Nitrogen is one of the most abundant elements in the biosphere, but its gaseous form is not biologically available to many organisms, including plants and animals. Diazotrophic microorganisms can convert atmospheric nitrogen into ammonia, a form that can be absorbed by plants in a process called biological nitrogen fixation (BNF). BNF is catalyzed by the enzyme nitrogenase, which not only reduces N2 to NH3 , but also reduces other substrates such as acetylene. The acetylene reduction assay (ARA) can be used to measure nitrogenase activity in diazotrophic organisms, either in symbiotic associations or in their free-living state. The technique uses gas chromatography to measure the reduction of acetylene to ethylene by nitrogenase in a simple, quick, and inexpensive manner. Here, we demonstrate how to: prepare nodulated soybean plants and culture free-living Azospirillum brasilense for the ARA, use the gas chromatograph to detect the ethylene formed, and calculate the nitrogenase activity based on the peaks generated by the chromatograph. The methods shown here using example organisms can be easily adapted to other nodulating plants and diazotrophic bacteria. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acetylene reduction assay in root nodules Basic Protocol 2: Acetylene reduction assay using diazotrophic bacteria Basic Protocol 3: Calculation of nitrogenase activity Support Protocol 1: Production of acetylene from calcium carbide Support Protocol 2: Calibration of the gas chromatograph Support Protocol 3: Total protein quantification.

Herbaspirillum seropedicae is a diazotrophic bacterium from the β-proteobacteria class that colonizes endophytically important gramineous species, promotes their growth through phytohormone-dependent stimulation and can express nif genes and fix nitrogen inside plant tissues. Due to these properties this bacterium has great potential as a commercial inoculant for agriculture. The H. seropedicae SmR1 genome is completely sequenced and annotated but despite the availability of diverse structural and functional analysis of this genome, studies involving small non-coding RNAs (sRNAs) has not yet been done. We have conducted computational prediction and RNA-seq analysis to select and confirm the expression of sRNA genes in the H. seropedicae SmR1 genome, in the presence of two nitrogen independent sources and in presence of naringenin, a flavonoid secreted by some plants. This approach resulted in a set of 117 sRNAs distributed in riboswitch, cis-encoded and trans-encoded categories and among them 20 have Rfam homologs. The housekeeping sRNAs tmRNA, ssrS and 4.5S were found and we observed that a large number of sRNAs are more expressed in the nitrate condition rather than the control condition and in the presence of naringenin. Some sRNAs expression were confirmed in vitro and this work contributes to better understand the post transcriptional regulation in this bacterium. H. seropedicae SmR1 express sRNAs in the presence of two nitrogen sources and/or in the presence of naringenin. The functions of most of these sRNAs remains unknown but their existence in this bacterium confirms the evidence that sRNAs are involved in many different cellular activities to adapt to nutritional and environmental changes.

The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.

There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear.

Plant growth stimulation by microorganisms that interact in a mutually beneficial manner remains poorly understood. Understanding the nature of plant-bacteria interactions may open new routes for plant productivity enhancement, especially cereal crops consumed by humans. Proteomic and metabolomic analyses are particularly useful for elucidating these mechanisms. A complete depiction of these mechanisms will prompt researchers to develop more efficient plant-bacteria associations. The success of microorganisms as biofertilizers may replace the current massive use of chemical fertilizers, mitigating many environmental and economic issues. In this review, we discuss the recent advances and current state of the art in proteomics and metabolomics studies involving grass-bacteria associations. We also discuss essential subjects involved in the bacterial plant-growth promotion, such, nitrogen fixation, plant stress, defense responses, and siderophore production.

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.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix(+)) or a fix mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized insitu laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix(-) strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indolealkaloid biosynthesis were more abundant in the roots colonized by the fix(-) strain, perhaps reflecting a plant defense response.

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

Biological aerosols (bioaerosol) are atmospheric particles that act as a dispersion unit of living organisms across the globe thereby affecting the biogeographic distribution of organisms. Despite their importance, there is virtually no knowledge about bioaerosols emitted by pristine forests. Here we provide the very first survey of the prokaryotic community of a bioaerosol collected inside pristine Amazon forest at 2 m above ground. Total atmospheric particles were collected at the Amazon Tall Tower Observatory, subjected to metagenomic DNA extraction and the prokaryotic diversity was determined by 16S rRNA gene amplicon sequencing. A total of 271,577 reads of 250 bp of the 16S rRNA gene amplicon were obtained. Only 27% of the reads could be classified using the 16S SILVA database. Most belonged to Proteobacteria, Actinobacteria and Firmicutes which is in good agreement with other bioaerosol studies. Further inspection of the reads using Blast searches and the 18S SILVA database revealed that most of the dataset was composed of Fungi sequences. The identified microbes suggest that the atmosphere may act as an important gateway to interchange bacteria between plants, soil and water ecosystems.

Rice is staple food of nearly half the world's population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growth and productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4% (1,014) genes were differentially regulated and of these 255 changed expression levels more than two times. Several of the repressed genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which transcripts of genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Three species of the β-Proteobacterial genus Herbaspirillum are able to fix nitrogen in endophytic associations with such important agricultural crops as maize, rice, sorghum, sugar-cane and wheat. In addition, Herbaspirillum rubrisubalbicans causes the mottled-stripe disease in susceptible sugar-cane cultivars as well as the red-stripe disease in some sorghum cultivars. The xylem of these cultivars exhibited a massive colonisation of mucus-producing bacteria leading to blocking the vessels. A cluster of eight genes ( bcs ) are involved in cellulose synthesis in Herbaspirillum rubrisubalbicans . Mutation of bcsZ , that encodes a 1,4-endoglucanase, impaired the exopolysaccharide production, the ability to form early biofilm and colonize sorghum when compared to the wild-type strain M1. This mutation also impaired the ability of Herbaspirillum rubrisubalbicans M1 to cause the red-stripe disease in Sorghum bicolor . We show cellulose synthesis is involved in the biofilm formation and as a consequence significantly modulates bacterial-plant interactions, indicating the importance of cellulose biosynthesis in this process.

Bacteria adjust the composition of their electron transport chain (ETC) to efficiently adapt to oxygen gradients. This involves differential expression of various ETC components to optimize energy generation. In Herbaspirillum seropedicae, reprogramming of gene expression in response to oxygen availability is controlled at the transcriptional level by three Fnr orthologs. Here, we characterised Fnr regulons using a combination of RNA-Seq and ChIP-Seq analysis. We found that Fnr1 and Fnr3 directly regulate discrete groups of promoters (Groups I and II, respectively), and that a third group (Group III) is co-regulated by both transcription factors. Comparison of DNA binding motifs between the three promoter groups suggests Group III promoters are potentially co-activated by Fnr3-Fnr1 heterodimers. Specific interaction between Fnr1 and Fnr3, detected in two-hybrid assays, was dependent on conserved residues in their dimerization interfaces, indicative of heterodimer formation in vivo. The requirements for co-activation of the fnr1 promoter, belonging to Group III, suggest either sequential activation by Fnr3 and Fnr1 homodimers or the involvement of Fnr3-Fnr1 heterodimers. Analysis of Fnr proteins with swapped activation domains provides evidence that co-activation by Fnr1 and Fnr3 at Group III promoters optimises interactions with RNA polymerase to fine-tune transcription in response to prevailing oxygen concentrations.

Mangroves are highly productive ecosystems located at the transition between the terrestrial and marine environments. Mangroves play an important role in carbon storage, nutrient cycling and support for the marine food web. Mangrove soils are formed by fine particles rich in organic carbon and are subject to constant fluctuations in oxygen, salinity and nutrient availability due to fresh water flux and tidal variations. Microbes play an important role in nutrient cycling in mangrove soils; however, studies on the mangrove soil microbiome are scarce. Here we compare the microbiome of pristine mangrove soil located in an environmentally protected area in Guaratuba, Southern Brazil, with the microbiome of mangrove soil affected by the presence of carbonaceaous debris eroding from an archeological site known as Sambaqui. We show that although the Sambaqui site has a major effect on soil chemistry, increasing the soil pH by 2.6 units, only minor changes in the soil microbiome were detected indicating resilience of the microbial community to pH variations. The high alpha diversity indexes and predicted metabolic potential suggest that the mangrove soil microbiome not only provides important ecological services but also may host a broad range of microbes and genes of biotechnological interest.

Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicansincreased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans riceinteractions were further characterized by proteomic approach.

Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicaeSmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.

Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

Herbaspirillum seropedicae is a nitrogen-fixing β-proteobacterium that associates with roots of gramineous plants. In silico analyses revealed that H. seropedicae genome has genes encoding a putative respiratory (NAR) and an assimilatory nitrate reductase (NAS). To date, little is known about nitrate metabolism in H. seropedicae, and, as this bacterium cannot respire nitrate, the function of NAR remains unknown. This study aimed to investigate the function of NAR in H. seropedicae and how it metabolizes nitrate in a low aerated-condition. RNA-seq transcriptional profiling in the presence of nitrate allowed us to pinpoint genes important for nitrate metabolism in H. seropedicae, including nitrate transporters and regulatory proteins. Additionally, both RNA-seq data and physiological characterization of a mutant in the catalytic subunit of NAR (narG mutant) showed that NAR is not required for nitrate assimilation but is required for: (i) production of high levels of nitrite, (ii) production of NO and (iii) dissipation of redox power, which in turn lead to an increase in carbon consumption. In addition, wheat plants showed an increase in shoot dry weight only when inoculated with H. seropedicae wild type, but not with the narG mutant, suggesting that NAR is important to H. seropedicae-wheat interaction.

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism.

In this study, a random mutant library of Herbaspirillum seropedicae SmR1 was constructed by Tn5 insertion and a mutant incapable of utilizing naringenin as a carbon source was isolated. The Tn5 transposon was found to be inserted in the fdeE gene (Hsero_1007), which encodes a monooxygenase. Two other mutant strains in fdeC (Hsero_1005) and fdeG (Hsero_1009) genes coding for a dioxygenase and a putative cyclase, respectively, were obtained by site-directed mutagenesis and then characterized. Liquid Chromatography coupled to mass spectrometry (LC-MS)/MS analyses of culture supernatant from the fdeE mutant strain revealed that naringenin remained unaltered, suggesting that the FdeE protein is involved in the initial step of naringenin degradation. LC-MS/MS analyses of culture supernatants from the wild-type (SmR1) and FdeC deficient mutant suggested that in H. seropedicae SmR1 naringenin is first mono-oxygenated by the FdeE protein, to produce 5,7,8-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H-chromen-4-one, that is subsequently dioxygenated and cleaved at the A-ring by the FdeC dioxygenase, since the latter compound accumulated in the fdeC strain. After meta-cleavage of the A-ring, the subsequent metabolic steps generate oxaloacetic acid that is metabolized via the tricarboxylic acid cycle. This bacterium can also modify naringenin by attaching a glycosyl group to the B-ring or a methoxy group to the A-ring, leading to the generation of dead-end products.