Roseli Wassem

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

Herbaspirillum-plant interactions: microscopical, histological and molecular aspects

Diazotrophic species in the genus Herbaspirillum (e.g. H. frisingense, H. rubrisubalbicans and H. seropedicae) associate with several economically important crops in the family Poaceae, such as maize (Zea mays), Miscanthus, rice (Oryza sativa), sorghum (Sorghum bicolor) and sugarcane (Saccharum sp.), and can increase their growth and productivity by a number of mechanisms, including nitrogen fixation. Hence, the improvement and use of these plant growth-promoting bacteria could provide economic and environmental benefits. We review the colonization processes of host plants by Herbaspirillum spp., including histological aspects and molecular mechanisms involved in these interactions, which may be epiphytic, endophytic, and even occasionally pathogenic. Herbaspirillum can recognize plant signals that modulate the expression of colonization traits and plant growth-promoting factors. Although a large proportion of herbaspirilla remain rhizospheric and epiphytic, plant-associated species in this genus are noted for their ability to colonize the plant internal tissues. Endophytic colonization by herbaspirilla begins with the attachment of the bacteria to root surfaces, followed by colonization at the emergence points of lateral roots and penetration through discontinuities of the epidermis; this appears to involve bacterial envelope structures, such as lipopolysaccharide (LPS), exopolysaccharide (EPS), adhesins and the type three secretion system (T3SS), but not necessarily the involvement of cell wall-degrading enzymes. Intercellular spaces are then rapidly occupied, proceeding to colonization of xylem and the aerial parts of the host plants. The response of the host plant includes both the recognition of the bacteria as non-pathogenic and the induction of systemic resistance to pathogens. Phytohormone signaling cascades are also activated, regulating the plant development. This complex molecular communication between some Herbaspirillum spp. and plant hosts can result in plant growth-promotion.

Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes

BackgroundThe rapid growth of the world’s population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB.ResultsWe performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes.ConclusionsPGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

Two roles for integration host factor at an enhancer‐dependent nifA promoter

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase. For the σ54 RNA polymerase, regulatory proteins bound to distally located enhancers engage the polymerase via DNA looping. The σ54‐dependent nifA promoter of Herbaspirillum seropedicae (Hs) is activated under nitrogen‐limiting growth conditions. Potential enhancers for the nitrogen control activators NTRC and NIFA and binding sites for integration host factor (IHF) and σ54‐holoenzyme were identified. DNA footprinting experiments showed that these sites functioned for protein binding. Their involvement in the promoter regulation was explored. In vitro, activation of the Hs nifA promoter by NTRC is stimulated by the DNA bending protein IHF. In marked contrast, activation by NIFA is greatly reduced by IHF, thus diminishing potentially destabilizing autoactivation of the nifA promoter by NIFA. Additionally, high levels of NIFA appear to limit NTRC‐dependent activation. This inhibition is IHF dependent. Therefore, IHF acts positively and negatively at the nifA promoter to restrict transcription activation to NTRC and one signal transduction pathway.

Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.

Differential gene expression of rice roots inoculated with the diazotroph Herbaspirillum seropedicae

Background and aimsRice (Oryza sativa L.) is the primary source of carbohydrate for the majority of the Worlds population. Herbaspirillum seropedicae is a diazotroph that lives within and on the surface of rice roots. It can promote the growth of rice, partly by supplying it with fixed nitrogen.MethodsTo better understand the rice–H. seropedicae interaction, cDNA libraries from rice roots either inoculated (RRCH) or uninoculated (RRSH) with the diazotroph were obtained and analysed.ResultsPotential differentially expressed genes identified from the libraries encoded a metallothionein-like protein type 1, a NOD26-like membrane integral protein ZmNIP2-1, a thionin family protein, an oryzain gamma chain precursor, stress-associated protein 1 (OsISAP1), probenazole-inducible protein PBZ1 and auxin- and ethylene-responsive genes. Differential expression was analysed by qRT-PCR for some of these genes and confirmed in most cases. The expression of stress- and defence-related genes coding for thionins, PBZ1 and OsISAP1 was repressed, while expression of a metallothionein gene was induced by inoculation with H. seropedicae. In contrast, expression of auxin-responsive genes was repressed, while expression of ethylene genes was either repressed or induced. The possible involvement of these and other genes in plant-bacterial interactions is discussed.ConclusionsThe decrease in expression of the defence-related proteins PBZ1 and thionins in the rice–H. seropedicae association, suggests that the bacteria modulate plant defence responses during colonisation. The expression of genes responsive to auxin and ethylene also appears to be regulated by the bacteria.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

Nitrogen fixation control in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a Gram-negative endophytic diazotroph that associates with important agricultural crops. Several studies have shown that this organism can contribute to plant growth suggesting potential for use as a biofertilizer. Nitrogen fixation in H. seropedicae is highly regulated both at the transcriptional and post-translational levels. Both of these regulatory levels respond to the ammonium availability in the external medium through a cascade of interacting proteins. The transcriptional regulation of the process also responds to oxygen, which is probably directly sensed by the transcriptional regulator NifA. Here, we review current knowledge of the regulation of nitrogen fixation in H. seropedicae. The signal transduction protein GlnK is a key regulator of nitrogen fixation at both the transcriptional and post-translational levels. In vitro analysis indicates that GlnK interacts with NifA and probably modulates its activity, thereby controlling nif expression. GlnK, together with the ammonium channel protein AmtB, also participates in the post-translational regulation of nitrogenase activity by an unidentified mechanism. This regulatory system efficiently controls nitrogen fixation according to prevailing fixed nitrogen and oxygen levels in H. seropedicae.

Naringenin regulates expression of genes involved in cell wall synthesis in Herbaspirillum seropedicae

ABSTRACT Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.