Acetylene Reduction Assay: A Measure of Nitrogenase Activity in Plants and Bacteria.

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.

Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria.

The plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE 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. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

In silico prediction and expression profile analysis of small non-coding RNAs in Herbaspirillum seropedicae SmR1.

BACKGROUND: Herbaspirillum seropedicae is a diazotrophic bacterium from the ß-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. RESULTS: 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. CONCLUSIONS: 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.

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria.

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 in-situ 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 indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

Proteomic and Metabolomic Analysis of Azospirillum brasilensentrC Mutant under High and Low Nitrogen Conditions.

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.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

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).

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites.

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.

Cellulose production increases sorghum colonization and the pathogenic potential of Herbaspirillum rubrisubalbicans M1.

Three species of the ß-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.

Hierarchical interactions between Fnr orthologs allows fine-tuning of transcription in response to oxygen in Herbaspirillum seropedicae.

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.

Herbaspirillum rubrisubalbicans, a mild pathogen impairs growth of rice by augmenting ethylene levels.

KEY MESSAGE: Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicans increased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans rice interactions were further characterized by proteomic approach. Herbaspirillum rubrisubalbicans is a well-known growth-promoting rhizobacteria that can also act as a mild phyto-pathogen. During colonisation of rice, RT-qPCR analyses showed that H. rubrisubalbicans up-regulates the methionine recycling pathway as well as phyto-siderophore synthesis genes. mRNA levels of ACC oxidase and ethylene levels also increased in rice roots but inoculation with H. rubrisubalbicans impaired growth of the rice plant. A proteomic approach was used to identify proteins specifically modulated by H. rubrisubalbicans in rice and amongst the differentially expressed proteins a V-ATPase and a 14-3-3 protein were down-regulated. Several proteins of H. rubrisubalbicans were identified, including the type VI secretion system effector Hcp1, suggesting that protein secretion play a role colonisation in rice. Finally, the alkyl hydroperoxide reductase, a primary scavenger of endogenous hydrogen peroxide was also identified. Monitoring the levels of reactive oxygen species in the epiphytic bacteria by flow cytometry revealed that H. rubrisubalbicans is subjected to oxidative stress, suggesting that the alkyl hydroperoxide reductase is an important regulator of redox homeostasis in plant-bacteria interactions.

RNA-seq analyses reveal insights into the function of respiratory nitrate reductase of the diazotroph Herbaspirillum seropedicae.

Herbaspirillum seropedicae is a nitrogen-fixing ß-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.

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.

BACKGROUND: Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation. RESULTS: The activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type. CONCLUSIONS: Fnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.

Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

Exopolysaccharide biosynthesis enables mature biofilm formation on abiotic surfaces by Herbaspirillum seropedicae.

H. seropedicae associates endophytically and epiphytically with important poaceous crops and is capable of promoting their growth. The molecular mechanisms involved in plant colonization by this microrganism are not fully understood. Exopolysaccharides (EPS) are usually necessary for bacterial attachment to solid surfaces, to other bacteria, and to form biofilms. The role of H. seropedicae SmR1 exopolysaccharide in biofilm formation on both inert and plant substrates was assessed by characterization of a mutant in the espB gene which codes for a glucosyltransferase. The mutant strain was severely affected in EPS production and biofilm formation on glass wool. In contrast, the plant colonization capacity of the mutant strain was not altered when compared to the parental strain. The requirement of EPS for biofilm formation on inert surface was reinforced by the induction of eps genes in biofilms grown on glass and polypropylene. On the other hand, a strong repression of eps genes was observed in H. seropedicae cells adhered to maize roots. Our data suggest that H. seropedicae EPS is a structural component of mature biofilms, but this development stage of biofilm is not achieved during plant colonization.

Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.

Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

Draft genome sequence of Herbaspirillum lusitanum P6-12, an endophyte isolated from root nodules of Phaseolus vulgaris.

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.

Characterization of a new Acidobacteria-derived moderately thermostable lipase from a Brazilian Atlantic Forest soil metagenome.

A clone (LP001) expressing a new lipase gene was isolated from a metagenomic library of the Brazilian Atlantic Forest soil. The DNA insert of LP001 was fully sequenced, and 38 ORFs were identified. Comparison of ORFs, %G + C content and gene organization with sequenced bacterial genomes suggested that the fosmid DNA insert belongs to an organism of the Acidobacteria phylum. Protein domain analysis and inactivation by transposon insertion showed that the protein encoded by ORF29 was responsible for the lipase activity and was named LipAAc. The purified LipAAc lipase was capable of hydrolyzing a broad range of substrates, showing the highest activity against p-nitrophenol (pNP) decanoate. The lipase was active over a pH range of 5.0-10.0 and was insensitive to divalent cations. LipAAc is moderately thermostable with optimum temperature between 50 and 60 °C and was thermally activated (80% activity increase) after 1 h incubation at 50 °C. Phylogenetic analysis suggested that the LipAAc is a member of family I of bacterial lipases and clusters with other moderately thermostable lipases of this group. Comparisons of the DNA insert of fosmid LP001 with other acidobacterial genomes and sequence database suggest that lipAAc gene has a fungal origin and was acquired by horizontal transfer.

Structural characterization of the RNA chaperone Hfq from the nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1.

The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qß replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the ß-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal α-helix followed by five twisted ß-strands, and a novel π-π stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution.