Summary
Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. ...The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. 11C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.
Significance Statement
The results indicate that, under the appropriate conditions, the C4 model grass species, Setaria viridis, can obtain 100% of its nitrogen needs from biological nitrogen fixation, as a result of inoculation with plant growth promoting bacteria. The work supports the future use of S. viridis as a model system to explore the mechanistic aspects of associative nitrogen fixation with the goal of transferring this knowledge to important crop species, such as corn.
Interest in the use of inoculants containing bacteria that promote plant growth is likely to increase in the coming years, due to higher costs of fertilizers, concerns over pollution and emphasis on ...sustainable agriculture. Although Brazil has a long tradition in research on nitrogen fixation in Azospirillum-grass associations, it has not led to recommendations of strains for use in commercial inoculants. In this study, we report the selection and evaluation of Azospirillum strains for the maize (Zea mays L.) and wheat (Triticum aestivum L.) crops, following protocols established by the Brazilian legislature, i.e. field experiments have to be performed in at least two different localities representing the crop growing regions, and for at least two seasons. In a first set of nine trials performed at Londrina and Ponta Grossa, southern Brazil, nine Azospirillum strains were evaluated after application to seeds as peat-based inoculants. A. brasilense strains Ab-V4, Ab-V5, Ab-V6 and Ab-V7 increased grain yields of maize by 662-823 kg ha⁻¹, or 24-30%, in relation to non-inoculated controls. Two A. lipoferum strains were tested in two of these experiments and promising results were also obtained. With wheat, A. brasilense strains Ab-V1, Ab-V5, Ab-V6 and Ab-V8 were the most effective, increasing yields by 312-423 kg ha⁻¹, or 13-18%. In a second trial set with eight field experiments at Londrina an Ponta Grossa, liquid and peat-based inoculants carrying a combination of A. brasilense strains Ab-V5 and Ab-V6 increased maize and wheat yields by 27% and 31%, respectively. Effects of inoculation were attributed to general increases in uptake of several macro and micronutrients and not specifically to biological nitrogen fixation. All experiments received only a low N-fertilizer starter at sowing (24 kg and 20 kg of N ha⁻¹ for the maize and wheat, respectively) and although yields can be globally considered low, they were compatible with Brazilian mean yields. This study resulted in the identification of the first Azospirillum strains authorized for the production of commercial inoculants in Brazil.
pH is frequently reported as the main driver for prokaryotic community structure in soils. However, pH changes are also linked to "spillover effects" on other chemical parameters (e.g., availability ...of Al, Fe, Mn, Zn, and Cu) and plant growth, but these indirect effects on the microbial communities are rarely investigated. Usually, pH also co-varies with some confounding factors, such as land use, soil management (e.g., tillage and chemical inputs), plant cover, and/or edapho-climatic conditions. So, a more comprehensive analysis of the direct and indirect effects of pH brings a better understanding of the mechanisms driving prokaryotic (archaeal and bacterial) community structures.
We evaluated an agricultural soil pH gradient (from 4 to 6, the typical range for tropical farms), in a liming gradient with confounding factors minimized, investigating relationships between prokaryotic communities (16S rRNA) and physical-chemical parameters (indirect effects). Correlations, hierarchical modeling of species communities (HMSC), and random forest (RF) modeling indicated that both direct and indirect effects of the pH gradient affected the prokaryotic communities. Some OTUs were more affected by the pH changes (e.g., some Actinobacteria), while others were more affected by the indirect pH effects (e.g., some Proteobacteria). HMSC detected a phylogenetic signal related to the effects. Both HMSC and RF indicated that the main indirect effect was the pH changes on the availability of some elements (e.g., Al, Fe, and Cu), and secondarily, effects on plant growth and nutrient cycling also affected the OTUs. Additionally, we found that some of the OTUs that responded to pH also correlated with CO
, CH
, and N
O greenhouse gas fluxes.
Our results indicate that there are two distinct pH-related mechanisms driving prokaryotic community structures, the direct effect and "spillover effects" of pH (indirect effects). Moreover, the indirect effects are highly relevant for some OTUs and consequently for the community structure; therefore, it is a mechanism that should be further investigated in microbial ecology.
Summary
Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Structures of siderophores ...produced by bacterial endophytes have not yet been elucidated. The aim of this work was to identify and characterize the siderophores produced by this bacterium. In a screening for mutants unable to produce siderophores we found a mutant that had a transposon insertion in a non‐ribosomal peptide synthase (NRPS) gene coding for a putative siderophore biosynthetic enzyme. The chemical structure of the siderophore was predicted using computational genomic tools. The predicted structure was confirmed by chemical analysis. We found that siderophores produced by H. seropedicae Z67 are a suite of amphiphilic lipopeptides, named serobactin A, B and C, which vary by the length of the fatty acid chain. We also demonstrated the biological activity of serobactins as nutritional iron sources for H. seropedicae. These are the first structurally described siderophores produced by endophytic bacteria.
Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as ...esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.
In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).
The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.
The rapid growth of the world's population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria ...(PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB.
We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes.
PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.
Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in ...a wide range of environments, including soil.
Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria.
Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.
Azospirillum brasilense is a diazotrophic microorganism capable of associating with roots of important grasses and cereals, promoting plant growth and increasing crop yields. Nitrogen levels and the ...Ntr regulatory system control the nitrogen metabolism in A. brasilense. This system comprises the nitrogen regulatory proteins GlnD, which is capable of adding uridylyl groups to the PII proteins, GlnB (PII-1) and GlnZ (PII-2), under limiting nitrogen levels. Under such conditions, the histidine kinase NtrB (nitrogen regulatory protein B) cannot interact with GlnB and phosphorylate NtrC (nitrogen regulatory protein C). The phosphorylated form of NtrC acts as a transcriptional activator of genes involved in the metabolism of alternative nitrogen sources. Considering the key role of NtrC in nitrogen metabolism in A. brasilense, in this work we evaluated the proteomic and metabolomic profiles of the wild-type FP2 strain and its mutant ntrC grown under high and low nitrogen. Analysis of the integrated data identifies novel NtrC targets, including proteins involved in the response against oxidative stress (i.e., glutathione S-transferase and hydroperoxide resistance protein), underlining the importance of NtrC to bacterial survival under oxidative stress conditions.
MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this ...study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.
The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play ...a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.
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•The Amazon aerosol microbiome was studied during distinct seasons and heights.•Seasons had a major impact on the Amazon aerosol microbiome.•Humidity and temperature were main drivers of Bacterial taxa present in the Amazon aerosol.•Airborne bacterial taxa were not directly related to those present in soil.
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