Cd2+, Cr6+, Cu2+, Fe2+, Pb2+, and Ni2+ using different assay techniques. Six isolates were selected as HMRs, of which two isolates, JJB5 and JJB11, exhibited the highest maximum tolerance concentrations (200- > 300 mg/L). Both isolates were the highest among six-HMR screened for their biodegradation potential of plastics low-density polyethylene, polystyrene, and polyvinyl chloride, recording the highest weight loss (15 ± 1.22 - 65 ± 1.2%) in their thin films. They also showed the highest biodegradability of the pesticides acetamiprid, chlordane, hexachlorocyclohexane, indoxacarb and lindane, indicating promising removal capacities (95.70-100%) for acetamiprid and indoxacarb using HPLC analysis. Additionally, the cell-free filtrate (CFF) of both isolates displayed the highest antimicrobial activity among the six-HMR screened against a variety of microbial test strains, recording the highest inhibition zone diameters (13.76 ± 0.66 - 26.0 ± 1.13 mm). GC‒MS analyses of the ethyl acetate extract of their CFFs revealed the presence of diverse chemical compounds with a multitude of remarkable biological activities. Based on their spore morphology and wall-chemotype, they were assigned to the nocardioform-actinomycetes. Furthermore, their phenotypic characteristics, together with 16S rRNA gene sequencing (OR121525-OR121526), revealed them as Nocardia harenae JJB5 and Amycolatopsis marina JJB11. Our results suggest that marine HMR actinomycetes are promising candidates for various biotechnological applications.

Biotechnological potential of actinomycetes in the 21st century: a brief review.

This brief review aims to draw attention to the biotechnological potential of actinomycetes. Their main uses as sources of antibiotics and in agriculture would be enough not to neglect them; however, as we will see, their biotechnological application is much broader. Far from intending to exhaust this issue, we present a short survey of the research involving actinomycetes and their applications published in the last 23 years. We highlight a perspective for the discovery of new active ingredients or new applications for the known metabolites of these microorganisms that, for approximately 80 years, since the discovery of streptomycin, have been the main source of antibiotics. Based on the collected data, we organize the text to show how the cosmopolitanism of actinomycetes and the evolutionary biotic and abiotic ecological relationships of actinomycetes translate into the expression of metabolites in the environment and the richness of biosynthetic gene clusters, many of which remain silenced in traditional laboratory cultures. We also present the main strategies used in the twenty-first century to promote the expression of these silenced genes and obtain new secondary metabolites from known or new strains. Many of these metabolites have biological activities relevant to medicine, agriculture, and biotechnology industries, including candidates for new drugs or drug models against infectious and non-infectious diseases. Below, we present significant examples of the antimicrobial spectrum of actinomycetes, which is the most commonly investigated and best known, as well as their non-antimicrobial spectrum, which is becoming better known and increasingly explored.

Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Description of an anaerobic actinobacterium, Kribbibacterium absianum gen. nov., sp. nov., a new member of the novel family Kribbibacteriaceae fam. nov., and reclassification of the genera Granulimonas and Leptogranulimonas.

Two rod-shaped, obligate anaerobic, Gram-stain-positive bacteria isolated from the pig faeces were designated YH-ols2216 and YH-ols2217T. Analysis of 16S rRNA gene sequences revealed that these isolates were most related to the members of the family Atopobiaceae, within the order Coriobacteriales, and Granulimonas faecalis KCTC 25474T with 92.0 and 92.5% similarities, respectively. The 16S rRNA gene sequence similarity within isolates was 99.9 %; and those between isolates YH-ols2216 and YH-ols2217T, and Atopobium minutum DSM 20586T, the type species of the type genus Atopobium within the family Atopobiaceae, were 88.5 and 88.7 %, respectively. Those between isolates and Coriobacterium glomerans PW2T, the type species of the type genus Coriobacterium within the family Coriobacteriaceae, were 88.7 and 89.1 %, respectively. The multi-locus sequence tree revealed that the isolates, alongside the genera Granulimonas and Leptogranulimonas, formed a distinct cluster between the families Atopobiaceae and Coriobacteriaceae. The average nucleotide identities and digital DNA-DNA hybridization values for the isolates and their most closely related strains ranged from 67.7 to 76.2 % and from 18.4 to 23.3 %, respectively. The main cellular fatty acids of the isolates were C18 : 0 DMA, C18 : 1 ω9c, C18 : 0 12OH, C18 : 0, and C16 : 0. The cell wall contained the peptidoglycan meso-diaminopimelic acid. Lactate was the main end-product of the isolates. The major polar lipids of isolate YH-ols2217T were aminophospholipid, aminolipids, and lipids. Menaquinones were not identified in the cells of the isolates. The DNA G+C contents of isolates YH-ols2216 and YH-ols2217T were 67.5 and 67.6 mol%, respectively. Considering these chemotaxonomic, phenotypic, and phylogenetic properties, Kribbibacteriaceae fam. nov. is proposed within the order Coriobacteriales. YH-ols2216 (=KCTC 25708=NBRC 116429) and YH-ols2217T (=KCTC 25709T=NBRC 116430T) represent a novel taxon within this new family and the name Kribbibacterium absianum gen. nov., sp. nov. is proposed. In addition, the genera Granulimonas and Leptogranulimonas are transferred to the family Kribbibacteriaceae fam. nov.

Genomic and physiological characterization of Kitasatospora sp. nov., an actinobacterium with potential for biotechnological application isolated from Cerrado soil.

An Actinobacteria - Kitasatospora sp. K002 - was isolated from the soil of Cerrado, a savanna-like Brazilian biome. Herein, we conducted a phylogenetic, phenotypic and physiological characterization, revealing its potential for biotechnological applications. Kitasatospora sp. K002 is an aerobic, non-motile, Gram-positive bacteria that forms grayish-white mycelium on solid cultures and submerged spores with vegetative mycelia on liquid cultures. The strain showed antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli. Genomic analysis indicated that Kitasatospora xanthocidica JCM 4862 is the closest strain to K002, with a dDDH of 32.8-37.8% and an ANI of 86.86% and the pangenome investigations identified a high number of rare genes. A total of 60 gene clusters of 22 different types were detected by AntiSMASH, and 22 gene clusters showed low similarity (< 10%) with known compounds, which suggests the potential production of novel bioactive compounds. In addition, phylogenetic analysis and morphophysiological characterization clearly distinguished Kitasatospora sp. K002 from other related species. Therefore, we propose that Kitasatospora sp. K002 should be recognized as a new species of the genus Kitasatospora - Kitasatospora brasiliensis sp. nov. (type strains = K002).

Stomatohabitans albus gen. nov., sp. nov., an oral living facultative anaerobic actinobacteria isolated form Steller sea lion, and proposal of Stomatohabitantaceae fam. nov. and Stomatohabitantales ord. nov.

Two novel actinobacteria, designated as SYSU M7M538T and SYSU M7M531, were isolated from oral of Eumetopias jubatus in Zhuhai Chimelong Ocean Kingdom, China. The cells of these microorganisms stained Gram-positive and were rod shaped. These strains were facultative anaerobic, and catalase-positive. Optimal growth occurred at 37 °C and pH 7.0 over 7 days of cultivation. Both strains possessed diphosphatidylglycerol, phosphatidylglycerol and phosphocholine as the major polar lipids. The main menaquinone was MK-9(H4). The major fatty acids were C16:0, C17:1w8c, C17:0, C18:1w9c and C18:0. Analyses of genome sequences revealed that the genome size of SYSU M7M538T was 2.1 Mbp with G + C content of 52.5 %, while the genome size of SYSU M7M531 was 2.3 Mbp with G + C content of 52.7 %. The ANI and 16S rRNA gene analysis results showed that the pairwise similarities between the two strains and other recognized Nitriliruptoria species were less than 64.9 % and 89.0 %, respectively. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains SYSU M7M538T and SYSU M7M531 formed a well-separated phylogenetic branch distinct from other orders of Nitriliruptoria. Based on the data presented here, these two strains are considered to represent a novel species of a novel genus, for which the name Stomatohabitans albus gen. nov., sp. nov., with the type strain SYSU M7M538T (=KCTC 59113T = GDMCC 1.4286T), are proposed. We also propose that these organisms represent a novel family named Stomatohabitantaceae fam. nov. of a novel order Stomatohabitantales ord. nov.