Development and application of ribosomal engineering in actinomycetes / 生物工程学报

Ribosomal engineering is a technique that can improve the biosynthesis of secondary metabolites in the antibiotics-resistant mutants by attacking the bacterial RNA polymerase or ribosome units using the corresponding antibiotics. Ribosomal engineering can be used to discover and increase the production of valuable bioactive secondary metabolites from almost all actinomycetes strains regardless of their genetic accessibility. As a consequence, ribosomal engineering has been widely applied to genome mining and production optimization of secondary metabolites in actinomycetes. To date, more than a dozen of new molecules were discovered and production of approximately 30 secondary metabolites were enhanced using actinomycetes mutant strains generated by ribosomal engineering. This review summarized the mechanism, development, and protocol of ribosomal engineering, highlighting the application of ribosomal engineering in actinomycetes, with the aim to facilitate future development of ribosomal engineering and discovery of actinomycetes secondary metabolites.

Anaerobic biodegradation of benzo(a)pyrene by a novel Cellulosimicrobium cellulans CWS2 isolated from polycyclic aromatic hydrocarbon-contaminated soil

Abstract Cellulosimicrobium cellulans CWS2, a novel strain capable of utilizing benzo(a)pyrene (BaP) as the sole carbon and energy source under nitrate-reducing conditions, was isolated from PAH-contaminated soil. Temperature and pH significantly affected BaP biodegradation, and the strain exhibited enhanced biodegradation ability at temperatures above 30 °C and between pH 7 and 10. The highest BaP removal rate (78.8%) was observed in 13 days when the initial BaP concentration was 10 mg/L, and the strain degraded BaP at constant rate even at a higher concentration (50 mg/L). Metal exposure experimental results illustrated that Cd(II) was the only metal ion that significantly inhibited biodegradation of BaP. The addition of 0.5 and 1.0 g/L glucose enhanced BaP biodegradation, while the addition of low-molecular-weight organic acids with stronger acidity reduced BaP removal rates during co-metabolic biodegradation. The addition of phenanthrene and pyrene, which were degraded to some extent by the strain, showed no distinct effect on BaP biodegradation. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the five rings of BaP opened, producing compounds with one to four rings which were more bioavailable. Thus, the strain exhibited strong BaP degradation capability and has great potential in the remediation of BaP-/PAH-contaminated environments.

Effect of sulfonylurea tribenuron methyl herbicide on soil Actinobacteria growth and characterization of resistant strains

ABSTRACT Repeated application of pesticides disturbs microbial communities and cause dysfunctions on soil biological processes. Granstar® 75 DF is one of the most used sulfonylurea herbicides on cereal crops; it contains 75% of tribenuron-methyl. Assessing the changes on soil microbiota, particularly on the most abundant bacterial groups, will be a useful approach to determine the impact of Granstar® herbicide. For this purpose, we analyzed Actinobacteria, which are known for their diversity, abundance, and aptitude to resist to xenobiotic substances. Using a selective medium for Actinobacteria, 42 strains were isolated from both untreated and Granstar® treated soils. The number of isolates recovered from the treated agricultural soil was fewer than that isolated from the corresponding untreated soil, suggesting a negative effect of Granstar® herbicide on Actinobacteria community. Even so, the number of strains isolated from untreated and treated forest soil was quite similar. Among the isolates, resistant strains, tolerating high doses of Granstar® ranging from 0.3 to 0.6% (v/v), were obtained. The two most resistant strains (SRK12 and SRK17) were isolated from treated soils showing the importance of prior exposure to herbicides for bacterial adaptation. SRK12 and SRK17 strains showed different morphological features. The phylogenetic analysis, based on 16S rRNA gene sequencing, clustered the SRK12 strain with four Streptomyces type strains (S. vinaceusdrappus, S. mutabilis, S. ghanaensis and S. enissocaesilis), while SRK17 strain was closely related to Streptomyces africanus. Both strains were unable to grow on tribenuron methyl as unique source of carbon, despite its advanced dissipation. On the other hand, when glucose was added to tribenuron methyl, the bacterial development was evident with even an improvement of the tribenuron methyl degradation. In all cases, as tribenuron methyl disappeared, two compounds were detected with increased concentrations. These by-products appeared to be persistent and were not degraded either chemically or by the studied strains. Based on these observations, we suggested that bacterial activity on carbon substrates could be directly involved in the partial breakdown of tribenuron methyl, by generating the required acidity for the first step of the hydrolysis. Such a process would be interesting to consider in bioremediation of neutral and alkaline tribenuron methyl-polluted soils.

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

ABSTRACT Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Biomineralization of a calcifying ureolytic bacterium Microbacterium sp. GM-1

Background: Biomineralization is a significant process performed by living organisms in which minerals are produced through the hardening of biological tissues. Herein, we focus on calcium carbonate precipitation, as part of biomineralization, to be used in applications for environmental protection, material technology, and other fields. A strain GM-1, Microbacterium sp. GM-1, isolated from active sludge, was investigated for its ability to produce urease and induce calcium carbonate precipitation in a metabolic process. Results: It was discovered that Microbacterium sp. GM-1 resisted high concentrations of urea up to 60 g/L. In order to optimize the calcification process of Microbacterium sp. GM-1, the concentrations of Ni2+ and urea, pH value, and culture time were analyzed through orthogonal tests. The favored calcite precipitation culture conditions were as follows: the concentration of Ni2+ and urea were 50 µM and 60 g/L, respectively, pH of 10, and culture time of 96 h. Using X-ray diffraction analysis, the calcium carbonate polymorphs produced by Microbacterium sp. GM-1 were proven to be mainly calcite. Conclusions: The results of this research provide evidence that Microbacterium sp. GM-1 can biologically induce calcification and suggest that strain GM-1 may play a potential role in the synthesis of new biominerals and in bioremediation or biorecovery.

Exploring plant growth-promotion actinomycetes from vermicompost and rhizosphere soil for yield enhancement in chickpea

Abstract The main objective of the present study was to isolate and characterize actinomycetes for their plant growth-promotion in chickpea. A total of 89 actinomycetes were screened for their antagonism against fungal pathogens of chickpea by dual culture and metabolite production assays. Four most promising actinomycetes were evaluated for their physiological and plant growth-promotion properties under in vitro and in vivo conditions. All the isolates exhibited good growth at temperatures from 20 °C to 40 °C, pH range of 7–11 and NaCl concentrations up to 8%. These were also found highly tolerant to Bavistin, slightly tolerant to Thiram and Captan (except VAI-7 and VAI-40) but susceptible to Benlate and Ridomil at field application levels and were found to produce siderophore, cellulase, lipase, protease, chitinase (except VAI-40), hydrocyanic acid (except VAI-7 and VAI-40), indole acetic acid and β-1,3-glucanase. When the four actinomycetes were evaluated for their plant growth-promotion properties under field conditions on chickpea, all exhibited increase in nodule number, shoot weight and yield. The actinomycetes treated plots enhanced total N, available P and organic C over the un-inoculated control. The scanning electron microscope studies exhibited extensive colonization by actinomycetes on the root surface of chickpea. The expression profiles for indole acetic acid, siderophore and β-1,3-glucanase genes exhibited up-regulation for all three traits and in all four isolates. The actinomycetes were identified as Streptomyces but different species in the 16S rDNA analysis. It was concluded that the selected actinomycetes have good plant growth-promotion and biocontrol potentials on chickpea.

Carbazole degradation in the soil microcosm by tropical bacterial strains

In a previous study, three bacterial strains isolated from tropical hydrocarbon-contaminated soils and phylogenetically identified as Achromobacter sp. strain SL1, Pseudomonas sp. strain SL4 and Microbacterium esteraromaticum strain SL6 displayed angular dioxygenation and mineralization of carbazole in batch cultures. In this study, the ability of these isolates to survive and enhance carbazole degradation in soil were tested in field-moist microcosms. Strain SL4 had the highest survival rate (1.8 x 107 cfu/g) after 30 days of incubation in sterilized soil, while there was a decrease in population density in native (unsterilized) soil when compared with the initial population. Gas chromatographic analysis after 30 days of incubation showed that in sterilized soil amended with carbazole (100 mg/kg), 66.96, 82.15 and 68.54% were degraded by strains SL1, SL4 and SL6, respectively, with rates of degradation of 0.093, 0.114 and 0.095 mg kg−1 h−1. The combination of the three isolates as inoculum in sterilized soil degraded 87.13% carbazole at a rate of 0.121 mg kg−1 h−1. In native soil amended with carbazole (100 mg/kg), 91.64, 87.29 and 89.13% were degraded by strains SL1, SL4 and SL6 after 30 days of incubation, with rates of degradation of 0.127, 0.121 and 0.124 mg kg−1 h−1, respectively. This study successfully established the survivability (> 106 cfu/g detected after 30 days) and carbazole-degrading ability of these bacterial strains in soil, and highlights the potential of these isolates as seed for the bioremediation of carbazole-impacted environments.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 x 10(9)-49.5 x 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R² values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 x 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Actinobacteriological research in India.

Actinobacteria are important sources of compounds for drug discovery and have attracted considerable pharmaceutical, chemical, agricultural and industrial interests. Actinobacteriological research is still in its infancy in India. Early work on actinobacteria started in the 20th century and mostly focused on studying the diversity, identification and screening for antibiotics, enzymes and enzyme inhibitors. Exploration of diverse habitats for the isolation of actinobacteria, have yielded till date 23 novel species. Screening of actinobacteria for antagonistic activity, has led to the discovery of four novel antibiotics. Research on enzymes mostly covered lipases, amylases, proteases, endoglucanases, α-galactosidases, pectin lyases, xylanases, L-asparaginases, L-glutaminase and cellulases. Research on exploiting actinobacteria for other purposes such as production of enzyme inhibitors, single cell protein, bioemulsifier and biosurfactants is still in the experimental stage. This review compiles the work done in last few years, with an emphasis on actinobacterial diversity and bioprospecting for pharmaceutically important compounds like antibiotics, enzymes and other important applications. The chemical creativity and biotechnological potential of Indian actinobacterial strains are yet to be fully explored. A national strategy is required consistent with the opportunities provided by CBD-Nagoya protocol.

Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10.

Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.

Alkalithermophilic actinomycetes in a subtropical area of Jujuy, Argentina / Actinomicetos termoalcalófilos del área subtropical de Jujuy, Argentina

The objective of this study was to examine the alkalithermophilic actinomycete communities in the subtropical environment of Jujuy, Argentina, characterized by sugarcane crops. Laceyella putida, Laceyella sacchari, Thermoactinomyces intermedius, Thermoactinomyces vulgaris and Thermoflavimicrobium dichotomicum were isolated on the media with novobiocin, from sugar cane plants and renewal rhizospheres, and grass and wood soils. Soil pH was almost neutral or lightly alkaline, except for grass soil acidified by lactic liquor. A smaller number of actinomycetes was found on the living plants and bagasse (recently obtained or stored according to the Ritter method) with respect to decomposed leaves on the soil. Thermophilic species of Laceyella, Thermoactinomyces, Thermoflavimicrobium, Saccharomonospora, Streptomyces and Thermononospora were isolated on the media without novobiocin, from composted sugar cane residues. Air captured near composted bagasse piles, contained alkalithermophilic actinomycete spores.

El objetivo de este trabajo fue examinar los actinomicetos termoalcalófilos presentes en el área subtropical de Jujuy, Argentina, caracterizada por el cultivo de la caña de azúcar. Se aislaron en medio con novobiocina las especies Laceyella putida, Laceyella sacchari, Thermoactinomyces intermedius, Thermoactinomyces vulgaris y Thermoflavimicrobium dichotomicum a partir de la rizósfera de plantas y de renuevos de caña de azúcar, así como de suelos de pastura y de monte natural. El pH de los suelos era casi neutro a ligeramente alcalino, excepto en un solo caso en que el suelo estaba acidificado por licor láctico. El número de actinomicetos encontrados sobre los tejidos vivos y en el bagazo recién obtenido o almacenado según el método de Ritter fue pequeño en comparación con el observado sobre las hojas en descomposición. L. sacchari predominó respecto de T. vulgaris. Se aislaron especies termoalcalófilas de Laceyella, Thermoactinomyces, Thermoflavimicrobium, Saccharomonospora, Streptomyces y Thermononospora de los residuos compostados de caña de azúcar utilizando medio sin novobiocina. El aire capturado cerca de pilas de bagazo en compostaje contenía esporos de estos organismos.

Soil microbial population and enzyme activity related to grazing pressure in alpine meadows of Nanda Devi Biosphere Reserve.

The present study aims to analyze the interaction of prevailing biotic pressure on soil environment with emphasis on its physicochemical and microbiological characteristics determining soil fertility status and thus supporting plant and animal biodiversity in Nanda Devi Biosphere Reserve (NDBR) which is located in northern part of Uttaranchal hills between 79 degrees 40'E to 80 degrees 05'E longitude and 30 degrees 17'N to 30 degrees 41'E latitude. The experimental results revealed that the physico-chemical characteristics (viz., moisture, pH, EC, C, N, P, K, CEC) of soil were maximum in moderately grazed meadow and minimum in intensively grazed meadow. Soil microbial analysis measured in terms of total viable count (TVC) exhibited grazing sensitivity trend being maximum population of bacteria > fungi > actinomycetes. The soil microbial population was positively correlated with soil respiration, dehydrogenase activity, acid phosphatase and microbial biomass, which exhibited uneven trend with grazing pressure. Soil from moderately grazed meadow showed highest microbial count and enzyme activities, whilst intensively grazed meadow showed lowest microbial count and enzyme activities. This depicts the beneficial role of prescribed grazing up to limited extent in management of soil fertility, which might have supported luxuriant growth of a variety of grasses.