M. G. Yates

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.

Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants

Abstract The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif(+) (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif(+) or Nif(-) strains.

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.

Genome Structure of the Genus Azospirillum

Azospirillum species are plant-associated diazotrophs of the alpha subclass of Proteobacteria. The genomes of five of the six Azospirillum species were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. The nifHDK operon was identified in the largest replicon.

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.

Expression and functional analysis of an N-truncated NifA protein of Herbaspirillum seropedicae

In Herbaspirillum seropedicae, an endophytic diazotroph, nif gene expression is under the control of the transcriptional activator NifA. We have over‐expressed and purified a protein containing the central and C‐terminal domains of the H. seropedicae NifA protein, N‐truncated NifA, fused to a His‐Tag sequence. This fusion protein was found to be partially soluble and was purified by affinity chromatography. Band shift and footprinting assays showed that the N‐truncated NifA protein was able to bind specifically to the H. seropedicae nifB promoter region. In vivo analysis showed that this protein activated the nifH promoter of Klebsiella pneumoniae in Escherichia coli only in the absence of oxygen and this activation was not negatively controlled by ammonium ions.

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.

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.