Engineered Streptomyces lividans Strains for Optimal Identification and Expression of Cryptic Biosynthetic Gene Clusters

Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover “cryptic” secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds

A prospective phase II study of L-asparaginase- CHOP plus radiation in newly diagnosed extranodal NK/T-cell lymphoma, nasal type

Purpose: To explore the efficacy and safety of L-asparaginase in newly-diagnosed extranodal nature killer (NK)/T -cell lymphoma (ENKTL), we conducted a prospective phase II study of L-asparaginase, cyclophosphamide, vincristine, doxorubicin and dexamethasone (CHOP-L) regimen in combination with radiotherapy. Patients and methods: Patients with newly diagnosed ENKTL and an ECOG performance status of 0 to 2 were eligible for enrollment. Treatment included 6-8 cycles of CHOP-L (cyclophosphamide, 750 mg/m(2) day 1; vincristine, 1.4 mg/m(2) day 1 (maximal dose 2 mg), doxorubicin 50 mg/m(2) day 1; dexamethasone 10 mg days 1-8; L-asparaginase 6000 u/m(2) days 2-8). Radiotherapy was scheduled after 4-6 cycles of CHOP-L regimen, depending on stage and primary anatomic site. The primary endpoint was complete response (CR) rate. Results: A total of 38 eligible patients were enrolled. The median age was 40.5 years (range, 15 to 71 years). Their clinical characteristics were male to female ratio, 24: 14; Ann Arbor stage I, 20; II, 11; III, 3; IV, 4. CR and overall response rates were 81.6% (95% CI, 69.3% to 93.9%) and 84.2%, respectively. With a median follow-up of 25 months, the 2-year overall survival, progression-free survival and disease-free survival rates were 80.1% (95% CI, 73.3% to 86.9%), 81% (95% CI, 74.5% to 87.5%) and 93.6% (95% CI, 89.3% to 97.9%), respectively. The major adverse events were myelosuppression, liver dysfunction, and digestive tract toxicities. Grade 3 to 4 leukopenia and neutropenia were 76.3% and 84.2%, respectively. No treatment-related death was observed. Conclusion: CHOP-L chemotherapy in combination with radiotherapy is a safe and highly effective treatment for newly diagnosed ENKTL.OncologyHematologySCI(E)9ARTICLEnull

Quantitative evaluation of precautions against the COVID-19 indoor transmission through human coughing

Abstract In this work, we focus on the dispersion of COVID-19-laden droplets using the transient computational fluid dynamics (CFD) modeling and simulation of the coughing process of virus carriers in an enclosure room, aiming to set up the basic prototype of popular precautionary strategies, i.e., face mask, upward ventilation, protective screen, or any combination thereof, against the indoor transmission of COVID-19 and other highly contagious diseases in the future. A multi-component Eulerian–Lagrangian CFD particle-tracking model with user-defined functions is utilized under 8 cases to examine the characteristics of droplet dispersion concerning the mass and heat transfer, droplet evaporation, air buoyancy, air convection, air-droplet friction, and turbulent dispersion. The result shows that implementing upward ventilation is the most effective measure, followed by wearing face masks. Protective screens can restrict the movement of the coughing droplets (though it will not reduce viral load). However, applying protective screens arranged with lean can be counterproductive in preventing the spread of COVID-19 when it is inappropriately placed with ventilation. The soundest solution is the combination of the face mask and upward ventilation, which can reduce the indoor infectious concentration by nearly 99.95% compared with the baseline without any precautionary strategies. With the resumption of school and work in the post-epidemic era, this study would provide intelligence-enhancing advice for the masses and rule-makers to curb the pandemic

A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿

Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development

Long-term deepened snow cover alters litter layer turnover rate in temperate steppes

The turnover of litter layer is a biogeochemical process fundamental to carbon and nutrient cycling, influencing seed germination, species coexisting, and carbon storage. Winter snow depth is undergoing increasing trend in Northern China, which has been shown to alter litter decomposition rate of individual species. However, it remains unknown how changes in snow depth affect the turnover rate of the whole litter layer, and whether the responses vary between different steppes. Most current litter decomposition studies are site-based or short-term treated, limiting the exploration of the long-term response of litter layer turnover in regional pattern. In this study, we selected six long-term (11-13 years) snow fence sites in Inner Mongolia, with three in the dry steppe and another three in the wet steppe, and investigated the responses of community-weighted litter residence time (LRT) to long-term increased snow treatment. We found that LRT increased by 0.02 year for every 10 cm increase in snow depth in the wet steppe, but was not affected in the dry steppe. The lack of effect of deepened snow on LRT in the dry steppe was attributed to the offset between the positive effect of the increased plant community-weighted height possibly via inhibiting photodegradation and the enhanced litter recalcitrance by producing higher proportion of stem litter, and the negative effect of the increased soil moisture via accelerating microbial decomposition. The significantly positive effect of the deepened snow on LRT in the wet steppe was mainly because the deepened snow increased grass biomass but had no effect on forb biomass, which reduced the degradability of the litter layer. Overall, our findings indicated that deepened snow changed plant community, which altered environmental conditions and enhanced litter recalcitrance, thereby increasing LRT in temperate steppes. However, this effect was diminished by enhancing microbial decomposition in the dry but not wet steppe, resulting in different overall responses of LRT to deep-snow in the two steppes. The slow litter turnover rate in the wet steppe might result in greater litter accumulation under future increased snow depth, which could be unfavourable for seed germination and alter plant diversity. A free plain language summary can be found within the Supporting Information of this article

Allocation and turnover of rhizodeposited carbon in different soil microbial groups

Recent advances in soil organic carbon (SOC) formation indicate that labile plant C is disproportionately important for stable SOC pool because it is utilized more efficiently by soil microbes. Rhizodeposited C, accounting for 5-20% of photosynthates, are highly bioavailable and readily metabolized substrates for soil microbes. However, it remains poorly understood how different microbial functional groups contribute to transforming rhizodeposited C to SOC. Here, we synthesized 23 studies performing (CO2)-C-13 pulse labelling of plants in conjunction with stable isotope probing of microbial phospholipid fatty acids, and explored the fate of rhizodeposited C in different soil microbial functional groups. Among microbial groups, fungi (25.3%) and gram-negative bacteria (GN; 23.5%) took up most of the rhizodeposition-derived C, while the relative abundance of fungi (13.7%) was less than a half of GN (32.7%). These results suggest that fungi have a higher capability of acquiring rhizodeposition-derived C, which may be due to their hyphal growth form allowing them to obtain rhizodeposited C more effectively. The mean turnover rates of rhizodeposited C in microbial groups ranged from 0.04 to 0.13 day(-1). We did not detect significant differences in the turnover rates of rhizodeposited C among microbial groups. Based on the distribution and turnover rate of rhizodeposited C in different microbial groups, we further revealed that GN (31.2%) and fungi (25.8%) produced the most microbial residues via rhizodeposition-derived C. Our results provided quantitative information about the roles of different microbial functional groups in competing for and processing rhizodeposition-derived C, which could help refine parameter estimation in modeling microbial transformation of plant C into SOC under global changes

The decline in plant biodiversity slows down soil carbon turnover under increasing nitrogen deposition in a temperate steppe

1. Nitrogen (N) deposition not only alters the physiological processes of individual plant, but also leads to world-wide biodiversity loss. However, little is known about how the hierarchical responses from individual physiological processes to plant community structure would have cascading effects on soil carbon (C) cycling. 2. Here, we assessed whether changes in plant chemical composition and community composition under increasing N input would affect the turnover rate of litter layer and soil C loss via heterotrophic respiration (R-h) in a temperate grassland. 3. We showed that more than a decade's N addition significantly decreased plant species richness, litter layer turnover rate and R-h. The C-13-NMR results showed that, for individual species, N addition either increased the abundance of recalcitrant C groups such as alkyl and methoxyl, or decreased labile C groups such as carbohydrate, resulting in decreases in carbohydrate C-to-methoxyl C ratio (CC/MC) for most species. Our data also showed that with the increase in N deposition, the abundance of relatively high degradable dominant species, such as Agropyron cristatum and Artimesia frigida declined rapidly, and the relatively recalcitrant species such as Potentilla bifurca and Leymus chinensis become dominant. Changes in individual species' chemical composition and plant community composition significantly decreased litter quality at community level, as indicated by the lower community-level CC/MC at higher N addition rates. 4. The result of step-AIC model selection further showed that plant diversity loss and the decrease in community-level CC/MC jointly explained the decrease in R-h after N addition best, and further relative importance partition result showed that these two factors respectively contributed 65.1% and 34.9% of the explained variation. 5. Overall, we demonstrated that changes in plant chemical composition and diversity loss due to N addition reduced the quality of plant C input to soil, which further slowed down litter layer turnover rate and inhibited soil heterotrophic respiration. Our study complements the intermediate links of how shifts in plant community structure regulate soil C cycle under global changes

Data from: The decline in plant biodiversity slows down soil carbon turnover under increasing nitrogen deposition in a temperate steppe

1. Nitrogen (N) deposition not only alters the physiological processes of individual plant, but also leads to worldwide biodiversity loss. However, little is known about how the hierarchical responses from individual physiological processes to plant community structure would have cascading effects on soil carbon (C) cycling. 2. Here, we assessed whether changes in plant chemistry and community composition under increasing N input would affect the turnover rate of litter layer and soil C loss via heterotrophic respiration (Rh) in a temperate grassland. 3. We showed that more than a decade’s N addition significantly decreased plant species richness, litter layer turnover rate and Rh. The 13C-NMR results showed that, for individual species, N addition either increased the abundance of recalcitrant C groups such as Alkyl and Methoxyl, or decreased labile C groups such as Carbohydrate, resulting in decreases in Carbohydrate C to Methoxyl C ratio (CC/MC) for most species. Our data also showed that with the increase in N deposition, the abundance of relatively high degradable dominant species, such as A. cristatum and A. frigida declined rapidly, and the relatively recalcitrant species such as P. bifurca and L. chinensis become dominate. Changes in individual species’ chemistry and plant community composition significantly decreased litter quality at community level, as indicated by the lower community level CC/MC at higher N addition rates. 4. The result of step-AIC model selection further found that plant diversity loss and the decrease in community level CC/MC jointly best explained the decrease in Rh after N addition, and further relative importance partition result showed that these two factors respectively contributed 65.1% and 34.9% of the explained variation. 5. Overall, we demonstrated that changes in plant chemistry and diversity loss due to N addition reduced the quality of plant C input to soil, which further slowed down litter layer turnover rate and inhibited soil heterotrophic respiration. Our study complements the intermediate links of how shifts in plant community structure regulates soil C cycle under global changes

Dataset_Yang_et al_2019

The file contains all the original data including soil heterotrophic respiration, litter layer residence time, species diversity, soil properties, community level plant chemical composition and microbial community composition