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Stevia rebaudiana is associated with the production of calorie-free steviol glycosides (SGs) sweetener, receiving worldwide interest as a sugar substitute for people with metabolic disorders. The aim of this investigation is to show the promising role of endophytic bacterial strains isolated from Stevia rebaudiana Egy1 leaves as a biofertilizer integrated with Azospirillum brasilense ATCC 29,145 and gibberellic acid (GA 3 ) to improve another variety of stevia (S. rebaudiana Shou-2) growth, bioactive compound production, expression of SGs involved genes, and stevioside content. Endophytic bacteria isolated from S. rebaudiana Egy1 leaves were molecularly identified and assessed in vitro for plant growth promoting (PGP) traits. Isolated strains Bacillus licheniformis SrAM2, Bacillus paralicheniformis SrAM3 and Bacillus paramycoides SrAM4 with accession numbers MT066091, MW042693 and MT066092, respectively, induced notable variations in the majority of PGP traits production. B. licheniformis SrAM2 revealed the most phytohormones and hydrogen cyanide (HCN) production, while B. paralicheniformis SrAM3 was the most in exopolysaccharides (EPS) and ammonia production 290.96 ± 10.08 mg/l and 88.92 ± 2.96 mg/ml, respectively. Treated plants significantly increased in performance, and the dual treatment T7 (B. paramycoides SrAM4 + A. brasilense) exhibited the highest improvement in shoot and root length by 200% and 146.7%, respectively. On the other hand, T11 (Bacillus cereus SrAM1 + B. licheniformis SrAM2 + B. paralicheniformis SrAM3 + B. paramycoides SrAM4 + A. brasilense + GA 3 ) showed the most elevation in number of leaves, total soluble sugars (TSS), and up-regulation in the expression of the four genes ent-KO, UGT85C2, UGT74G1 and UGT76G1 at 2.7, 3.3, 3.4 and 3.7, respectively. In High-Performance Liquid Chromatography (HPLC) analysis, stevioside content showed a progressive increase in all tested samples but the maximum was exhibited by dual and co-inoculations at 264.37% and 289.05%, respectively. It has been concluded that the PGP endophytes associated with S. rebaudiana leaves improved growth and SGs production, implying the usability of these strains as prospective tools to improve important crop production individually or in consortium., (© 2024. The Author(s).)

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 CH 2 and CH 3 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., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anil Kumar Tripathi reports financial support and equipment, drugs, or supplies were provided by Banaras Hindu University. Anil Kumar Tripathi reports a relationship with Banaras Hindu University that includes: employment. None If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., Competing Interests: The authors declare no conflict of interest.

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.., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 σ 2 and σ 4 , 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 σ 2 -σ 4 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., (© 2022 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2022. Published by Elsevier Inc.)

Strain Az39 T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20-38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5-0.9 to 1.8-2.2 µm. They contain C 16 : 0 , C 18 : 0 and C 18 : 1  ω 7 c / ω 6 c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum , showing a close relationship with Azospirillum baldaniorum Sp245 T , Azospirillum brasilense Sp7 T and Azospirillum formosense CC-Nfb-7 T . Housekeeping gene analysis revealed that Az39 T , together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245 T , A. brasilense Sp7 T and A. formosense CC-Nfb-7 T . Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Az39 T and the aforementioned type strains revealed values below 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39 T , Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5 %. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39 T , along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39 T (=LBPCV39 T =BR 148428 T =CCCT 22.01 T ).

Non-legume plants such as rice and maize can form beneficial associations with plant growth-promoting bacteria (PGPB) such as Herbaspirillum seropedicae and Azospirillum brasilense. Several studies have shown that these PGPB promote plant growth via multiple mechanisms. Our current understanding of the molecular aspects and signaling between plants like rice and PGPB like Herbaspirillum seropedicae is limited. In this study, we used an experimental system where H. seropedicae could colonize the plant roots and promote growth in wild-type rice. Using this experimental setup, we identified 1688 differentially expressed genes (DEGs) in rice roots, 1 day post-inoculation (dpi) with H. seropedicae. Several of these DEGs encode proteins involved in the flavonoid biosynthetic pathway, defense, hormone signaling pathways, and nitrate and sugar transport. We validated the expression pattern of some genes via RT-PCR. Next, we compared the DEGs identified in this study to those we previously identified in rice roots during associations with another PGPB, Azospirillum brasilense. We identified 628 genes that were differentially expressed during both associations. The expression pattern of these genes suggests that some of these are likely to play a significant role(s) during associations with both H. seropedicae and A. brasilense and are excellent targets for future studies., (© 2022. The Author(s).)

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 CO 2 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% CO 2  + 75% methane (CH 4 ) 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 CO 2 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 CO 2 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., (© 2022 The Society for Applied Microbiology.)

The use of inoculants carrying diazotrophic and other plant growth-promoting bacteria plays an essential role in the Brazilian agriculture, with a growing use of microorganism-based bioproducts. However, in the last few years, some farmers have multiplied microorganisms in the farm, known as "on farm" production, including inoculants of Bradyrhizobium spp. for soybean (Glycine max L. Merrill.) and Azospirillum brasilense for corn (Zea mays L.) or co-inoculation in soybean. The objective was to assess the microbiological quality of such inoculants concerning the target microorganisms and contaminants. In the laboratory, 18 samples taken in five states were serial diluted and spread on culture media for obtaining pure and morphologically distinct colonies of bacteria, totaling 85 isolates. Molecular analysis based on partial sequencing of the 16S rRNA gene revealed 25 genera of which 44% harbor species potentially pathogenic to humans; only one of the isolates was identified as Azospirillum brasilense, whereas no isolate was identified as Bradyrhizobium. Among 34 isolates belonging to genera harboring species potentially pathogenic to humans, 12 had no resistance to antibiotics, six presented intrinsic resistance, and 18 presented non-intrinsic resistance to at least one antibiotic. One of the samples analyzed with a shotgun-based metagenomics approach to check for the microbial diversity showed several genera of microorganisms, mainly Acetobacter (~ 32% of sequences) but not the target microorganism. The samples of inoculants produced on farm were highly contaminated with non-target microorganisms, some of them carrying multiple resistances to antibiotics., (© 2021. Sociedade Brasileira de Microbiologia.)

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 Ab KGSADH variants we tested, three variants with replacement at the Arg281 site of Ab KGSADH showed enhanced enzymatic activities. In particular, the Ab KGSADH R281Y variant exhibited improved catalytic efficiency by 2.5-fold compared with the wild type.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7 days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

An Egyptian rhizobacterium Azospirillum sp. isolated from Sadat city was able to produce indole acetic acid (IAA) up to (30.59 µg/ml). The isolate was identified biochemically and by 16S rRNA sequencing which showed 99.9% similarity to Azospirillum brasilense. The new isolate has been registered in Genbank with accession number MH179119.1. Extracted IAA was used as reducing or stabilizing agent of sliver nanoparticles (AgNPs). Successful fabrication of biogenic IAA-AgNPs was confirmed by Fourier Transform Infrared Spectrophotometer (FTIR) analysis of IAA which showed absorbance peak at 3434.78 cm -1 due to the N-H stretch of primary amines. Highly resolution Transmission Electron Microscopy (HR-TEM) showed AgNPs coating or capping with IAA in spherical shaped with size ranged from 6.01 to 44.02 nm. Energy dispersive X-ray (EDX) analysis revealed that Ag + ions were attached to the surface of IAA-AgNPs particles. HR-TEM examination showed cell wall damage of Citrobacter freundii cells after exposure to IAA-AgNPs leading to cell death. In vivo results showed that C. freundii infection of rats induced significant increase in liver and kidney functions and deleterious histopathological alteration in rat's tissues. However, treatment by extracted IAA and IAA-AgNPs could normalize the biochemical and histopathological alterations occurred in infected rats. This is the first study to prove that IAA extracted from Azospirillum brasilense is a hopeful capping agent for NPs which has potential to protect against pathogenic infections, nontoxic and/or safe on rat's metabolisms., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

To evaluate the natural occurrence of the plant growth-promoting bacterium Azospirillum brasilense and petunia plants, local strains were isolated and characterized by biochemical and molecular methods. Three strains were assessed in greenhouse conditions using Petunia × hybrida Ultra™. Treatments: Plants without bacterial inoculation or chemical fertilization; fertilized with NPK and KNO 3 ; and independently inoculated with the strains 2A1, 2A2, and 2E1 by submerging their roots in a bacterial suspension (~10 6  CFU·ml -1 ). Root length, dry weight of roots and shoots, leaf area, leaf greenness, and nutrient content were evaluated. The number of days from transplanting to the opening of the first flower and the number of flowers per plant were also determined. As a result, five isolates were characterized as A. brasilense, showing the capacity to produce indoles and siderophores, to solubilize phosphate, nitrogenase activity, and nifH-PCR amplification. In general, all the parameters of the plant assay were improved in plants inoculated with A. brasilense, with variations among the strains, as well as the onset of flowering and the number of flowers per plant, compared with uninoculated or fertilized plants. This is the first report on the natural occurrence of A. brasilense in petunia with the capacity to improve plant growth and flowering., (© 2021 Wiley-VCH GmbH.)

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 OD 600 (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 15 N 2 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.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Iron (Fe), an essential element for plant growth, is abundant in soil but with low bioavailability. Thus, plants developed specialized mechanisms to sequester the element. Beneficial microbes have recently become a favored method to promote plant growth through increased uptake of essential micronutrients, like Fe, yet little is known of their mechanisms of action. Functional mutants of the epiphytic bacterium Azospirillum brasilense, a prolific grass-root colonizer, were used to examine mechanisms for promoting iron uptake in Zea mays. Mutants included HM053, FP10, and ipdC, which have varying capacities for biological nitrogen fixation and production of the plant hormone auxin. Using radioactive iron-59 tracing and inductively coupled plasma mass spectrometry, we documented significant differences in host uptake of Fe 2+/3+ correlating with mutant biological function. Radioactive carbon-11, administered to plants as 11 CO 2 , provided insights into shifts in host usage of 'new' carbon resources in the presence of these beneficial microbes. Of the mutants examined, HM053 exhibited the greatest influence on host Fe uptake with increased plant allocation of 11 C-resources to roots where they were transformed and exuded as 11 C-acidic substrates to aid in Fe-chelation, and increased C-11 partitioning into citric acid, nicotianamine and histidine to aid in the in situ translocation of Fe once assimilated.

Azospirillum brasilense is a non-photosynthetic member of the family Rhodospirillaceae. Some strains of this bacterium are reported to produce bacterioruberin type of carotenoids, which are generally produced by halophilic or psychrophilic bacteria. Since no other member of Rhodospirillaceae produces bacterioruberin type of carotenoids, we investigated the presence of genes involved in bacterioruberin and spirilloxanthin biosynthetic pathways in A. brasilense Cd. Although genes encoding the spirilloxanthin pathway were absent, homologs of several genes (crtC and crtF) involved in the biosynthesis of bacterioruberins were present in the genome of A. brasilense Cd. However, the homolog of CruF responsible for the final step in bacterioruberin biosynthesis could not be found. We also characterized the carotenoids of A. brasilense Cd using thin-layer chromatography, high-performance liquid chromatography, absorption spectra and high-resolution mass spectrometry (HRMS). Resolution of the methanol extract of carotenoids in ultra-performance liquid chromatography showed nine peaks, out of which six peaks showed absorption spectra characteristic of carotenoids. HRMS of each peak produced 1-14 fragments with different m/z values. Two of these fragments were identified as 19'-hydroxyfucoxanthinol and 8'-apoalloxanthinal, which are the carotenoids found in aquatic microalgae., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO 3 or NH 4 Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

The bacterium Azospirillum brasilense can swim and swarm owing to the work of polar and lateral flagella. Its major surface glycopolymers consist of lipopolysaccharides (LPS) and Calcofluor-binding polysaccharides (Cal + phenotype). Motility and surface glycopolymers are important for the interactions of plant-associated bacteria with plants. The facultative plant endophyte A. brasilense Sp245 produces two antigenically different LPS, LpsI, and LpsII, containing identical O-polysaccharides. Previously, using vector pJFF350 for random Omegon-Km mutagenesis, we constructed a mutant of Sp245 named KM018 that still possessed flagella, although paralyzed. The mutant was no longer able to produce Calcofluor-binding polysaccharides and LpsII. Because of the limited experimental data on the genetic aspects of surface glycopolymer production and flagellar motility in azospirilla, the aim of this study was to identify and examine in more detail the coding sequence of strain Sp245, inactivated in the mutant. We found that pJFF350 was integrated into a coding sequence for a putative integral membrane protein of unknown function (AZOBR_p60025) located in the sixth plasmid of Sp245. To clarify the role of the putative protein, we cloned AZOBR_p60025 in the expression vector pRK415 and used it for the genetic complementation of mutant KM018. The SDS-PAGE, immunodiffusion, and linear immunoelectrophoresis analyses showed that in strain KM018 (pRK415-p60025), the wild-type LpsI + LpsII + profile was restored. The complemented mutant had a Cal + phenotype and it was capable of swimming and swarming motility. Thus, the AZOBR_p60025-encoded protein significantly affects the composition of the major cell-surface glycopolymers and the single-cell and social motility of azospirilla.

Background: Small RNAs (sRNAs) are non-coding RNAs known to regulate various biological functions such as stress adaptation, metabolism, virulence as well as pathogenicity across a wide range of bacteria, mainly by controlling mRNA stabilization or regulating translation. Identification and functional characterization of sRNAs has been carried out in various plant growth-promoting bacteria and they have been shown to help the cells cope up with environmental stress. No study has been carried out to uncover these regulatory molecules in the diazotrophic alpha-proteobacterium Azospirillum brasilense Sp245 to date., Results: Expression-based sRNA identification (RNA-seq) revealed the first list of ~ 468 sRNA candidate genes in A. brasilense Sp245 that were differentially expressed in nitrogen starvation versus non-starved conditions. In parallel, in silico tools also identified 2 of the above as candidate sRNAs. Altogether, putative candidates were stringently curated from RNA-seq data based on known sRNA parameters (size, location, secondary structure, and abundance). In total, ~ 59 significantly expressed sRNAs were identified in this study of which 53 are potentially novel sRNAs as they have no Rfam and BSRD homologs. Sixteen sRNAs were randomly selected and validated for differential expression, which largely was found to be in congruence with the RNA-seq data., Conclusions: Differential expression of 468 A. brasilense sRNAs was indicated by RNA-seq data, a subset of which was confirmed by expression analysis. Four of the significantly expressed sRNAs were not observed in nitrogen starvation while 16 sRNAs were found to be exclusively expressed in nitrogen depletion. Putative candidate sRNAs identified have potential mRNA targets primarily involved in stress (abiotic and biotic) adaptability; regulation of bacterial cellular, biological and molecular pathways such as nitrogen fixation, polyhydroxybutyrate synthesis, chemotaxis, biofilm formation and transcriptional regulation. In addition to directly influencing bacteria, some of these sRNAs also have targets influencing plant-microbe interactions through adhesion of bacteria to plant roots directly, suppressing host response, inducing plant defence and signalling.

Azospirillum brasilense Az19 is a plant-beneficial bacterium capable of protecting plants from the negative effects of drought. The objective of this study was to determine and analyze the genomic sequence of strain Az19 as a means of identifying putative stress-adaptation mechanisms. A high-quality draft genome of ca. 7 Mb with a predicted coding potential of 6710 genes was obtained. Phylogenomic analyses confirmed that Az19 belongs to the brasilense clade and is closely related to strains Az39 and REC3. Functional genomics revealed that the denitrification pathway of Az19 is incomplete, which was in agreement with a reduced growth on nitrate under low O 2 concentrations. Putative genes of the general stress response and oxidative stress-tolerance, as well as synthesis of exopolysaccharides, carotenoids, polyamines and several osmolytes, were detected. An additional poly-beta-hydroxybutyrate (PHB) synthase coding gene was found in Az19 genome, but the accumulation of PHB did not increase under salinity. The detection of exclusive genes related to DNA repair led to discover that strain Az19 also has improved UV-tolerance, both in vitro and in planta. Finally, the analysis revealed the presence of multiple kaiC-like genes, which could be involved in stress-tolerance and, possibly, light responsiveness. Although A. brasilense has been a model for the study of beneficial plant-associated rhizobacteria, the evidence collected in this current study suggests, for the first time in this bacterial group, an unexpected possibility of adaptation to the phyllosphere., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense , a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes ( crtE1 and crtE2 ) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase ( ads ) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals. IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae , do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production., (Copyright © 2020 American Society for Microbiology.)

The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d-rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII - mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415-p60126 into KM139. In the complemented mutant KM139 (pRK415-p60126), the wild-type LPSI + LPSII + profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415-p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI + LpsII - strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI + LpsII + strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 10 5 CFU/ml (4.5 ng/μl ADN) or 10 2 CFU/ml (0.88 ng/μl DNA) or in a minimal concentration of 0.098 ng/μl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers., (Copyright © 2019 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Azospirillum brasilense is a plant growth-promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA , however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C 4 -dicarboxylates in A. brasilense , respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana , with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

Aims: The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions., Methods and Results: We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500 nmol l -1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium., Conclusions: Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity., Significance and Impact of the Study: This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism., (© 2019 The Society for Applied Microbiology.)

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG., (© 2019 Federation of European Biochemical Societies.)

The O-polysaccharide (O-antigen) of Vibrio cholerae O14 was studied using chemical analyses and 1D and 2D NMR spectroscopy. The following structure of the repeating unit of the O-antigen was established: where GlcpN(SHb) indicates 2-deoxy-2-[(S)-3-hydroxybutanoylamino]-d-glucose. We found that Vibrio cholerae O14 is similar to that of O-polysaccharide of Azospirillum brasilense S17, which has been reported earlier. Moreover, we predicted functions of all the genes in the O-antigen gene cluster according to the structure established. Our study enriches the existing O-antigen database of Vibrio cholerae, and further facilitates the bacterial serotype identification., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacteria Azospirillum brasilense may swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf). They also construct sessile biofilms on various interfaces. As compared to the wild-type strain Sp245, the previously characterized Fla - Laf - flhB1 mutant Sp245.1063 accumulated less biomass in mature biofilms, which also were susceptible to the forces of hydrodynamic shear. In this study, we compared biofilms formed by strain Sp245 and its previously constructed derivatives on the interfaces between a minimal (malate-salt medium, or MSM) or rich (LB) liquid growth medium and a hydrophilic (glass) or hydrophobic (polystyrene) solid surface under static or dynamic conditions. In all experimental settings, the alterations in Sp245.1063's mature biofilm traits were partially (in MSM) or completely (in LB) rescued in the complemented mutant Sp245.1063 (pRK415-150177), which received the pRK415-borne coding sequence for the putative FlhB1 protein of the flagellar type III secretion system. Although Laf were not found in the biofilms of azospirilla, Fla was present on the biofilm cells of the complemented mutant Sp245.1063 (pRK415-150177) and other studied strains, which had normal flagellation on liquid and solid nutritional media. Accordingly, mature biofilms of these strains contained more biomass and were significantly more resistant to shaking at 140 rpm, as compared to the biofilms of the flagella-free mutant bacteria. These data proved that the polar flagellum of A. brasilense Sp245 plays a significant positive role in biofilm biomass increase and in biofilm stabilization.

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 10 4 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 10 3  cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sorghum [Sorghum bicolor (L.) Moench] is a multipurpose grass cultivated in drylands due to its adaptation to drought. However the characteristics of sorghum-associated bacteria are not known in the Brazilian drylands. The aim of this study was to isolate and evaluate the plant growth promotion potential bacteria from field-grown sorghum under two irrigation and manure application levels in a Brazilian semi-arid reagion. Sorghum was irrigated with 3 or 1 mm day -1 and fertilized or not with liquid goat manure. Bacteria were obtained from surface-disinfected roots applying two nitrogen-free semi-solid media. The bacteria were evaluated for the presence of nifH gene, 16S rRNA sequences, calcium-phosphate solubilization, production of auxins and siderophores and for sorghum growth promotion. We obtained 20 out of 24 positive bacteria for nifH. The isolates were classified as in six different genera. All isolates produced auxins "in vitro", six bacteria produced siderophores and three Enterobacteriaceae solubilized calcium-phosphate. At least ten bacteria resulted in the increased total N content in the sorghum shoots, comparable to fertilization with 50 mg N plant -1 week -1 and to inoculation with Azospirillum brasilense Ab-V5. Enterobacter sp. ESA 57 was the best sorghum plant-growth promoting bacteria isolated in this study.

The genome of Azospirillum brasilense encodes five RpoH sigma factors: two OxyR transcription regulators and three catalases. The aim of this study was to understand the role they play during oxidative stress and their regulatory interconnection. Out of the 5 paralogs of RpoH present in A. brasilense , inactivation of only rpoH1 renders A. brasilense heat sensitive. While transcript levels of rpoH1 were elevated by heat stress, those of rpoH3 and rpoH5 were upregulated by H 2 O 2 Catalase activity was upregulated in A. brasilense and its rpoH :: km mutants in response to H 2 O 2 except in the case of the rpoH5 :: km mutant, suggesting a role for RpoH5 in regulating inducible catalase. Transcriptional analysis of the katN , katA I, and katA II genes revealed that the expression of katN and katA II was severely compromised in the rpoH3 :: km and rpoH5 :: km mutants, respectively. Regulation of katN and katA II by RpoH3 and RpoH5, respectively, was further confirmed in an Escherichia coli two-plasmid system. Regulation of katA II by OxyR2 was evident by a drastic reduction in growth, KatAII activity, and katA II:: lacZ expression in an oxyR2 :: km mutant. This study reports the involvement of RpoH3 and RpoH5 sigma factors in regulating oxidative stress response in alphaproteobacteria. We also report the regulation of an inducible catalase by a cascade of alternative sigma factors and an OxyR. Out of the three catalases in A. brasilense , those corresponding to katN and katA II are regulated by RpoH3 and RpoH5, respectively. The expression of katA II is regulated by a cascade of RpoE1→RpoH5 and OxyR2. IMPORTANCE In silico analysis of the A. brasilense genome showed the presence of multiple paralogs of genes involved in oxidative stress response, which included 2 OxyR transcription regulators and 3 catalases. So far, Deinococcus radiodurans and Vibrio cholerae are known to harbor two paralogs of OxyR, and Sinorhizobium meliloti harbors three catalases. We do not yet know how the expression of multiple catalases is regulated in any bacterium. Here we show the role of multiple RpoH sigma factors and OxyR in regulating the expression of multiple catalases in A. brasilense Sp7. Our work gives a glimpse of systems biology of A. brasilense used for responding to oxidative stress., (Copyright © 2018 American Society for Microbiology.)

This study reports the introduction of egfp or mCherry markers to the Sp245, Sp7, and M40 wild-type strains of Azospirillum brasilense and the hhkB (encoding for a putative hybrid histidine kinase) minus mutant an isogenic strain of A. brasilense Sp245 to monitor colonization of wheat (Triticum aestivum). Two plasmids were constructed: (1) the pJMS-2 suicide plasmid derived from pSUP202 and harboring the mCherry gene expressed under the constitutive kanamycin resistance promoter to create a cis tag and (2) the broad-range plasmid pMP2449-5 that carries the mCherry gene under the lac promoter, which is derived from the plasmid pMP2444; to create the in trans tag. The stability of the plasmids encoding egfp and mCherry were confirmed in vitro for seven days of bacterial growth, and then, the A. brasilense strains harboring the plasmids were studied under nonselective conditions for adherence to seeds and, at seven or 14 days post-inoculation, for wheat root colonization. The utility of the labeled strains was proven by observation, using fluorescence microscopy and confocal laser scanning microscopy (CLSM) in wheat plants inoculated with the labeled strains and compared with the CFU g -1 for seed and wheat root. The method was suitable for observation of the in situ formation of mini-colonies, enabled visualization of bacterial colonization sites on large root fragments, and showed adherence to germinated seeds and root colonization of all strains by cell counts and direct microscopic examination. Thus, we are able to quantify the structures of the biofilms formed by each strain., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Indole-3-acetic acid (IAA) is one of the most important molecules produced by Azospirillum sp., given that it affects plant growth and development. Azospirillum brasilense strains Sp245 and Az39 (pFAJ64) were pre-incubated in MMAB medium plus 100 mg/mL L-tryptophan and treated with or exposed to the following (a) abiotic and (b) biotic stress effectors: (a) 100 mM NaCl or Na 2 SO 4, 4.0% (w/v) PEG 6000, 0.5 mM H 2 O 2, 0.1 mM abscisic acid, 0.1 mM 1-aminocyclopropane 1-carboxylic acid, 45 °C or daylight, and (b) 4.0% (v/v) filtered supernatant of Pseudomonas savastanoi (Ps) or Fusarium oxysporum (Fo), 0.1 mM salicylic acid (SA), 0.1 mM methyl jasmonic acid (MeJA), and 0.01% (w/v) chitosan (CH). After 30 and 120 min of incubation, biomass production, cell viability, IAA concentration (µg/mL), and ipdC gene expression were measured. Our results show that IAA production increases with daylight or in the presence of PEG 6000 , ABA, SA, CH, and Fo. On the contrary, exposure to 45 °C or treatment with H 2 O 2, NaCl, Na 2 SO 4, ACC, MeJA, and Ps decrease IAA biosynthesis. In this report, growth and IAA biosynthesis in A. brasilense under biotic and abiotic stress conditions are discussed from the point of view of their role in bacterial lifestyle and their potential application as bioproducts.

Several Gram-negative soil bacteria have the ability to differentiate into dormant cysts when faced with harsh environmental conditions. For example, when challenged with nutrient deprivation or desiccation, the plant-growth-promoting bacterium Azospirillum brasilense differentiates from a replicative and motile rod-shaped vegetative cell into a non-motile dormant spherical cyst. Currently, little is known about either the metabolic differences that exist between vegetative and cyst cell types, or about aspects of cyst physiology that allow dormant cells to survive harsh conditions. Here we compared transcriptomic profiles of vegetative and encysted A. brasilense. We observed that approximately one fifth of the A. brasilense transcriptome undergoes changes in expression between replicative vegetative cells and non-replicative cysts. A dramatic alteration in expression of genes involved in cell wall or cell membrane biogenesis was observed, which is congruent with changes in exopolysaccharide and lipid composition that occur between these cell types. Encysted cells also exhibited repressed mRNA abundance of genes involved in amino acid biosynthesis, ribosomal biogenesis and translation. We further observed that cysts create an anaerobic/micro-aerobic environment, as evidenced by repressed expression of oxidative phosphorylation genes coupled with increased expression of nitrate/nitrite reduction and nitrogen fixation genes.

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327-HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1:: Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase-response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.

Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the visualization of these responses are detailed in this unit. These assays are simple and can be readily implemented with minimum set ups. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla - Laf - fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.