RESUMO
Microorganisms are known to be a promising source of biopigments because they are easy to obtain, can be produced on a commercial scale, and are environmentally friendly. Therefore, the aim of this work was to characterize a brown pigment (BP) produced by HM053 in NFbHPN-lactate medium. The BP was extracted from the pellet (BPP) or supernatant (BPS), in the presence (BPPTrp, BPSTrp) or absence (BPPw, BPSw) of tryptophan (Trp). The UV-vis results were similar among all BP samples and compared with commercial melanin used as a standard, and the maximum absorption was observed around 200-220 nm. FTIR spectra showed that BP and commercial melanin had slight differences, with a small band between 3000-2840 cm- 1, related to C-H in the CH2 and CH3 aliphatic groups, which is not observed in the commercial melanin. Between BPP and BPS showed a different structure with bands in the region 1230-1070 cm- 1 related to groups C-O. The thermogravimetric curves for BPSw and BPSTrp showed similar behavior, with 4 stages of mass loss. The similarity between BPPw and BPPTrp with 2 stages of mass loss was also observed. Scanning electron microscopy results showed morphological differences between BPP and BPS, where BPP had a physical structure more homogeneous and a regular flat surface, while the BPS physical structure did not seem homogeneous and the surface was uneven with some spherical structures as commercial melanin.
Assuntos
Azospirillum brasilense , Melaninas , Triptofano , Triptofano/metabolismo , Triptofano/química , Melaninas/química , Melaninas/metabolismo , Azospirillum brasilense/metabolismo , Azospirillum brasilense/química , Azospirillum brasilense/genética , Pigmentos Biológicos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Meios de Cultura/químicaRESUMO
We assessed, in a field experiment, the effects of arbuscular mycorrhizal fungi (Rhizophagus intraradices) and plant growth-promoting bacteria (Azospirillum brasilense) on the soil biological activity and the growth of key pioneer species used in the revegetation of coal-mining areas undergoing recovery. We applied four inoculation treatments to the pioneer plant species (Lablab purpureus, Paspalum notatum, Crotalaria juncea, Neonotonia wightii, Stylosanthes guianensis, Andropogon gayanus and Trifolium repens) used in the recovery process: NI (Control - Non-inoculated), AZO (A. brasilense), AMF (R. intraradices), and co-inoculation of AZO and AMF. On the 75th and 180th days, we measured plant dry mass, mycorrhizal colonization, N and P concentration, and accumulation in plant tissue. We collected soil to quantify glomalin content and soil enzyme activity. After 180 days, we did a phytosociological characterization of the remaining spontaneous plants.The both microorganisms, singly or co-inoculated, promoted increases in different fractions of soil glomalin, acid phosphatase activity, and fluorescein diacetate activity at 75 and 180 days. The inoculation was linked to higher plant biomass production (62-89%) and increased plant P and N accumulation by 34-75% and 70-85% at 180 days, compared with the non-inoculated treatment. Among the pioneer species sown Crotalaria juncea produced the highest biomass at the 75th and 180th days (67% and 76% of all biomass), followed by Lablab purpureus (3% and 0.5%), while the other species failed to establish. At 180 days, we observed twenty spontaneous plant species growing in the area, primarily from the Poaceae family (74%). That suggests that the pioneer species present in the area do not hinder the ecological succession process. Inoculation of R. intraradices and A. brasilense, isolated or combined, increases soil biological activity, growth, and nutrient accumulation in key pioneer plant species, indicating the potential of that technique for the recovery of lands degraded by coal mining.
Assuntos
Azospirillum brasilense , Minas de Carvão , Micorrizas , Microbiologia do Solo , Solo , Micorrizas/fisiologia , Micorrizas/crescimento & desenvolvimento , Solo/química , Azospirillum brasilense/metabolismo , Azospirillum brasilense/crescimento & desenvolvimento , Glomeromycota/fisiologia , Glomeromycota/crescimento & desenvolvimento , Desenvolvimento Vegetal , Nitrogênio/metabolismo , Nitrogênio/análiseRESUMO
Azospirillum brasilense is a non-photosynthetic α-Proteobacteria, belongs to the family of Rhodospirillaceae and produces carotenoids to protect itself from photooxidative stress. In this study, we have used Resonance Raman Spectra to show similarity of bacterioruberins of Halobacterium salinarum to that of A. brasilense Cd. To navigate the role of genes involved in carotenoid biosynthesis, we used mutational analysis to inactivate putative genes predicted to be involved in carotenoid biosynthesis in A. brasilense Cd. We have shown that HpnCED enzymes are involved in the biosynthesis of squalene (C30), which is required for the synthesis of carotenoids in A. brasilense Cd. We also found that CrtI and CrtP desaturases were involved in the transformation of colorless squalene into the pink-pigmented carotenoids. This study elucidates role of some genes which constitute very pivotal role in biosynthetic pathway of carotenoid in A. brasilense Cd.
Assuntos
Azospirillum brasilense , Carotenoides , Esqualeno , Carotenoides/metabolismo , Azospirillum brasilense/metabolismo , Azospirillum brasilense/genética , Esqualeno/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Análise Espectral RamanRESUMO
Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.
Assuntos
Azospirillum brasilense , Proteínas de Bactérias , Quimiotaxia , Nitratos , Raízes de Plantas , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Azospirillum brasilense/fisiologia , Nitratos/metabolismo , Raízes de Plantas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.
Assuntos
Azospirillum brasilense , Cucumis sativus , Pisum sativum , Rhizobium leguminosarum , Plântula , Solanum lycopersicum , Solanum lycopersicum/microbiologia , Solanum lycopersicum/crescimento & desenvolvimento , Cucumis sativus/microbiologia , Cucumis sativus/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , Rhizobium leguminosarum/crescimento & desenvolvimento , Rhizobium leguminosarum/metabolismo , Azospirillum brasilense/crescimento & desenvolvimento , Azospirillum brasilense/metabolismo , Pisum sativum/microbiologia , Pisum sativum/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Quimiotaxia , Exsudatos de Plantas/química , Exsudatos de Plantas/metabolismoRESUMO
MicroRNA (miRNA) is a class of non-coding RNAs. They play essential roles in plants' physiology, as in the regulation of plant development, response to biotic and abiotic stresses, and symbiotic processes. This work aimed to better understand the importance of maize's miRNA during Azospirillum-plant interaction when the plant indole-3-acetic acid (IAA) production was inhibited with yucasin, an inhibitor of the TAM/YUC pathway. Twelve cDNA libraries from a previous Dual RNA-Seq experiment were used to analyze gene expression using a combined analysis approach. miRNA coding genes (miR) and their predicted mRNA targets were identified among the differentially expressed genes. Statistical differences among the groups indicate that Azospirillum brasilense, yucasin, IAA concentration, or all together could influence the expression of several maize's miRNAs. The miRNA's probable targets were identified, and some of them were observed to be differentially expressed. Dcl4, myb122, myb22, and morf3 mRNAs were probably regulated by their respective miRNAs. Other probable targets were observed responding to the IAA level, the bacterium, or all of them. A. brasilense was able to influence the expression of some maize's miRNA, for example, miR159f, miR164a, miR169j, miR396c, and miR399c. The results allow us to conclude that the bacterium can influence directly or indirectly the expression of some of the identified mRNA targets, probably due to an IAA-independent pathway, and that they are somehow involved in the previously observed physiological effects.
Assuntos
Azospirillum brasilense , MicroRNAs , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Zea mays/metabolismo , Ácidos Indolacéticos/metabolismo , Plantas/metabolismo , MicroRNAs/genética , RNA Mensageiro/metabolismoRESUMO
Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense, chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense. We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization.
Assuntos
Azospirillum brasilense , Quimiotaxia , Quimiotaxia/fisiologia , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras , Citoplasma/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/genéticaRESUMO
Increased soil salinity is one of the main concerns in agriculture and food production, and it negatively affects plant growth and crop productivity. In order to mitigate the adverse effects of salinity stress, plant biostimulants (PBs) have been indicated as a promising approach. Indeed, these products have a beneficial effect on plants by acting on primary and secondary metabolism and by inducing the accumulation of protective molecules against oxidative stress. In this context, the present work is aimed at comparatively investigating the effects of microbial (i.e., Azospirillum brasilense) and plant-derived biostimulants in alleviating salt stress in tomato plants by adopting a multidisciplinary approach. To do so, the morphological and biochemical effects were assessed by analyzing the biomass accumulation and root characteristics, the activity of antioxidant enzymes and osmotic stress protection. Furthermore, modifications in the metabolomic profiles of both leaves and root exudates were also investigated by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS). According to the results, biomass accumulation decreased under high salinity. However, the treatment with A. brasilense considerably improved root architecture and increased root biomass by 156% and 118% in non-saline and saline conditions, respectively. The antioxidant enzymes and proline production were enhanced in salinity stress at different levels according to the biostimulant applied. Moreover, the metabolomic analyses pointed out a wide set of processes being affected by salinity and biostimulant interactions. Crucial compounds belonging to secondary metabolism (phenylpropanoids, alkaloids and other N-containing metabolites, and membrane lipids) and phytohormones (brassinosteroids, cytokinins and methylsalicylate) showed the most pronounced modulation. Overall, our results suggest a better performance of A. brasilense in alleviating high salinity than the vegetal-derived protein hydrolysates herein evaluated.
Assuntos
Azospirillum brasilense , Solanum lycopersicum , Solanum lycopersicum/metabolismo , Azospirillum brasilense/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Raízes de Plantas/metabolismo , Plantas/metabolismo , Estresse SalinoRESUMO
Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: ⢠Genes required for glucose utilization in Azospirillum lipoferum were identified. ⢠A gene cassette encoding glucose utilization was constructed. ⢠Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..
Assuntos
Azospirillum brasilense , Azospirillum , Oryza , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Glucose/metabolismoRESUMO
The current study aimed to scale up the favorable bio-stimulants for enhancing the growth and breeding strategies of Stevia rebaudiana to increase sugar productivity. Inoculation of 45-day-old S. rebaudiana plantlets with Bacillus cereus and Azospirillum brasilense alone or in combination for 30 days allowed comparisons among their effects on enhancement and improvement of plant growth, production of bioactive compounds and expression of steviol glycoside genes. B. cereus SrAM1 isolated from surface-sterilized Stevia rebaudiana leaves was molecularly identified using 16s rRNA and tested for its ability to promote plant growth. Beneficial endophytic B. cereus SrAM1 induced all plant growth-promoting traits, except solubilization of phosphate, therefore it showed high effectiveness in the promotion of growth and production of bioactive compounds. Treatment of plants with B. cereus SrAM1 alone revealed carbohydrates content of 278.99 mg/g, total soluble sugar of 114.17 mg/g, total phenolics content of 34.05 mg gallic acid equivalent (GAE)/g dry weight) and total antioxidants activity of 32.33 mg (A.A)/g dry weight). Thus, plantlets inoculated with B. cereus SrAM1 alone exhibited the greatest responses in physiological and morphological parameters, but plantlets inoculated with B. cereus SrAM1 + A. brasilense showed a maximal upregulation of genes responsible for the biosynthesis of steviol glycosides (Kaurene oxidase, ent-KO; UDP-dependent glycosyl transferases of UGT85C2, UGT74G1, UGT76G1). Taken together, the used bacterial strains, particularly B. cereus SrAM1 could significantly improve the growth of S. rebaudiana via dynamic interactions in plants.
Assuntos
Azospirillum brasilense , Diterpenos do Tipo Caurano , Stevia , Antioxidantes/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bacillus cereus/genética , Diterpenos do Tipo Caurano/metabolismo , Ácido Gálico/farmacologia , Regulação da Expressão Gênica de Plantas , Glucosídeos/metabolismo , Glicosídeos/metabolismo , Biologia Molecular , Fosfatos/metabolismo , Melhoramento Vegetal , Folhas de Planta/metabolismo , RNA Ribossômico 16S , Stevia/metabolismo , Açúcares/metabolismo , Transferases/genética , Difosfato de Uridina/metabolismoRESUMO
Although most cultivated soils have high levels of total phosphorus (P), the levels of bioavailable inorganic P (Pi) are insufficient. The application of plant-growth-promoting rhizobacteria (PGPR) is an eco-friendly strategy for P utilization; however, PGPR-mediated plant responses that enhance Pi acquisition remain unexplored. Here, we investigated the effect of Azospirillum brasilense on Arabidopsis adaptation to Pi deficiency. Results showed that A. brasilense inoculation alleviated Pi-deficiency-induced growth inhibition and anthocyanin accumulation and increased the total P content in Arabidopsis plants. A comprehensive analysis of root morphology revealed that A. brasilense increased root hair density and length under Pi-limited conditions. We further demonstrated that A. brasilense enhanced the acid phosphatase activity and upregulated the expression of several Pi transporter genes, such as PHOSPHATE1 (PHO1), PHOSPHATE TRANSPORTER 1:(PHT1:1) and PHT1;4. However, A. brasilense did not enhance the growth o total P content in pht1;1, pht1;4 and pht1;1pht1;4 mutants. Moreover, A. brasilense could not increase the P content and PHT1;1 expression in the root hairless mutant rsl4rsl2, because of the occurrence of low-Pi-induced PHT1;1 and PHT1;4 in root hairs. These results indicate that A. brasilense can promote root hair development and enhance acid phosphatase activity and Pi transporter expression levels, consequently improving the Pi absorption capacity and conferring plant tolerance to Pi deficiency.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Azospirillum brasilense , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Azospirillum brasilense/metabolismo , Fosfatos/metabolismo , Fósforo/metabolismo , Raízes de Plantas/metabolismoRESUMO
The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.
Assuntos
Azospirillum brasilense , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ2 and σ4 , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ2 -σ4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10.
Assuntos
Azospirillum brasilense , Fator sigma , Aminoácidos/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Homeostase , Fator sigma/químicaRESUMO
AIMS: This study assessed, at the physiological and molecular levels, the effect of biogas on indole-3-acetic acid (IAA) biosynthesis by Azospirillum brasilense as well as the impact of this bacterium during CO2 fixation from biogas by Chlorella vulgaris and Scenedesmus obliquus. METHODS AND RESULTS: IpdC gene expression, IAA production and the growth of A. brasilense cultured under air (control) and biogas (treatment) were evaluated. The results demonstrated that A. brasilense had a better growth capacity and IAA production (105.7 ± 10.3 µg ml-1 ) when cultured under biogas composed of 25% CO2 + 75% methane (CH4 ) with respect to the control (72.4 ± 7.9 µg ml-1 ), although the ipdC gene expression level was low under the stressful condition generated by biogas. Moreover, this bacterium was able to induce a higher cell density and CO2 fixation rate from biogas by C. vulgaris (0.27 ± 0.08 g l-1 d-1 ) and S. obliquus (0.22 ± 0.08 g l-1 d-1 ). CONCLUSIONS: This study demonstrated that A. brasilense has the capacity to grow and actively maintain its main microalgal growth-promoting mechanism when cultured under biogas and positively influence CO2 fixation from the biogas of C. vulgaris and S. obliquus. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings broaden research in the field of Azospirillum-microalga interactions and the prevalence of Azospirillum in environmental and ecological topics in addition to supporting the uses of plant growth-promoting bacteria to enhance biotechnological strategies for biogas upgrading.
Assuntos
Azospirillum brasilense , Chlorella vulgaris , Microalgas , Atmosfera , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Biocombustíveis , Dióxido de Carbono/metabolismo , Chlorella vulgaris/metabolismo , Ácidos Indolacéticos/metabolismo , Microalgas/metabolismoRESUMO
3-Hydroxypropionic acid (3HP) is a platform chemical and can be converted into other valuable C3-based chemicals. Because a large amount of glycerol is produced as a by-product in the biodiesel industry, glycerol is an attractive carbon source in the biological production of 3HP. Although eight 3HP-producing aldehyde dehydrogenases (ALDHs) have been reported so far, the low conversion rate from 3-hydroxypropionaldehyde (3HPA) to 3HP using these enzymes is still a bottleneck for the production of 3HP. In this study, we elucidated the substrate binding modes of the eight 3HP-producing ALDHs through bioinformatic and structural analysis of these enzymes and selected protein engineering targets for developing enzymes with enhanced enzymatic activity against 3HPA. Among ten AbKGSADH variants we tested, three variants with replacement at the Arg281 site of AbKGSADH showed enhanced enzymatic activities. In particular, the AbKGSADHR281Y variant exhibited improved catalytic efficiency by 2.5-fold compared with the wild type.
Assuntos
Azospirillum brasilense , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Gliceraldeído/análogos & derivados , Glicerol/metabolismo , Engenharia Metabólica , Propano/metabolismo , Engenharia de ProteínasRESUMO
The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.
Assuntos
Azospirillum brasilense , Chlorella , Microalgas , Sistemas de Secreção Tipo VI , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Microalgas/genética , Microalgas/metabolismo , Simbiose , Sistemas de Secreção Tipo VI/metabolismoRESUMO
Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.
Assuntos
Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Frutose/farmacologia , Glicerol/farmacologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura , Frutose/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicerol/metabolismo , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Fasciclin domain proteins (FDP) are found in all domains of life, but their biological role and regulation are not clearly understood. While studying the proteome of a mutant (Car1) of Azospirillum brasilense Sp7 with a Tn5 insertion in the gene encoding an anti-sigma factor (ChrR1), we found that FDP was maximally expressed. To study the biological role of this FDP, we inactivated fdp in A. brasilense Sp7 and in its Car1 mutant, which rendered them sensitive to methylene blue (MB) and toluidine blue (TB) in the presence of light. The transcription of fdp was also strongly upregulated by an ECF sigma factor (RpoE1) and photooxidative stress. The fdp null mutants of A. brasilense Sp7 and its Car1 mutant produced relatively fewer carotenoids and showed reduced flocculation. The reduced ability of fdp null mutants to flocculate was partly due to their reduced ability to produce carotenoids as inhibition of carotenoid synthesis by diphenylamine reduced their flocculation ability by 15-20%. Hence, FDP plays an important role in protecting A. brasilense Sp7 against photo-oxidative stress by supporting carotenoid accumulation and cell aggregation.
Assuntos
Azospirillum brasilense/fisiologia , Proteínas de Bactérias/metabolismo , Luz , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carotenoides/metabolismo , Biologia Computacional , Floculação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Filogenia , Domínios Proteicos , Fator sigma/genética , Fator sigma/metabolismoRESUMO
The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.
Assuntos
Azospirillum brasilense , Bacillus thuringiensis , Compostos Orgânicos Voláteis , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bacillus thuringiensis/genética , Raízes de Plantas/microbiologia , Compostos Orgânicos Voláteis/metabolismo , Zea mays/metabolismo , Zea mays/microbiologiaRESUMO
Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.