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).

The comparative plastisphere microbial community profile at Kung Wiman beach unveils potential plastic-specific degrading microorganisms.

Background: Plastic waste is a global environmental issue that impacts the well-being of humans, animals, plants, and microorganisms. Microplastic contamination has been previously reported at Kung Wiman Beach, located in Chanthaburi province along with the Eastern Gulf of Thailand. Our research aimed to study the microbial population of the sand and plastisphere and isolate microorganisms with potential plastic degradation activity. Methods: Plastic and sand samples were collected from Kung Wiman Beach for microbial isolation on agar plates. The plastic samples were identified by Fourier-transform infrared spectroscopy. Plastic degradation properties were evaluated by observing the halo zone on mineral salts medium (MSM) supplemented with emulsified plastics, including polystyrene (PS), polylactic acid (PLA), polyvinyl chloride (PVC), and bis (2-hydroxyethyl) terephthalate (BHET). Bacteria and fungi were identified by analyzing nucleotide sequence analysis of the 16S rRNA and internal transcribed spacer (ITS) regions, respectively. 16S and ITS microbiomes analysis was conducted on the total DNA extracted from each sample to assess the microbial communities. Results: Of 16 plastic samples, five were identified as polypropylene (PP), four as polystyrene (PS), four as polyethylene terephthalate (PET), two as high-density polyethylene (HDPE), and one sample remained unidentified. Only 27 bacterial and 38 fungal isolates were found to have the ability to degrade PLA or BHET on MSM agar. However, none showed degradation capabilities for PS or PVC on MSM agar. Notably, Planococcus sp. PP5 showed the highest hydrolysis capacity of 1.64 ± 0.12. The 16S rRNA analysis revealed 13 bacterial genera, with seven showing plastic degradation abilities: Salipiger, Planococcus, Psychrobacter, Shewanella, Jonesia, Bacillus, and Kocuria. This study reports, for the first time of the BHET-degrading properties of the genera Planococcus and Jonesia. Additionally, The ITS analysis identified nine fungal genera, five of which demonstrated plastic degradation abilities: Aspergillus, Penicillium, Peacilomyces, Absidia, and Cochliobolus. Microbial community composition analysis and linear discriminant analysis effect size revealed certain dominant microbial groups in the plastic and sand samples that were absent under culture-dependent conditions. Furthermore, 16S and ITS amplicon microbiome analysis revealed microbial groups were significantly different in the plastic and sand samples collected. Conclusions: We reported on the microbial communities found on the plastisphere at Kung Wiman Beach and isolated and identified microbes with the capacity to degrade PLA and BHET.

Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

Potential of different species of actinobacteria in the management of Meloidogyne javanica.

Root-knot nematodes (RKN) are one of the most harmful soil-borne plant pathogens in the world. Actinobacteria are known phytopathogen control agents. The aim of this study was to select soil actinobacteria with control potential against the RKN (Meloidogyne javanica) in tomato plants and to determine mechanisms of action. Ten isolates were tested and a significant reduction was observed in the number of M. javanica eggs, and galls 46 days after infestation with the nematode. The results could be explained by the combination of different mechanisms including parasitism and induction of plant defense response. The M. javanica eggs were parasited by all isolates tested. Some isolates reduced the penetration of juveniles into the roots. Other isolates using the split-root method were able to induce systemic defenses in tomato plants. The 4L isolate was selected for analysis of the expression of the plant defense genes TomLoxA, ACCO, PR1, and RBOH1. In plants treated with 4L isolate and M. javanica, there was a significant increase in the number of TomLoxA and ACCO gene transcripts. In plants treated only with M. javanica, only the expression of the RBOH1 and PR1 genes was induced in the first hours after infection. The isolates were identified using 16S rRNA gene sequencing as Streptomyces sp. (1A, 3F, 4L, 6O, 8S, 9T, and 10U), Kribbella sp. (5N), Kitasatospora sp. (2AE), and Lentzea sp. (7P). The efficacy of isolates from the Kitasatospora, Kribbella, and Lentzea genera was reported for the first time, and the efficacy of Streptomyces genus isolates for controlling M. javanica was confirmed. All the isolates tested in this study were efficient against RKN. This study provides the opportunity to investigate bacterial genera that have not yet been explored in the control of M. javanica in tomatoes and other crops.

Combinatorial Biosynthesis of 3-O-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A. pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A. pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.

Microbacterium aquilitoris sp. nov. and Microbacterium gwkjiense sp. nov., isolated from beach.

Three Gram-reaction-positive bacterial strains, designated KSW-18T, KSW2-22, and KSW4-11T, were isolated from seawater, and two dried seaweed samples collected at Gwakji Beach in Jeju, Republic of Korea, respectively, and their taxonomic positions were examined by a polyphasic approach. The 16S rRNA gene phylogeny showed that strain KSW4-11T was tightly associated with Microbacterium oleivorans NBRC 103075T, while strains KSW-18T and KSW2-22 formed a distinctive subline at the base of a clade including the above two strains. The three isolates showed high sequence similarity with one another (99.7-99.9%; 1-4 nt differences) and Microbacterium oleivorans (99.8-99.9%; 1-3 nt differences). The chemotaxonomic features were typical for the genus Microbacterium; Lysine as the diagnostic diamino acid and N-glycolylated muramic acid of the peptidoglycans, the predominant menaquinones of MK-11, MK-10 and MK-12, the major fatty acids of anteiso-C15:0 and anteiso-C17:0, and the major polar lipids including diphosphatidylglycerol, phosphatidylglycerol, and two or three unidentified glycolipids. In core genome-based phylogenetic tree, strains KSW-18T and KSW2-22 were closely associated with Microbacterium oleivorans NBRC 103075T, while strain KSW4-11T formed a distinctive subline at the base of a clade including the above three strains, in contrast to the 16S rRNA gene tree. Strains KSW-18T and KSW2-22 shared an OrthoANIu of 98.6% and a digital DNA-DNA hybridization of 87.6% with each other, representing that they were strains of a species, while the OrthoANIu and digital DNA-DNA hybridization values between strains KSW-18T and KSW4-11T, and between both of these isolates and all members of the genus Microbacterium were ≤86.5% and ≤30.7%, respectively. The analyses of overall genomic relatedness indices and phenotypic distinctness support that the three isolates represent two new species of the genus Microbacterium. Based on the results obtained here, Microbacterium aquilitoris sp. nov. (type strain KSW-18T = KCTC 49623T = NBRC 115222T) and Microbacterium gwkjiense sp. nov. (type strain KSW4-11T = KACC 23321T = DSM 116380T) are proposed.

Speluncibacter jeojiensis gen. nov. sp. nov., a novel bacterium of the order Mycobacteriales isolated from a cave and a proposal of Speluncibacteraceae fam. nov.

Two Gram-stain-positive, aerobic, non-spore-forming, non-motile, irregular rod-shaped actinobacteria, designated as D2-41T and D3-21, were isolated from soil samples collected in a natural cave in Jeju, Republic of Korea. Both of the isolates were shown to share 100 % 16S rRNA sequence identity. The cell wall contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H2). The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid, an unidentified aminoglycolipid, an unidentified phospholipid and two unidentified lipids. The predominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH). Mycolic acids of C30-C38 were present. The 16S rRNA gene trees showed that the organisms occupied a distinct position remotely located from recognized genera within the order Mycobacteriales, albeit with the 16S rRNA gene similarities of 97.0-97.1 % with Rhodococcus olei, Rhodococcus rhodnii and Rhodococcus triatomae. The genome sizes and DNA G+C contents of strains D2-41T and D3-21 were 4.77-4.88 Mbp and 69.8 mol%, respectively. Both of the isolates shared an average nucleotide identity of 99.4 % and digital DNA-DNA hybridization of 95.2 % to each other, revealing that strains D2-41T and D3-21 belonged to the same species. In the core genome-based phylogenomic tree, both of the isolates were found to be closely associated with members of the genus Tomitella. However, strains D2-41T and D3-21 revealed the highest amino acid identity values (mean 66.5 %, range 66.2-67.0 % with the genus Prescottella of the family Nocardiaceae, followed by the genus Tomitella (mean 64.1 %, range 63.6-64.7 %) of the family Tomitellaceae. Based on the combined data obtained here, the novel isolates belong to a new genus of the new family for which the name Speluncibacter jeojiensis gen. nov. sp. nov. is proposed, with Speluncibacteraceae fam. nov. The type strain is strain D2-41T (=KACC 17930T=DSM 101875T).

Bacterial community and culturable actinomycetes of Phyllostachys viridiglaucescens rhizosphere.

During the course of development plants form tight interactions with microorganisms inhabiting their root zone. In turn, rhizosphere bacteria, in particular members of the phylum Actinomycetota, positively influence the host plant by increasing access to essential nutrients and controlling the pathogenic microorganism's population. Herein, we report the characterisation of the rhizosphere associated actinobacteria community of Phyllostachys viridiglaucescens growing in the Nikitsky Botanical Garden (Crimean Peninsula, Ukraine). The overall composition of the bacterial community was elucidated by 16S rRNA gene amplicon sequencing followed by isolation of culturable microorganisms with the focus on actinomycetes. The metagenomic approach revealed that the representatives of phylum Actinomycetota (57.1%), Pseudomonadota (20.0%), and Acidobacteriota (12.2%) were dominating in the studied microbiome with Ilumatobacter (phylum Actinomycetota) (13.1%) being the dominant genus. Furthermore, a total of 159 actinomycete isolates, belonging to eight genera of Streptomyces, Micromonospora, Nonomuraea, Arthrobacter, Actinomadura, Kribbella, Cellulosimicrobium, and Mumia, were recovered from P. viridiglaucescens rhizosphere. The isolated species were tested for antimicrobial activity. 64% of isolates were active against at least one bacterial test-culture and 7.5% against fungal test culture. In overall, the rhizosphere bacterial communities act as a great source of actinobacterial diversity with the high potential for production of new bioactive compounds.

Simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata with glucose as carbon source under aerobic conditions.

Previous researches have recognized the vital role of Tetrasphaera elongata in enhanced biological phosphorus removal systems, but the underlying mechanisms remain under-investigated. To address this issue, this study investigated the metabolic characteristics of Tetrasphaera elongata when utilizing glucose as the sole carbon source. Results showed under aerobic conditions, Tetrasphaera elongata exhibited a glucose uptake rate of 136.6 mg/(L·h) and a corresponding phosphorus removal rate of 8.6 mg P/(L·h). Upregulations of genes associated with the glycolytic pathway and oxidative phosphorylation were observed. Noteworthily, the genes encoding the two-component sensor histidine kinase and response regulator transcription factor exhibited a remarkable 28.3 and 27.4-fold increase compared with the group without glucose. Since these genes play a pivotal role in phosphate-specific transport systems, collectively, these findings shed light on a potential mechanism for simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata under aerobic conditions, providing fresh insights into phosphorus removal from wastewaters.

Catheter-related bloodstream infection caused by Tsukamurella tyrosinosolvens identified by secA1sequencing in an immunocompromised child: a case report.

BACKGROUND: Tsukamurella spp. are obligate aerobic, gram-positive, non-motile, and slightly acid-fast bacilli belonging to the Actinomycetes family. They share many characteristics with Nocardia, Rhodococcus, Gordonia, and the rapidly growing Mycobacterium species. Therefore, standard testing may misidentify Tsukamurella spp. as another species. Accurate and rapid diagnosis is critical for proper infection management, but identification of this bacterium is difficult in the standard laboratory setting. CASE PRESENTATION: A bloodstream infection caused by a gram-positive bacterium and related to a central venous catheter was identified in an immunocompromised 2-year-old girl. Tsukamurella tyrosinosolvens was identified by modified secA1 sequencing. Antibiotic treatment and removal of the central venous catheter resolved the infection. Inappropriate management of the catheter during an overnight stay outside of the hospital was considered as a possible source of infection. CONCLUSIONS: SecA1 sequencing may be a useful diagnostic tool in the identification of T. tyrosinosolvens. Providing proper central venous catheter care instructions to patients, their families, and medical staff is important for infection prevention.

Development of a multiplex real-time PCR method for the detection of Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens in soybean seeds.

Multiplex real-time PCR with TaqMan® probes has been developed for the simultaneous detection of soybean pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens. The method specificity has been confirmed using 25 strains of target bacteria and 18 strains of other bacteria common to soybean seeds as endophytes. The multiplex real-time PCR developed has been shown to have high sensitivity - a positive result was achieved at 0.01 ng/µl of DNA for both target organisms, and at 100 CFU/ml of bacteria in soybean seed homogenate. The robustness of the multiplex real-time PCR developed has been verified by the detection of the pathogens in 25 commercial seed stocks, in comparison with previously published PCR protocols. In all tests, three seed stocks were positive and 22 were negative. The multiplex real-time PCR can be applied in diagnostic practice for the simultaneous detection of two important pathogens of leguminous plants.

Microbacterium nymphoidis sp. nov. and Microbacterium festucae sp. nov., two novel species with high plant-promoting potential isolated from wetland plants in China.

Two novel plant growth-promoting, rod-shaped, Gram-positive and non-motile rhizobacteria, W1NT and W2RT, were isolated from wetland plants Festuca elata and Nymphoides peltatum, respectively, in China. The results of the 16S rRNA sequence alignment analysis showed that they were related to Microbacterium, with the highest similarity to Microbacterium ketosireducens (98.7 %) and Microbacterium laevaniformans (98.5 %) for strain W1NT, and to Microbacterium terricola (98.1 %) and Microbacterium marinum (98.0 %) for strain W2RT. Phylogenetic analyses based on 16S rRNA gene sequences and 92 conserved concatenated proteins suggested that the two strains belong to the genus Microbacterium and were placed in two separate novel phylogenetic clades. The genome sizes of the two strains were 3.2 and 3.7 Mb, and the G+C contents were 71.7 and 68.5 mol%, respectively. The comparative genome results showed that the average nucleotide identity values between W1NT and W2RT and other species ranged from 73.5 to 83.6 %, and the digital DNA-DNA hybridization values ranged from 19.7 to 26.8 %. These two strains show physiological and biochemical features that differ from those of closely related species. Rhamnose, galactose and glucose were present in the characteristic sugar fractions of strains W1NT and W2RT. The peptidoglycan of strains W1NT and W2RT contained the amino acids ornithine, alanine and aspartic acid. C15 : 0 anteiso, C17 : 0 anteiso and C16 : 0 iso were the predominant cellular fatty acids in W1NT and W2RT. Phosphatidylglycerol and diphosphatidylglycerol are major polar lipid components. Strain W1NT not only formed bacterial biofilms but also had the ability to solubilize phosphorus and produce indole-3-acetic acid. Strain W2RT had siderophore-producing and lignin-degrading properties. Based on their genetic and phenotypic characteristics, strains W1NT and W2RT were classified as novel bacteria in the genus Microbacterium and designated as Microbacterium festucae sp. nov. (type strain W1NT=ACCC 61807T=GDMCC 1.2966T=JCM 35339T) and Microbacterium nymphoidis sp. nov. (type strain W2RT=ACCC 61808T=GDMCC 1.2967T=JCM 35340T).

Systematic investigation of the emerging pathogen of Tsukamurella species in a Chinese tertiary teaching hospital.

Tsukamurella species have been clinically regarded as rare but emerging opportunistic pathogens causing various infections in humans. Tsukamurella pneumonia has often been misdiagnosed as pulmonary tuberculosis due to its clinical presentation resembling tuberculosis-like syndromes. Tsukamurella species have also been confused in the laboratory with other phylogenetic bacteria, such as Gordonia. This study aimed to investigate the clinical, microbiological, and molecular characteristics; species distribution; and antimicrobial susceptibility of Tsukamurella species. Immunodeficiency and chronic pulmonary disease appeared to be risk factors for Tsukamurella pneumonia, and the presence of bronchiectasis and pulmonary nodules on imaging was highly correlated with this infection. The study confirmed that groEL (heat shock protein 60) and secA (the secretion ATPase) genes are reliable for identifying Tsukamurella species. Additionally, the ssrA (stable small RNA) gene showed promise as a tool for discriminating between different Tsukamurella species with the shortest sequence length. In terms of antimicrobial susceptibility, quinolones, trimethoprim/sulfamethoxazole, amikacin, minocycline, linezolid, and tigecycline demonstrated potent in vitro activity against Tsukamurella isolates in our study. The study also proposed a resistance mechanism involving a substitution (S91R) within the quinolone-resistance-determining region of the gyrA gene, which confers resistance to levofloxacin and ciprofloxacin. Furthermore, we found that disk diffusion testing is not suitable for testing the susceptibilities of Tsukamurella isolates to ciprofloxacin, imipenem, and minocycline. In conclusion, our systematic investigation may contribute to a better understanding of this rare pathogen. Tsukamurella species are rare but emerging human pathogens that share remarkable similarities with other mycolic acid-containing genera of the order Actinomycetales, especially Mycobacterium tuberculosis. Consequently, misdiagnosis and therapeutic failures can occur in clinical settings. Despite the significance of accurate identification, antimicrobial susceptibility, and understanding the resistance mechanism of this important genus, our knowledge in these areas remains fragmentary and incomplete. In this study, we aimed to address these gaps by investigating promising identification methods, the antimicrobial susceptibility patterns, and a novel quinolone resistance mechanism in Tsukamurella species, utilizing a collection of clinical isolates. The findings of our study will contribute to improve diagnosis and successful management of infections caused by Tsukamurella species, as well as establishing well-defined performance and interpretive criteria for antimicrobial susceptibility testing.

Comparison of bacterial and fungal communities structure and dynamics during chicken manure and pig manure composting.

Composting is a sustainable and eco-friendly technology that turns animal waste into organic fertilizers. It remains unclear whether differences exist in the structure of microbial communities during different livestock manure composting. This study analyzed the dynamic change of bacterial and fungal communities, metabolic function, and trophic mode during chicken manure (CM) and pig manure (PM) composting based on 16S rRNA and ITS sequencing. Environmental factors were investigated for their impact on microbial communities. During composting, bacterial diversity decreased and then increased, while fungal diversity slightly increased and then decreased. Saccharomonospora and Aspergillus were the dominant genera and key microorganisms in CM and PM, respectively, which played crucial roles in sustaining the stability of the ecological network structure in the microbial ecology and participating in metabolism. Saccharomonospora gradually increased, while Aspergillus increased at first and then decreased. PM had better microbial community stability and more keystone taxa than CM. In CM and PM, the primary function of bacterial communities was metabolism, while saprotroph was the primary trophic mode of fungal communities. Dissolved organic carbon (DOC) was the primary factor influencing the structure and function of microbial communities in CM and PM. In addition to DOC, pH and moisture were important factors affecting the fungal communities in CM and PM, respectively. These results show that the succession of bacteria and fungi in CM and PM proceeded in a similar pattern, but there are still some differences in the dominant genus and their responses to environmental factors.

Complete genome sequence analysis of plant growth-promoting bacterium, Isoptericola sp. AK164 isolated from the rhizosphere of Avicennia marina growing at the Red Sea coast.

Isoptericola sp. AK164 is a Gram-positive, aerobic bacterial genus from the family Promicromonosporaceae, isolated from the root rhizosphere of Avicennia marina. AK164 significantly enhanced the growth of the Arabidopsis thaliana plant under normal and saline conditions. These bacteria can produce ACC deaminase and several enzymes playing a role in carbohydrate hydrolyses, such as cellulose, hemicellulose, and chitin degradation, which may contribute to plant growth, salt tolerance, and stress elevation. The genome sequence AK164 has a single circular chromosome of approximately 3.57 Mbp with a GC content of 73.53%. A whole genome sequence comparison of AK164 with type strains from the same genus, using digital DNA-DNA hybridization and average nucleotide identity calculations, revealed that AK164 might potentially belong to a new species of Isoptericola. Genome data and biochemical analyses indicate that AK164 could be a potential biostimulant for improving agriculture in submerged saline land.

Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes.

CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.

Agromyces larvae sp. nov., a novel actinobacterium isolated from gut of larvae of Protaetia brevitarsis seulensis.

A novel actinobacterium strain, designated CFWR-12T, was isolated from the larval gut of Protaetia brevitarsis seulensis grown at the National Institute of Agricultural Sciences, Wanju-gun, Republic of Korea, and its taxonomic position was evaluated. Strain CFWR-12T was aerobic, Gram-stain-positive and non-motile. Growth occurred at 10-40 °C, pH 6.0-9.0 and 0-4 % (w/v) NaCl, with optimal growth at 28-30 °C, pH 7.0 and in the absence of NaCl. Strain CFWR-12T showed high 16S rRNA gene sequence similarity to Agromyces intestinalis KACC 19306T (99.0 %) and Agromyces protaetiae FW100M-8T (97.9 %). The genome sequence of strain CFWR-12T was 4.01 Mb in size with a high G+C content of 71.2 mol%. The values of average nucleotide identity and digital DNA-DNA hybridization between strain CFWR-12T and A. intestinalis KACC 19306T were 89.8 and 39.1 %, respectively, which were the highest among the closely related Agromyces species. The predominant cellular fatty acids (>10 %) were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0, and the major respiratory quinones (>10 %) were MK-11 and MK-12. The polar lipids were composed of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and an unidentified lipid while the peptidoglycan type was identified to be B1. Data based on chemotaxonomic, phylogenetic, phenotypic and genomic evidence demonstrated that strain CFWR-12T represents a novel species of the genus Agromyces, for which the name Agromyces larvae sp. nov. is proposed. The type strain is strain CFWR-12T (=KACC 19307T= NBRC 113047T).

Streptomyces biostimulants: an effective sustainable approach to reduce inorganic N input and maintain high yield of wheat crop in different soil types.

AIMS: This study aimed to isolate and characterize endophytic plant growth-promoting (PGP) actinomycetes from the wild medicinal plant Zygophyllum album. METHODS AND RESULTS: Eight actinomycetes were isolated, identified, and screened for their PGP activities to improve the growth and production of wheat plants under low N-inputs. Based on 16S rRNA analysis, the isolated actinobacteria showed high diversity and had multiple in vitro PGP attributes. In pot experiments, Streptomyces sp. NGB-Act4 and NGB-Act6 demonstrated the highest significant PGP activities to enhance the growth of wheat plants under reduced N-inputs. Under various field conditions (high-fertility clay soils and low-fertility sandy soils), in combination with 50% N-dose, the two streptomycetes showed significant increases in grain N% and grain yield of the wheat crop compared with the 50% N-fertilized treatment. Irrespective of soil type, wheat plants inoculated with strain NGB-Act4 produced grain yield and grain N% significantly greater than or comparable to the full N-dose treatment. CONCLUSIONS: This is the first field report on the successful use of endophytic streptomycetes as an effective strategy to improve wheat yield and reduce the use of synthetic N fertilizers.

Microbiome assembly mechanism and functional potential in enhanced biological phosphorus removal system enriched with Tetrasphaera-related polyphosphate accumulating organisms.

Tetrasphaera-related polyphosphate accumulating organisms (PAOs) are the key functional guilds for enhanced biological phosphorus removal (EBPR) systems. Their biomass enrichment can be enhanced by the nitrification inhibitor allylthiourea (ATU). However, the underlying assembly mechanism and the functional potential of the EBPR microbiome regulated by ATU are unclear. This study investigates the effect of ATU on microbiome assembly and functional potential by closely following the microbiota dynamics in an EBPR system enriched with Tetrasphaera-related PAOs for 288-days before, during and after ATU addition. The results showed that ATU addition increased microbiota structural similarity and compositional convergence, and enhanced determinism in the assembly of EBPR microbiome. During exposure to ATU, Tetrasphaera-related PAOs were governed by homogeneous selection and the dominant species revealed by 16S rRNA gene-based phylogenetic analysis shifted from clade III to clade I. Meanwhile, ATU supply promoted significant enrichment of functional genes involved in phosphate transport (pit) and polyphosphate synthesis and degradation (ppk1 and ppk2), whereas both Nitrosomonas and ammonia monooxygenase-encoding genes (amoA/B/C) assignable to this group of nitrifying bacteria decreased. Moreover, ATU addition relieved the significant abundance correlation between filamentous bacteria Ca. Promineofilum and denitrifying Brevundimonas (FDR-adjusted P < 0.01), damaging their potential synergic or cooperative interactions, thus weakening their competitiveness against Tetrasphaera-related PAOs. Notably, ATU withdrawn created opportunistic conditions for the unexpected explosive growth and predominance of Thiothrix filaments, leading to a serious bulking event. Our study provides new insights into the microbial ecology of Tetrasphaera-related PAOs in EBPR system, which could guide the establishment of an efficient microbiota for EBPR.