Maria B. R. Steffens

Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

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.

ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG

Nitrogen fixation in some diazotrophic bacteria is regulated by mono‐ADP‐ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP‐ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the PII proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH‐modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane‐associated after an ammonium shock, and both this membrane sequestration and ADP‐ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP‐ribosyl‐removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a PII family member. They also suggest a model for control of ADP‐ribosylation that is likely to be applicable to all diazotrophs that exhibit such post‐translational regulation of nitrogenase.

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.

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

In 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.

Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes

Maize is one of the most important crops worldwide, and in Brazil, the state of Paraná stands as its largest producer. The crop demands high inputs of N fertilizers, therefore all strategies aiming to optimize the grain production with lower inputs are very relevant. Endophytic bacteria have a high potential to increment maize grain yield by means of input via biological nitrogen fixation and/or plant growth promotion, in this last case increasing the absorption of water and nutrients by the plants. In this study, we established a collection of 217 endophytic bacteria, isolated from roots of four lineages and three hybrid genotypes of maize, and isolated in four different N-free culture media. Biochemical―comprising growth in different carbon sources, intrinsic tolerance to antibiotics, and biochemical tests for catalase, nitrate reductase, urease, and growth in N-free media in vitro―and genetic characterization by BOX-PCR revealed great variability among the isolates. Both commercial hybrids and homozygous lineages were broadly colonized by endophytes, and sequencing of the 16S rRNA gene revealed the presence of bacteria belonging to the genera Pantoea, Bacillus, Burkholderia, and Klebsiella. Qualitative differences in endophytic colonization were detected between lineages and hybrid genotypes.

In vitro interactions between the PII proteins and the nitrogenase regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase-activating glycohydrolase (DraG) in Azospirillum brasilense.

The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of PII uridylylation status and the PII effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when PII proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the PII ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity.

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.

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.