Fast-growing Strains Research Articles

Biocrude Production Using a Novel Cyanobacterium: Pilot-Scale Cultivation and Lipid Extraction via Hydrothermal Liquefaction.

The use of renewable energy to reduce fossil fuel consumption is a key strategy to mitigate pollution and climate change, resulting in the growing demand for new sources. Fast-growing proprietary cyanobacterial strains of Fremyella diplosiphon with an average life cycle of 7-10 days, and a proven capacity to generate lipids for biofuel production are currently being studied. In this study, we investigated the growth and photosynthetic pigmentation of a cyanobacterial strain (SF33) in both greenhouse and outdoor bioreactors, and produced biocrude via hydrothermal liquefaction. The cultivation of F. diplosiphon did not significantly differ under suboptimal conditions (p < 0.05), including in outdoor bioreactors with growth differences of less than 0.04 (p = 0.035) among various batches. An analysis of the biocrude's components revealed the presence of fatty acid biodiesel precursors such as palmitic acid and behenic acid, and alkanes such as hexadecane and heptadecane, used as biofuel additives. In addition, the quantification of value-added photosynthetic pigments revealed chlorophyll a and phycocyanin concentrations of 0.0011 ± 5.83 × 10-5 μg/μL and 7.051 ± 0.067 μg/μg chlorophyll a. Our results suggest the potential of F. diplosiphon as a robust species that can grow at varying temperatures ranging from 13 °C to 32 °C, while producing compounds for applications ranging from biofuel to nutritional supplements. The outcomes of this study pave the way for production-level scale-up and processing of F. diplosiphon-derived biofuels and marketable bioproducts. Fuel produced using this technology will be eco-friendly and cost-effective, and will make full use of the geographical location of regions with access to brackish waters.

Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes

The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ5(6)→Δ4(5) double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β-hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β-hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes (MSMEG_1604, MSMEG_5228 and MSMEG_5233) encoding known enzymes exhibiting 3β-hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3β-hydroxysteroid dehydrogenase/Δ5(6)→Δ4(5) isomerase.

Generation of myostatin gene-edited blotched snakehead (Channa maculata) using CRISPR/Cas9 system

Blotched snakehead (Channa maculata) is one of the most important freshwater fish in China. Genome editing is of great potential in generating new snakehead strains with valuable economic traits without integration of exogenous genes. Myostatin (Mstn) inhibits the development and growth of skeletal muscle in vertebrates, including teleosts. Mstn mutations induced by genome editing can stimulate muscle growth and development, therefore Mstn is an appropriate candidate to breed the fast-growing snakehead strain. For this study, one orthologous Mstn gene was characterized in C. maculata, designated as CmMstn, which possessed the relatively conserved gene and protein structure. CmMstn had the highest expression level and sexual dimorphism in muscles. Two CRISPR/Cas9 target sites were designed to generate knockout mutants within the mature mRNA sequence of CmMstn located in Exon I and Exon II. 1 nL mixture solution of 200 pg gRNA and 300 pg Cas9 protein was directly microinjected into each snakehead embryonic yolk at 1⁓4 cell stage. Mstn was efficiently edited by CRISPR/Cas9 system in shakehead, and all designed gRNAs effectively worked at their target sites with high mutagenesis efficiencies, from 40.0% to 53.3%. The majority of mutations were frame-shift and resulted in premature termination of transcription and disruption of the molecular function of CmMstn protein. Furthermore, we obtained four founders (F0) of snakehead with the mutated Mstn gene in their primordial germ cells, and nineteen F1 offspring of the F0 mutants mated with wild-type individuals were displayed to carry Mstn null alleles. It is the first application of genome editing technology in blotched snakehead, and CRISPR/Cas9 system successfully edit the primordial germ cells. Our findings will accelerate our understanding of the role of Mstn gene in snakehead growth, and speed up the cultivation of the fast-growing snakehead strain.

Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens.

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age.

Generation of fast growth Nile tilapia (Oreochromis niloticus) by myostatin gene mutation

Tilapia is the second most prolific species grown in aquaculture after carp, and is widely grown in >100 countries. Myostatin (MSTN) has been proved to be a negative regulator of skeletal muscle growth. Mutation of MSTN gene resulted in significant increase in both body size and muscle mass in vertebrates, mostly with a species-specific effect. To generate a new strain in Nile tilapia (Oreochromis niloticus) with fast growth traits, the molecular characterization, gene expression and functional studies of mstn gene were extensively investigated. Phylogenetic and synteny analyses indicated that mstnb, but not mstna, might be the orthologous gene of mammalian MSTN gene. Expression pattern analyzed by qRT-PCR demonstrated that mstnb is abundant in muscle, while mstna is dominantly expressed in the brain. Fluorescence in situ hybridization (FISH) analysis also demonstrated that mstnb is exclusively expressed in the basement membrane of white skeletal muscle fibers, further suggesting the possible role of mstnb gene in tilapia muscle growth. To verify the roles of mstnb in skeletal muscle growth, we obtain the mstnb−/− mutant using CRISPR/Cas9 gene editing technology. Compared with the wild type (WT) fish, the mstnb−/− mutants showed a typical double-muscle phenotype with increased muscle mass, sticking out between head and dorsal fin, from 5 months after hatching (mah). Morphological observation revealed an excessive proliferation of white muscle fibers at 7 mah. The average body weights (21.67 ± 8.26 g), body heights (4.46 ± 0.65 cm) and body widths (2.36 ± 0.53 cm) of the mstnb−/− fish were 49.45%, 32.74% and 37.21% higher than those of the mstnb+/+ fish (body weight, 14.50 ± 8.60 g; body height, 3.36 ± 0.45 cm; body width, 1.72 ± 0.18 cm) at 5 mah. The results showed that the growth performance parameters of the mstnb−/− fish, including weight gain rate (1.99 times), condition factor (1.77 times) and specific growth rate (1.23 times), were significantly higher than the mstnb+/+ fish under laboratory conditions at 5 mah. In addition, the parameters of WGR, CF, SGR and FE were significantly increased in mstnb−/− Nile tilapia than those of mstnb+/+ Nile tilapia and GIFT tilapia for a 90-day feeding trial under the wild natural environment. Interestingly, sexually dimorphic effects on muscle growth have been found with an increase of growth performance of skeletal muscle in mstn−/− XY fish, but not mstn−/− XX fish at 7 mah. Taken together, this study, for the first time, proved that mstn gene mutation might promote the generation of new fast-growing male strain of Nile tilapia.

Bacillus subtilis M6 improves intestinal barrier, antioxidant capacity and gut microbial composition in AA broiler.

Bacillus subtilis can secret a variety of substances to improve human and animal gut health via inhibiting the proliferation of pathogenic bacteria. In this study, a fast-growing and stress-resistant strain of Bacillus subtilis M6 (B. subtilis M6) were isolated, which showed a strong antibacterial activity to E. coli K88, S. typhimurium ATCC14028, and S. aureus ATCC25923 in vitro. In vivo studies showed that B. subtilis M6 can significantly improve the average daily gain (ADG) using an AA broiler model. Dietary B. subtilis M6 improved the intestinal morphology. The villus height of jejunum and ileum were significantly increased. The concentration of malondialdehyde (MDA) in the ileal mucosa was significantly reduced in B. subtilis M6 treatment group, which suggested the oxidative stress of the ileum was significantly relieved. Though the β diversity of treatments was not significantly, B. subtilis M6 improved the composition of intestinal microbes, especially at the level of caecum genus, the dominant genus was changed from Ruminococcus to Akkermansia, which indicated the change of intestinal carbohydrate nutrition. In conclusion, these data indicate that the B. subtilis M6 shows a probiotic potential to improve intestinal health via altering gut microbiota.

Fast-growing phototrophic microorganisms and the productivity of phototrophic cultures.

Fast-growing cyanobacterial and microalgal strains are considered to be a promising resource to overcome current productivity barriers of phototrophic cultivation. The purpose of this communication, however, is to argue that a high maximal growth rate itself is not a sufficient or necessary property for high phototrophic productivity. Rather, the light-limited specific growth rate of a phototrophic microorganism is a product of several factors, including the rate of light absorption, the photosynthetic efficiency, and the maximal biomass yield per mol photons. It is suggested that, in addition to the maximal growth rate, reports on fast-growing strains should also assess photosynthetic efficiency and maximal biomass yield as predictors of culture productivity. The arguments within the communication are underpinned by a theoretical analysis of a light-limited chemostat, compared to its heterotrophic counterpart. It is shown that for the light-limited chemostat maximal productivity occurs at low dilution rates.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Mixed-sex Nile tilapia (Oreochromis niloticus) can perform competitively with mono-sex stocks in cage production

All-male tilapia stocks are widely used by farmers to supply both domestic and international markets with homogenous, large sized fish (500 g+). While a number of strategies are possible, hormonal treatment of fry with 17α-methyltestosterone (MT) is the most common method used by commercial farmers due to its low cost and ease of application. However, contrasting to its current widespread use, the implications of MT in tilapia farming have raised concerns especially from public and environmental perspectives. Therefore, in this study we tested the impact of stocking a mixed-sex fast growing strain of Nile tilapia (Oreochromis niloticus) fry at high density and then grading out females at 4 or 8 weeks intervals during grow-out and compared final production with a standard MT-treated mono-sex system. From a production perspective, the strategies to remove females at 4 or 8 weeks were successful as no differences in harvest weight, survival and feed conversion rate were observed when compared to the MT-treated group. Similarly, no differences at harvest were obtained in terms of external appearance, Fulton's condition factor, gonadosomatic index, fillet yield, fat-somatic index and visceral-somatic index (%) between MT-monosex group and the groups where females were removed (4 or 8 weeks). However, a financial analysis of this approach showed that the additional costs (fry, feed and labour) involved in the mixed-sex strategy resulted in lower profits. This could be mitigated if a proportion of the removed females could be sold at a premium price as potential broodstock. In the model presented, sales of 13% of the removed females for broodfish at current Thai prices, or a premium of at least 8% for non-sex-reversed final product would be sufficient for the mixed-sex system to return a higher profit than the mono-sex system. The latter strategy could also enable further social licence through use of small fish in nutritional and outgrower initiatives.

Dynamic proteome allocation regulates the profile of interaction of auxotrophic bacterial consortia.

Microbial ecosystems are composed of multiple species in constant metabolic exchange. A pervasive interaction in microbial communities is metabolic cross-feeding and occurs when the metabolic burden of producing costly metabolites is distributed between community members, in some cases for the benefit of all interacting partners. In particular, amino acid auxotrophies generate obligate metabolic inter-dependencies in mixed populations and have been shown to produce a dynamic profile of interaction that depends upon nutrient availability. However, identifying the key components that determine the pair-wise interaction profile remains a challenging problem, partly because metabolic exchange has consequences on multiple levels, from allocating proteomic resources at a cellular level to modulating the structure, function and stability of microbial communities. To evaluate how ppGpp-mediated resource allocation drives the population-level profile of interaction, here we postulate a multi-scale mathematical model that incorporates dynamics of proteome partition into a population dynamics model. We compare our computational results with experimental data obtained from co-cultures of auxotrophic Escherichia coli K12 strains under a range of amino acid concentrations and population structures. We conclude by arguing that the stringent response promotes cooperation by inhibiting the growth of fast-growing strains and promoting the synthesis of metabolites essential for other community members.

Poly-cis-isoprene Degradation by Nocardia sp. BSTN01 Isolated from Industrial Waste.

The natural and synthetic rubber (NR and SR) products are made up of poly-cis-isoprene which are estimated as one of the major solid-wastes and need to be cleared through bacterial bioremediation. The present research reports isolation and characterization of a gram-positive, non-spore forming, filamentous actinomycete Nocardia sp. BSTN01 from the waste of a rubber processing industry. We found NR- and SR-dependent growth of BSTN01 over a period of time. BSTN01 has been found to degrade NR by 55.3% and SR by 45.9% in 6weeks. We have found an increase in the total protein of BSTN01 cells up to 623.6 and 573.9µg/ml for NR and SR, respectively, after 6weeks of growth in rubber-supplemented MSM medium. Scanning electron microscopy revealed adhesive growth of BSTN01 on the surface of NR and SR. Formation of aldehyde groups due to the degradation was indicated by Schiff's test and confirmed by FTIR-ATR analysis. The genome sequence of BSTN01 revealed the gene responsible for rubber degradation. The presence of lcp gene and structural analysis of the latex clearing protein further confirmed the reliability. Studies on quantification of rubber degradation capability by the isolated strain prove it to be an efficient degrader of NR and SR. This study revealed the genome sequence and structural analysis of the proteins responsible for degradation of rubber. A new fast-growing Nocardia strain can degrade both NR and SR with higher efficiency and have future potential for rubber solid-waste management either alone or in consortia.

A Fast-Growing Oleaginous Strain of Coelastrella Capable of Astaxanthin and Canthaxanthin Accumulation in Phototrophy and Heterotrophy.

Considering the importance of microalgae as a promising feedstock for the production of both low- and high-value products, such as lipids and pigments, it is desirable to isolate strains which simultaneously accumulate these two types of products and grow in various conditions in order to widen their biotechnological applicability. A novel freshwater strain from the genus Coelastrella was isolated in Belgium. Compared to other Coelastrella species, the isolate presented rapid growth in phototrophy, dividing 3.5 times per day at a light intensity of 400 µmol·m−2·s−1 and 5% CO2. In addition, nitrogen depletion was associated with the accumulation of astaxanthin, canthaxanthin, and fatty acids, which reached ~30% of dry weight, and a majority of SFAs and MUFAs, which are good precursors for biodiesel. This strain also accumulated astaxanthin and canthaxanthin in heterotrophy. Although the content was very low in this latter condition, it is an interesting feature considering the biotechnological potential of the microalgal heterotrophic growth. Thus, due to its rapid growth in the light, its carotenogenesis, and its fatty acids characteristics, the newly identified Coelastrella strain could be considered as a potential candidate for biorefinery purposes of both low- and high-values products.

Machine learning-informed and synthetic biology-enabled semi-continuous algal cultivation to unleash renewable fuel productivity

Algal biofuel is regarded as one of the ultimate solutions for renewable energy, but its commercialization is hindered by growth limitations caused by mutual shading and high harvest costs. We overcome these challenges by advancing machine learning to inform the design of a semi-continuous algal cultivation (SAC) to sustain optimal cell growth and minimize mutual shading. An aggregation-based sedimentation (ABS) strategy is then designed to achieve low-cost biomass harvesting and economical SAC. The ABS is achieved by engineering a fast-growing strain, Synechococcus elongatus UTEX 2973, to produce limonene, which increases cyanobacterial cell surface hydrophobicity and enables efficient cell aggregation and sedimentation. SAC unleashes cyanobacterial growth potential with 0.1 g/L/hour biomass productivity and 0.2 mg/L/hour limonene productivity over a sustained period in photobioreactors. Scaling-up the SAC with an outdoor pond system achieves a biomass yield of 43.3 g/m2/day, bringing the minimum biomass selling price down to approximately $281 per ton.

Production of Scenedesmus almeriensis Using Pilot-Scale Raceway Reactors Located inside a Greenhouse

The microalga S. almeriensis was produced in spring and summer using 80 m2 raceway reactors located inside a greenhouse in Almería, Spain. This microalga was selected because it is a fast-growing and robust strain with potential applications in the production of functional foods and feeds or as a high-value agricultural biostimulant. Overall, the biomass productivity obtained in summer was 24.9 ± 0.9 g·m−2·day−1. This value was higher than that obtained in spring, with an average value of 21.4 ± 1.3 g·m−2·day−1 (p &lt; 0.05). The Fv/Fm value of the cultures at the stationary phase was comparable and around 0.6, which is the optimum of this strain. No major differences in the macromolecular composition of the biomass were observed between seasons, with an average protein, lipid, ash, and carbohydrate content of 37.9, 4.6, 10.8, and 46.7%, respectively. The data reported herein were used to validate a previously described model. The differences between the experimental and the predicted biomass productivities were below 5% in spring and 8% in summer, although a larger dataset is needed to validate the model. Overall, results supported the robustness of the selected strain and its utilisation in different industrial sectors.

Comparison in Growth, Feeding, and Metabolism Between a Fast-Growing Selective Strain and a Cultured Population of Pearl Oyster (Pinctada fucata martensii)

Pearl oyster (Pinctada fucata martensii) is the main species cultured for marine pearls in the world. A breeding program was carried out for desirable production traits, including high growth rate, and a fast-growing selective strain of pearl oysters was established. In the current study, we compared the growth characteristics between a selective strain and a cultured population of P. f. martensii in Beihai, Guangxi Province, China. Large size (SL) and small size (SS) individuals of the selective strain were selected, and the differences of physiological and metabolic indexes, such as feeding, respiration, excretion, and enzyme activities between SL and SS and cultured population (CL), were also compared. The results showed that at the age of 6 months, pearl oysters of the selective strain were 14.61% larger than CL, and the proportion of SL (30–40 mm) was 59%, which was two times higher than CL (28%). SL with a rapid growth rate had a high clearance rate (CR), and the CR of SL was about 1.8 times higher than that of CL and 5 times higher than that of SS. In addition, the activities of digestive enzymes (amylase, pepsin, and lipase) and growth-related carbonic anhydrase enzymes in SL were higher than those in the other two groups (p &amp;lt; 0.05). SS with a slow growth rate had higher oxygen consumption (OCR) and ammonia excretion (AER) rates than SL and CL (p &amp;lt; 0.05). Our results suggest that the rapid growth of the selective strain P. f. martensii can be attributed to increased energy intake and reduced energy consumption.

A Polyphasic Characterisation of Tetradesmus almeriensis sp. nov. (Chlorophyta: Scenedesmaceae)

The microalga Tetradesmus almeriensis, previously known as Scenedesmus almeriensis, has been isolated and cultivated as a highly productive, fast-growing strain known as a natural source of different products of commercial interest, including bioactive compounds such as lutein. This strain produces up to 40 g·m−2·day−1 of lutein under optimal conditions and is highly recommendable for outdoor production in temperate and warm climates, showing maximal performance at temperatures up to 35 °C with no photo-inhibition taking place with irradiances greater than 1000 μE·m−2·s−1. Morphological and molecular data allow its assignment to the Chlorophycean genus Tetradesmus. The new species can be distinguished from similar Tetradesmus taxa due to its unique combination of features that are seen under light microscopy. We present herein a robust and comprehensive phylogenetic analysis of T. almeriensis, together with several additional Scenedesmaceae species, using a combination of maximum likelihood and Bayesian approaches. Our results confirm T. almeriensis as a distinct species consistently clustering with other Scenedesmaceae.

Metabolic stability of freshwater Nitzschia palea strains under silicon stress associated with triacylglycerol accumulation

Si is an essential nutrient for diatom growth. It is involved in frustule formation and is directly linked to the cell cycle. Despite numerous studies analyzing Si starvation in diatoms, the interaction between Si and central metabolism remains poorly understood. Accordingly, we analyzed the metabolite profiles of two freshwater strains of Nitzschia palea, BR006 and BR022 (a fast and a slow-growing strain, respectively), under different Si concentrations (0, 0.25, 1.25, and 5 mM) in different harvesting points. The strains exhibited similar metabolic responses under Si starvation, such as high pigment and total lipid, as well as high primary metabolite accumulation; only ornithine level was low in both strains in the Si-starved condition. However, the response of each strain differed under different Si concentrations. Triacylglycerol (TAG) accumulation in cells was inversely proportional to growth rate. Unlike BR006, slow-growing BR022 cells exhibited high TAG production even under high Si conditions. These results suggest that under optimal Si conditions, in the fast-growing BR006 strain, carbon from photosynthesis is directed toward new cell formation, while in the slow-growing BR022 cells, carbon is stored as TAG as a priority.

Nitrogen Source Preference and Growth Carbon Costs of Leucaena leucocephala (Lam.) de Wit Saplings in South African Grassland Soils.

Leucaena leucocephala (Fabaceae) is native to Central America and has invaded many climatic regions of the tropics. In South Africa, the species is categorized as an emerging or incipient weed used as fodder, timber, firewood and in erosion control on degraded habitats. The species is common along the eastern subtropical regions of KwaZulu-Natal (KZN) Province, where it invades grasslands, savannas and edges of forests. Soils of these ecosystems are characterized as nutrient deficient and acidic. Using a pot trial, we determined the effects of the nutrient addition treatments on microbial symbiosis, N nutrition and biomass accumulation of L. leucocephala under greenhouse conditions. After 180 days of growth, plants were harvested, and their utilization of N derived from the atmosphere and from the soil was quantified through determination of δ15N values. L. leucocephala maintained growth and N nutrition by relying on both atmospheric- and soil-derived N across all soil treatments. The NDFA was significantly higher in high P (N1 + P, N2 + P and N3 + P) soils. L. leucocephala was able to nodulate with intermediate and fast-growing strains from the Mesorhizobium and Rhizobium genus in N2 + P grown plants. This shows that L. leucocephala possesses traits that are successful in acquiring nutrients, especially in nutrient limited conditions, by establishing plant symbiosis with multiple bacteria and relying on extracting N from the soil and from the atmosphere through the symbiosis.

Phenotypic Recovery of a Heterobasidion Isolate Infected by a Debilitation-Associated Virus Is Related to Altered Host Gene Expression and Reduced Virus Titer

The fungal genus Heterobasidion includes forest pathogenic species hosting a diverse group of partitiviruses. They include the host debilitating Heterobasidion partitivirus 13 strain an1 (HetPV13-an1), which was originally observed in a slowly growing H. annosum strain 94233. In this study, a relatively fast-growing sector strain 94233-RC3 was isolated from a highly debilitated mycelial culture of 94233, and its gene expression and virus transcript quantities as well as the genomic sequence of HetPV13-an1 were examined. The sequence of HetPV13-an1 genome in 94233-RC3 was identical to that in the original 94233, and thus not the reason for the partial phenotypic recovery. According to RNA-seq analysis, the HetPV13-an1 infected 94233-RC3 transcribed eight genes differently from the partitivirus-free 94233-32D. Three of these genes were downregulated and five upregulated. The number of differentially expressed genes was considerably lower and the changes in their expression were small compared to those of the highly debilitated original strain 94233 with the exception of the most highly upregulated ones, and therefore viral effects on the host transcriptome correlated with the degree of the virus-caused debilitation. The amounts of RdRp and CP transcripts of HetPV13-an1 were considerably lower in 94233-RC3 and also in 94233 strain infected by a closely related mildly debilitating virus HetPV13-an2, suggesting that the virus titer would have a role in determining the effect of HetPV13 viruses on their hosts.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications.

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidateas a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 μL) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 μM) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughputgene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.