Role of PII proteins in nitrogen fixation control of Herbaspirillum seropedicae strain SmR1

<p>Abstract</p> <p>Background</p> <p>The PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In <it>Herbaspirillum seropedicae</it>, two PII-like proteins (GlnB and GlnK), encoded by the genes <it>glnB </it>and <it>glnK</it>, were identified. The <it>glnB </it>gene is monocistronic and its expression is constitutive, while <it>glnK </it>is located in the <it>nlmAglnKamtB </it>operon and is expressed under nitrogen-limiting conditions.</p> <p>Results</p> <p>In order to determine the involvement of the <it>H. seropedicae glnB </it>and <it>glnK </it>gene products in nitrogen fixation, a series of mutant strains were constructed and characterized. The <it>glnK^-</it>mutants were deficient in nitrogen fixation and they were complemented by plasmids expressing the GlnK protein or an N-truncated form of NifA. The nitrogenase post-translational control by ammonium was studied and the results showed that the <it>glnK </it>mutant is partially defective in nitrogenase inactivation upon addition of ammonium while the <it>glnB </it>mutant has a wild-type phenotype.</p> <p>Conclusions</p> <p>Our results indicate that GlnK is mainly responsible for NifA activity regulation and ammonium-dependent post-translational regulation of nitrogenase in <it>H. seropedicae</it>.</p

Identification and characterization of PhbF: A DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1

<p>Abstract</p> <p>Background</p> <p><it>Herbaspirillum seropedicae </it>SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism.</p> <p>Results</p> <p>In this work we describe the characterization of the PhbF protein from <it>H. seropedicae </it>SmR1 which was purified and characterized after expression in <it>E. coli</it>. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in <it>H. seropedicae </it>SmR1. <it>In silico </it>analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using <it>lacZ </it>fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes.</p> <p>Conclusions</p> <p>Our results indicate that <it>H. seropedicae </it>SmR1 PhbF regulates expression of <it>phb</it>-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival <it>in planta</it>.</p

A“Dirty” Footprint: Macroinvertebrate diversity in Amazonian Anthropic Soils

International audienceAmazonian rainforests, once thought to be pristine wilderness, are increasingly known to have been widely inhabited, modified, and managed prior to European arrival, by human populations with diverse cultural backgrounds. Amazonian Dark Earths (ADEs) are fertile soils found throughout the Amazon Basin, created by pre-Columbian societies with sedentary habits. Much is known about the chemistry of these soils, yet their zoology has been neglected. Hence, we characterized soil fertility, macroinvertebrate communities, and their activity at nine archeological sites in three Amazonian regions in ADEs and adjacent reference soils under native forest (young and old) and agricultural systems. We found 673 morphospecies and, despite similar richness in ADEs (385 spp.) and reference soils (399 spp.), we identified a tenacious pre-Columbian footprint, with 49% of morphospecies found exclusively in ADEs. Termite and total macroinvertebrate abundance were higher in reference soils, while soil fertility and macroinvertebrate activity were higher in the ADEs, and associated with larger earthworm quantities and biomass. We show that ADE habitats have a unique pool of species, but that modern land use of ADEs decreases their populations, diversity, and contributions to soil functioning. These findings support the idea that humans created and sustained high-fertility ecosystems that persist today, altering biodiversity patterns in Amazonia

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

Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense

AbstractIn Azospirillum brasilense ADP-ribosylation of dinitrogenase reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG). The PII proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation

Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte &#946;-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2). Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH) and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp) and modE2 (372 bp) genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae