Soil Isolates Research Articles

Antiproliferative and antimicrobial compounds from Streptomyces levis: supported by in vitro and in silico molecular docking approach

The present work describes the investigation of secondary metabolites produced by rhizospheric soil isolate Streptomyces levis with respect to partial purification, bioactivity, structural elucidation, and docking study. MIC (Minimum Inhibitory Concentration) of the fraction was found to be 16 µg/mL, 32 µg/mL, and 8 µg/mL against S. aureus, E. coli, and C. albicans respectively. The fraction exhibited an IC50 of 90.48 μg/mL and 59.10 µg/mL against L929 and HeLa cell lines respectively. Spectroscopic analysis showed abundant of Isofucosterol 3-O-[6-O-(9-Octadecanoyl)-b-D-glucopyranoside], and Viniferal. These compounds were docked against receptor molecules E6, E7, Caspase-3, and Bcl2. The in-silico results suggested that all the secondary metabolites showed higher docking scores than the standard compound 5-Fluorouracil. Molecular docking study and published literature suggested that Isofucosterol 3-O-[6-O-(9-Octadecanoyl)-b-D-glucopyranoside] inactivated viral oncoprotein E7 and arrested the cell cycle from entering in S phase. Viniferal induced apoptosis by inactivation of Bcl2.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Metagenomic nanopore sequencing for exploring the nature of antimicrobial metabolites of Bacillus haynesii

Multidrug-resistant (MDR) pathogens are a rising global health worry that imposes an urgent need for the discovery of novel antibiotics particularly those of natural origin. In this context, we aimed to use the metagenomic nanopore sequence analysis of soil microbiota coupled with the conventional phenotypic screening and genomic analysis for identifying the antimicrobial metabolites produced by promising soil isolate(s). In this study, whole metagenome analysis of the soil sample(s) was performed using MinION™ (Oxford Nanopore Technologies). Aligning and analysis of sequences for probable secondary metabolite gene clusters were extracted and analyzed using the antiSMASH version 2 and DeepBGC. Results of the metagenomic analysis showed the most abundant taxa were Bifidobacterium, Burkholderia, and Nocardiaceae (99.21%, followed by Sphingomonadaceae (82.03%) and B. haynesii (34%). Phenotypic screening of the respective soil samples has resulted in a promising Bacillus isolate that exhibited broad-spectrum antibacterial activities against various MDR pathogens. It was identified using microscopical, cultural, and molecular methods as Bacillus (B.) haynesii isolate MZ922052. The secondary metabolite gene analysis revealed the conservation of seven biosynthetic gene clusters of antibacterial metabolites namely, siderophore lichenicidin VK21-A1/A2 (95% identity), lichenysin (100%), fengycin (53%), terpenes (100%), bacteriocin (100%), Lasso peptide (95%) and bacillibactin (53%). In conclusion, metagenomic nanopore sequence analysis of soil samples coupled with conventional screening helped identify B. haynesii isolate MZ922052 harboring seven biosynthetic gene clusters of promising antimicrobial metabolites. This is the first report for identifying the bacteriocin, lichenysin, and fengycin biosynthetic gene clusters in B. haynesii MZ922052.

Emergence of colistin-resistant Stenotrophomonas maltophilia with high virulence in natural aquatic environments

The presence of Stenotrophomonas maltophilia in aquatic environments poses great health risks to immunocompromised individuals because of its multidrug resistance and resultant high mortality. However, a significant gap exists in the isolation and understanding of colistin-resistant S. maltophilia in aquatic environments. In this study, nine colistin-resistant S. maltophilia strains isolated from natural lakes were explored, and their phylogenetic relationship, biofilm formation, virulence, and antibiotic resistance profiles and underlying genetic determinants were assessed. After genome analysis, besides known multi-locus sequence typing (MLST) of ST532, new assigned ST965 and ST966 which phylogenetically clustered into soil isolates were found firstly. All the isolates exhibited resistance to multiple antibiotics, including aminoglycosides, beta-lactams, tetracyclines, and even colistin, with the highest minimum inhibitory concentration (MIC) against colistin reaching 640 mg/L. Comparative genomic analysis revealed aph(3′)-Iic, blaL1, tetT, phoP, mcr-3, arnA, pmrE, and efflux pump genes as the genetic determinants underlying this multidrug resistance. Notably, the biofilm-forming capacities of the newly discovered ST965 and ST966 isolates were significant stronger than those of the known ST532 isolates (p < 0.01), resulting in the death of over 50 % of the Galleria mellonella population within 1 day of injection. The ST965 isolates demonstrated the highest virulence against G. mellonella, followed by the ST966 isolates and ST532 isolates which was phylogenetically clustered with clinical isolates, indicating that the novel S. maltophilia strains of ST965 and ST966 may pose considerable health risks to humans. Our findings provide insights into colistin-resistant S. maltophilia in aquatic environments and raise concerns about the health risks posed by the newly assigned sequence types of colistin-resistant S. maltophilia with potential high virulence in natural aquatic environments.

Genome analysis and description of Tunturibacter gen. nov. expands the diversity of Terriglobia in tundra soils.

Increased temperatures in Arctic tundra ecosystems are leading to higher microbial respiration rates of soil organic matter, resulting in the release of carbon dioxide and methane. To understand the effects of this microbial activity, it is important to better characterize the diverse microbial communities in Arctic soil. Our goal is to refine our understanding of the phylogenetic diversity of Terriglobia, a common but elusive group within the Acidobacteriota phylum. This will help us link this diversity to variations in carbon and nitrogen usage patterns. We used long-read Oxford Nanopore MinION sequences in combination with metagenomic short-read sequences to assemble complete Acidobacteriota genomes. This allowed us to build multi-locus phylogenies and annotate pangenome markers to distinguish Acidobacteriota strains from several tundra soil isolates. We identified a phylogenetic cluster containing four new species previously associated with Edaphobacter lichenicola. We conclude that this cluster represents a new genus, which we have named Tunturibacter. We describe four new species: Tunturibacter lichenicola comb. nov., Tunturibacter empetritectus sp. nov., Tunturibacter gelidoferens sp. nov., and Tunturibacter psychrotolerans sp. nov. By uncovering new species and strains within the Terriglobia and improving the accuracy of their phylogenetic placements, we hope to enhance our understanding of this complex phylum and shed light on the mechanisms that shape microbial communities in polar soils.

Spread of antibiotic-resistant actinobacteria in soils exposed to various types of anthropogenic impact

The work investigated the spectra of resistance to a certain set of antibiotics and the frequency of occurrence of resistant strains among typical soil bacteria – actinomycetes. Equal samples of isolates from sod-podzolic soil were used, selected at sites typifying various types of economic activity, namely: the production areas of a plywood factory and a chemical plant, the territory of a medical institution, and a solid waste collection and storage site. A total of 58 isolates with features characteristic of actinomycetes of the genus Streptomyces of the sections Cinereus, Helvolo-Flavus and Albus were obtained. It has been established that various types of economic activity lead to specific changes in the antibiotic resistance, which characterizes the soil of a natural grass-grass meadow (back-ground). Isolates with resistance to nalidixic acid, amoxicillin and ceftriaxone were found in the sample taken on the territory of the chemical plant more often than in the soil of the background site. The proportion of strains resistant to nalidixic acid, lincomycin and azithromycin was significantly (P&lt;0.05) higher in the soil samples taken at the MSW collection site and the territory of the medical institution, while those resistant to nalidixic acid and amoxicillin were in the sample from the plywood factory Groups of antibiotics (quinolones – nalidixic acid, and β-lactams – amoxicillin and ceftriaxone) were identified, the resistance to which in streptomyces isolates from soils involved in economic activity was significantly higher than in soil isolates from the background territory. The data obtained in the work indicate the need to monitor the spread of antibiotic resistance in soils that are not only at risk of accumulation of residual concentrations of antibiotics, but also other consequences of anthropogenic influences.

KARAKTERISASI SELULASE TERMOSTABIL ASAL ISOLAT BAKTERI TANAH PERTANIAN PASANG SURUT

Cellulase is an enzyme that can break up β - (1→4) glycosidic bonds of cellulose. Generally, cellulases are classified into three major groups, i.e., endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1 .91), and β glucosidase (EC 3.2.1.21). Meanwhile, five isolates of tidal swamp soil have been isolated; thus, in this research step, we are acquainted with cellulase activity and the characterization of cellulase enzymes on various temperatures, pH, and specific substrates. The activity of an enzyme was measured using the modified Miller and Bradford methods to detect the specific characteristics of the isolate. The results showed that five isolates namely ATP-1 BKP-5, HAMML-2, PKL-2, and SPBKK-3 have activities 0.025 nKat.ml-1, 0.032 nKat.ml-1, 0.296 nKat.ml-1, 0.114 nKat.ml-1, and 0.087 nKat.ml-1, respectively. Furthermore, cellulase from isolates ATP 1, BKP-5, and PKL 2 have an optimum activity at pH 7,0; 50 oC. The isolate HAMML-2 has an optimum activity at pH 5,0; 60 oC. SPBK 3 has an optimum activity at pH 5,0; 50 oC. All of those isolates can degrade CMC, Avicell, and Whatman paper No.1 and retain the activity relative to the temperatures 50 and 60 0C as the characteristics of the thermostable enzyme.

Wild Animals in Captivity: An Analysis of Parasite Biodiversity and Transmission among Animals at Two Zoological Institutions with Different Typologies.

We have conducted a 10-year-long coprological study of the animals housed in two zoological institutions (ZooAquarium and Faunia, Madrid, Spain) to assess the parasite biodiversity, prevalence, and their relation with host class, diet, and enclosure type (soil type and level of isolation from wild fauna). A total of 4476 faecal samples from 132 mammal species and 951 samples from 86 avian species were examined. The results indicated that only 12.8% of avian species had parasites at least once during the study period, whereas 62.1% of mammal species tested positive. Predominantly, protists (Entamoeba, flagellates, and ciliates) and nematodes (mainly Trichuris) were identified in the findings. Carnivorous species were primarily infected by nematodes, while herbivorous and omnivorous species were mainly infected by protists. The number of infected herbivorous and omnivorous species was significantly greater than carnivorous species. Differences were observed based on soil type (artificial, natural, mixed) and isolation level (isolated/accessible), but these differences were not statistically significant. Several parasites (Entamoeba spp., Giardia spp., Balantidoides coli, Trichuris spp.) could potentially be transmitted between humans and some mammals and birds. Regular animal analyses and a personnel health program in the institutions would minimise transmission risks between zoo animals, wildlife, and humans.

A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes.

Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.

Phylogenetic analysis of Acanthamoeba isolated from soil samples and nasal cavities of patients with malignancy: a public health concern in the northwest of Iran.

The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it.

Recovery and genetic characterization of clinically-relevant ST2 carbapenem-resistant Acinetobacter baumannii isolates from untreated hospital sewage in Zhejiang Province, China

The global transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant and grave threat to human health. To investigate the potential relationship between hospital sewage and the transmission of CRAB within healthcare facilities, isolates of Acinetobacter spp. obtained from untreated hospital sewage samples were subjected to antimicrobial susceptibility tests, genome sequencing, and bioinformatic and phylogenetic tree analysis, and that data were matched with those of the clinical isolates. Among the 70 Acinetobacter spp. sewage isolates tested, A. baumannii was the most prevalent and detectable in 5 hospitals, followed by A. nosocomialis and A. gerneri. Worryingly, 57.14 % (40/70) of the isolates were MDR, with 25.71 % (18/70) being resistant to carbapenem. When utilizing the Pasteur scheme, ST2 was the predominant type among these CRAB isolates, with Tn2006 (ΔISAba1-blaOXA-23-ATPase-yeeB-yeeA-ΔISAba1) and Tn2009 (ΔISAba1-blaOXA-23-ATPase-hp-parA-yeeC-hp-yeeB-ΔISAba1) being the key mobile genetic elements that encode carbapenem resistance. Seven A. gerneri isolates which harbored Tn2008 (ISAba1-blaOXA-23 -ATPase) and the blaPER-1 gene were also identified. Besides, an A. soil isolate was found to exhibit high-level of meropenem resistance (MIC ≥128 mg/L) and harbor a blaNDM-1 gene located in a core genetic structure of ISAba125-blaNDM-1-ble-trpF-dsbC-cutA. To investigate the genetic relatedness between isolates recovered from hospital sewage and those collected from ICUs, a phylogenetic tree was constructed for 242 clinical isolates and 9 sewage isolates. The results revealed the presence of two evolutionary clades, each containing isolates from both ICU and sewage water, suggesting that CRAB isolates in untreated sewage water were also the transmission clones or closely related evolutionary isolates recoverable in hospital settings. Findings in this work confirm that hospital sewage is a potential reservoir of CRAB.

Nanosponge hydrogel of octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate of Alcaligenes faecalis

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate (ODHP) was extracted in a previous study from the culture broth of soil isolate Alcaligenes faecalis MT332429 and showed a promising antimycotic activity. This study was aimed to formulate ODHP loaded β-cyclodextrins (CD) nanosponge (NS) hydrogel (HG) to control skin fungal ailments since nanosponges augment the retention of tested agents in the skin. Box-Behnken design was used to produce the optimized NS formulation, where entrapment efficiency percent (EE%), polydispersity index (PDI), and particle size (PS) were assigned as dependent parameters, while the independent process parameters were polyvinyl alcohol % (w/v %), polymer-linker ratio, homogenization time, and speed. The carbopol 940 hydrogel was then created by incorporating the nanosponges. The hydrogel fit Higuchi’s kinetic release model the best, according to in vitro drug release. Stability and photodegradation studies revealed that the NS-HG remained stable under tested conditions. The formulation also showed higher in vitro antifungal activity against Candida albicans compared to the control fluconazole. In vivo study showed that ODHP-NS-HG increased survival rates, wound contraction, and healing of wound gap and inhibited the inflammation process compared to the other control groups. The histopathological examinations and Masson’s trichrome staining showed improved healing and higher records of collagen deposition. Moreover, the permeability of ODHP-NS-HG was higher through rats’ skin by 1.5-folds compared to the control isoconazole 1%. Therefore, based on these results, NS-HG formulation is a potential carrier for enhanced and improved topical delivery of ODHP. Our study is a pioneering research on the development of a formulation for ODHP produced naturally from soil bacteria.Key points• Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate was successfully formulated as a nanosponge hydrogel and statistically optimized.• The new formula exhibited in vitro good stability, drug release, and higher antifungal activity against C. albicans as compared to the fluconazole.• Ex vivo showed enhanced skin permeability, and in vivo analysis showed high antifungal activity as evidenced by measurement of various biochemical parameters and histopathological examination.

Evaluation of the Tensile and Puncture Properties of Geotextiles Influenced by Soil Moisture under Freezing Conditions.

Freezing conditions under different humidity will influence the mechanical properties of geotextiles, leading to the gradual fracture of geotextiles. It brings hidden danger to the whole isolation, reinforcement and protection of rock and soil. It is particularly important to study the tensile and puncture properties of geotextiles considering low temperature and moisture content. In this paper, a series of tensile and puncture tests of geotextiles are performed under different low temperatures (0, -3, -6, -9, and -12 °C) and at different moisture content levels (0, 5, 10, 30, 50, and 80%). From the microscopic perspective, the failure mechanism considering the low temperature and moisture content was explained comprehensively. Experimental results indicate that with a decrease in freezing temperature, the tensile strength of geotextiles increases as a parabolic function while the elongation at failure decreases as an exponential function. Additionally, the puncture strength of geotextiles presented a parabolic increase with the decreasing temperature. Under the freezing temperature environment, the higher moisture content of geotextiles can generate a higher puncture strength increment. This research contributes to a more comprehensive understanding of the tensile and puncture properties of geotextile materials considering low temperature and moisture content. It can provide important guidance for the design of slopes, the reinforcement of earthen dams, and roadbed reinforcement with geotextiles in cold regions.

Production of Phyto hormone from Phosphate solubilising soil isolates

The transformation of insoluble phosphorous into soluble form is carried out by a number of microbes present in the soil. Phosphorous is essential for growth and productivity of plants in terms of cell division, photosynthesis and development of good root system and utilization of carbohydrates. Phosphorous deficiency results in the leaves turning brown accompanied by small leaves, weak stem and slow development. The large fraction of soil microbes can dissolve insoluble phosphorous in the soil and make them available to plants for its growth and development. In the present work we isolated Phosphate solubilising bacteria from rhizospheric soil from Betel Wine plant and promising strain were subjected for phytohormone (IAA) production.

Phylogeographic characterization of Burkholderia pseudomallei isolated from Bangladesh.

Burkholderia pseudomallei possesses a diverse set of genes which encode a vast array of biological functions reflecting its clinical, ecological and phenotypic diversity. Strain variation is linked to geographic location as well as pattern of land uses. This soil-dwelling Gram-negative pathogen causes melioidosis, a tropical disease endemic in northern Australia and Southeast Asian regions including Bangladesh. Phylogeographic analyses of B. pseudomallei isolates by molecular typing techniques could be used to examine the diversity of this organism as well as to track melioidosis epidemics. In this study, 22 B. pseudomallei isolates, of which 20 clinical and two soil isolates were analyzed, utilizing Real-time PCR assay and multilocus sequence typing (MLST). The sequences were then submitted to PubMLST database for analysis and construction of phylogenetic tree. A total of 12 different sequence types (STs) that includes four novel STs were identified for the first time. Strains having STs 1005, 1007 and 56 were the most widespread STs frequently isolated in Bangladesh. ST 1005, ST 56, ST 1007 and ST 211 have been detected not only in Bangladesh but are also present in many Southeast Asian countries. ST 1005 was detected in both soil and clinical samples of Gazipur. Most prevalent, ST 56 has been previously reported from Myanmar, Thailand, Cambodia and Vietnam, confirming the persistence of the genotype over the entire continent. Further large-scale study is necessary to find out the magnitude of the infection and its different reservoirs in the environment along with phylogeographic association.

Bacterial Diversity and Occurrences in Cassava Effluent-Contaminated Soil Collected From Ozubulu, Anambra State, Nigeria

Environmental degradation due to indiscriminate disposal of refuse has a tremendous effect on microbial activities in the soil. Most soil in Nigeria has been subjected to different kinds of pollutants resulting from anthropogenic activities which have become a major threatening factor to the quality of soil. This study was undertaken to evaluate bacterial diversity and occurrences in cassava effluent contaminated soil from Ozubulu, Anambra State. A total of 30 composite soil samples were aseptically collected from cassava effluent disposal site using a sterile soil auger. The predominant bacterial isolates that aided nutrients cycling were appropriately characterized, and their diversities in both impacted and non-impacted soil samples were enumerated. Furthermore, the bacterial isolates were characterized based on morphology, microscopy, and biochemical characteristics. There was a significant reduction in the occurrences of the bacterial isolates in the impacted soil, and Gram negative rods such as Pseudomonas, Klebsiella and Micrococcus (Gram positive) species were the bacterial isolates. The study has revealed that cassava effluent affects microbial distribution in the soil. Keywords: Bacterial, Diversity, Occurrences, Cassava, Effluent-Contaminated and Soil

Study on the Influence of a Rubber-Modified Soil Isolation Layer on the Isolation Performance of Frame Structures with Different Foundation Forms

In order to investigate the seismic performance of a rubber-modified soil isolation layer, a three-dimensional finite element model was constructed using finite element analysis software, utilizing a two-story frame structure as the engineering background. Nonlinear dynamic time history analysis and comparisons were performed against the seismic performance of the structure. The evaluation was based on several parameters, including the contact area of the base, the thickness of the rubber-particle-modified soil isolation layer, ground motion records with varying amplitudes, and seismic frequency spectrum characteristics. The research findings indicate that the implementation of a rubber-modified soil isolation layer effectively mitigates the peak acceleration, horizontal displacement, and shear stress of the frame structure. This not only enhances the seismic performance of the structure but also enlarges the contact area of the base. Increasing the thickness of the rubber-modified soil isolation layer will effectively decrease the peak acceleration, horizontal displacement, and shear stress of the structure during seismic events. The effectiveness of the isolation provided by the rubber-modified soil layer improves as the intensity of the ground motion record increases.

Effect of biosurfactant on thiocyanate degradation by automobile service station soil isolates Brachybacterium sp. and Bacillus albus

This study investigated the effect of biosurfactants on the microbial degradation of thiocyanate (SCN−), with the aim of finding a sustainable solution for the treatment of industrial wastes containing thiocyanate and related compounds. Isolates VK8 and VT7, screened from amongst 40 automobile service station soil isolates, showed promising growth in the presence of high thiocyanate concentration (500 mg/L). Biosurfactant BS1-S1W from isolate S1W (Brucella intermedia) enhanced the degradation of SCN− by Brachybacterium sp. (VT7) and Bacillus albus (VK8) by 92% and 95%, respectively, at 200 mg/L. Biosurfactant BS2-VO6 from isolate VO6-2 (Brevundimonas naejangsanensis) caused increment in the reduction of SCN- by 88% and 80%, respectively. FTIR and LC-MS analysis of the purified biosurfactants confirmed their glycolipid nature. Enhanced effect of biosurfactant was more pronounced at higher KSCN concentration. Little is known about the effects of microbial products on the enhancement of microbial biodegradation of SCN−. Production and application of biosurfactant from B. naejangsanensis has not been reported earlier and hence can lead to more effective biological agents of enhanced biomedical and environmental relevance. This is the first report of the application of Brachybacterium and B. albus for thiocyanate degradation and the effect of biosurfactant on the same.

BIOSYNTHESIS, PARTIAL PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR L-ASPARAGINASE FROM NOVEL BACILLUS SUBTILIS SUBSP. SPIZIZENII TU-B-10 ISOLATED FROM GCU GARDEN SOIL MICROFLORA

This study focused on L-asparaginase (LA), which breaks down L-asparagine into ammonia and aspartic acid and is known for its anti-carcinogenic properties. A bacterial strain from GCU garden soil was isolated, characterized, and screened for LA production using various assays. Physical and nutritional parameters for maximum LA biosynthesis were optimized by employing one factor at one time (OFAT) optimization strategy. Partial purification of the enzyme was followed by the determination of its various biochemical properties. The phylogenetic analysis has confirmed the close relation of soil isolate SHF-11 to Bacillus subtilis subsp. spizizenii TU-B-10. A twofold increase in enzyme activity with 23.05 IU/ml corresponding to 100 IU/mg of protein was achieved using 2% inoculum size, initial pH 7, agitation at 200 rpm, incubation time, and temperature of 72 hrs and 37 °C, respectively under submerged fermentation consuming 0.1% sucrose as carbon source and 1.5% asparagine as an inducer in the presence of 0.5% tryptone and 0.25% yeast extract as nitrogen source. SDS-PAGE analysis showed that the enzyme exhibited a protein band of almost 40 kDa. The highest activity of LA from Bacillus subtilis subsp. spizizenii TU-B-10 (BsLA) was observed at pH 8 and 37 ºC. EDTA was found to be a strong inhibitor of BsLA activity at the final concentration of 0.1 M, while Mg+2 ions were found to be a strong activator of BsLA. By optimizing purification parameters, more potential and specific preparations of LA are likely to come up that can meet industrial and biomedical standards.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.