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Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)
Souza, André L.F.;Chubatsu, Leda S.;Souza, Emanuel M.;Pedrosa, Fábio O.;Monteiro, Rose A.;Rego, Fabiane G.M.;Rigo, Liu U.;
Genetics and Molecular Biology , 2008, DOI: 10.1590/S1415-47572008000400022
Abstract: in prokaryotes molybdenum is taken up by a high-affinity abc-type transporter system encoded by the modabc genes. the endophyte β-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.
Initial performance of maize in response to NPK fertilization combined with Herbaspirillum seropedicae
Baldotto, Marihus Altoé;Baldotto, Lílian Estrela Borges;Santana, Rogério Batista;Marciano, Cláudio Roberto;
Revista Ceres , 2012, DOI: 10.1590/S0034-737X2012000600015
Abstract: the inoculation with plant growth-promoting bacteria can be a technological approach useful for increasing the production of maize. the objective of this study was to evaluate the initial performance of maize in response to application of doses of npk combined with the inoculation of the diazotrophic bacteria herbaspirillum seropedicae in an greenhouse experiment. the experiment consisted of six fertilizer levels: 0, 25, 50, 75, 100 and 200% of the recommended dose of npk applied to maize inoculated and non-inoculated with h. seropedicae. at 30 days after the treatment application, the growth characteristics and nutritional status of the plants were evaluated. plant development was influenced by fertilization, but it was enhanced by combination with the bacteria, which resulted in significant increases in the dry mass of shoots (7%) and leaf area (9%) when compared with non-inoculated plants. the results showed increases in the concentration of n (11%), p (30%) and k (17%) of maize plants in response to bacterial inoculation together with npk compared with plants that were applied fertilize only. the greater consistency and stability response of the host plant to bacterization in the presence of chemical fertilizer indicate a promissory biotechnological approach for improving the initial growth and adaptation of maize to the cultivation environment.
Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae
Schmidt, M.A.;Souza, E.M.;Baura, V.;Wassem, R.;Yates, M.G.;Pedrosa, F.O.;Monteiro, R.A.;
Brazilian Journal of Medical and Biological Research , 2011, DOI: 10.1590/S0100-879X2011007500004
Abstract: herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. in the present study, we have investigated the attachment to and internal colonization of phaseolus vulgaris roots by the h. seropedicae wild-type strain smr1 and by a strain of h. seropedicae expressing a red fluorescent protein (dsred) to track the bacterium in the plant tissues. two-day-old p. vulgaris roots were incubated at 30°c for 15 min with 6 x 108 cfu/ml h. seropedicae smr1 or ram4. three days after inoculation, 4 x 104 cells of endophytic h. seropedicae smr1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 106 cfu/g. the identity of the recovered bacteria was confirmed by amplification and sequencing of the 16srrna gene. furthermore, confocal microscopy of p. vulgaris roots inoculated with h. seropedicae ram4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that h. seropedicae ram4 is capable of colonizing the roots of the dicotyledon p. vulgaris. determination of dry weight of common bean inoculated with h. seropedicae smr1 suggested that this bacterium has a negative effect on the growth of p. vulgaris.
Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players  [PDF]
Evandro F. Tirapelle, Marcelo Müller-Santos, Michelle Z. Tadra-Sfeir, Marco A. S. Kadowaki, Maria B. R. Steffens, Rose A. Monteiro, Emanuel M. Souza, Fabio O. Pedrosa, Leda S. Chubatsu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075066
Abstract: Herbaspirillum seropedicae is a diazotrophic ？-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.
Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides  [PDF]
Eduardo Balsanelli, Thalita Regina Tuleski, Valter Antonio de Baura, Marshall Geoffrey Yates, Leda Satie Chubatsu, Fabio de Oliveira Pedrosa, Emanuel Maltempi de Souza, Rose Adele Monteiro
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0077001
Abstract: 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.
Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses  [PDF]
Fábio O. Pedrosa ,Rose Adele Monteiro,Roseli Wassem,Leonardo M. Cruz,Ricardo A. Ayub,Nelson B. Colauto,Maria Aparecida Fernandez,Maria Helena P. Fungaro,Edmundo C. Grisard,Mariangela Hungria,Humberto M. F. Madeira,Rubens O. Nodari,Clarice A. Osaku,Maria Luiza Petzl-Erler,Hernán Terenzi,Luiz G. E. Vieira,Maria Berenice R. Steffens,Vinicius A. Weiss,Luiz F. P. Pereira,Marina I. M. Almeida,Lysangela R. Alves,Anelis Marin,Luiza Maria Araujo,Eduardo Balsanelli,Valter A. Baura,Leda S. Chubatsu,Helisson Faoro,Augusto Favetti,Geraldo Friedermann,Chirlei Glienke,Susan Karp,Vanessa Kava-Cordeiro,Roberto T. Raittz,Humberto J. O. Ramos,Enilze Maria S. F. Ribeiro,Liu Un Rigo,Saul N. Rocha,Stefan Schwab,Anilda G. Silva,Eliel M. Souza,Michelle Z. Tadra-Sfeir,Rodrigo A. Torres,Audrei N. G. Dabul,Maria Albertina M. Soares,Luciano S. Gasques,Ciela C. T. Gimenes,Juliana S. Valle,Ricardo R. Ciferri,Luiz C. Correa,Norma K. Murace,Jo？o A. Pamphile,Eliana Valéria Patussi,Alberto J. Prioli,Sonia Maria A. Prioli,Carmem Lúcia M. S. C. Rocha,Olívia Márcia N. Arantes,Márcia Cristina Furlaneto,Leandro P. Godoy,Carlos E. C. Oliveira,Daniele Satori,Laurival A. Vilas-Boas,Maria Angélica E. Watanabe,Bibiana Paula Dambros,Miguel P. Guerra,Sandra Marisa Mathioni,Karine Louise Santos,Mario Steindel,Javier Vernal,Fernando G. Barcellos,Rubens J. Campo,Ligia Maria O. Chueire,Marisa Fabiana Nicolás,Lilian Pereira-Ferrari,José L. da Concei？？o Silva,Nereida M. R. Gioppo,Vladimir P. Margarido
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002064
Abstract: 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.
Initial performance of maize in response to NPK fertilization combined with Herbaspirillum seropedicae Desempenho inicial do milho, em resposta à aduba o NPK combinada com a inocula o de Herbaspirillum seropedicae  [cached]
Marihus Altoé Baldotto,Lílian Estrela Borges Baldotto,Rogério Batista Santana,Cláudio Roberto Marciano
Revista Ceres , 2012,
Abstract: The inoculation with plant growth-promoting bacteria can be a technological approach useful for increasing the production of maize. The objective of this study was to evaluate the initial performance of maize in response to application of doses of NPK combined with the inoculation of the diazotrophic bacteria Herbaspirillum seropedicae in an greenhouse experiment. The experiment consisted of six fertilizer levels: 0, 25, 50, 75, 100 and 200% of the recommended dose of NPK applied to maize inoculated and non-inoculated with H. seropedicae. At 30 days after the treatment application, the growth characteristics and nutritional status of the plants were evaluated. Plant development was influenced by fertilization, but it was enhanced by combination with the bacteria, which resulted in significant increases in the dry mass of shoots (7%) and leaf area (9%) when compared with non-inoculated plants. The results showed increases in the concentration of N (11%), P (30%) and K (17%) of maize plants in response to bacterial inoculation together with NPK compared with plants that were applied fertilize only. The greater consistency and stability response of the host plant to bacterization in the presence of chemical fertilizer indicate a promissory biotechnological approach for improving the initial growth and adaptation of maize to the cultivation environment. A inocula o de bactérias promotoras de crescimento de plantas pode ser uma abordagem tecnológica útil para aumentar a produ o de milho. O objetivo deste trabalho foi avaliar o desempenho inicial de plantas de milho, em resposta à aplica o de doses de NPK combinadas com a inocula o da bactéria diazotrófica Herbaspirillum seropedicae, em experimento em casa de vegeta o. A matriz experimental consistiu em seis níveis de aduba o: 0, 25, 50, 75, 100 e 200 % da dose de NPK recomendada, aplicados em plantas de milho inoculadas e n o inoculadas com H. seropedicae. Aos 30 dias após a aplica o dos tratamentos, foram avaliadas as características de crescimento e nutricionais das plantas. O crescimento do milho foi influenciado pela aduba o, mas foi refor ado pela combina o com a bactéria, que resultou em aumentos significativos na matéria seca da parte aérea (7 %) e área foliar (9 %), quando comparado com as das plantas n o inoculadas. Os resultados indicaram incrementos no acúmulo de N (11 %), P (30 %) e K (17 %), em plantas de milho, em resposta à inocula o bacteriana em conjunto com NPK, quando comparado com os das plantas que apenas receberam aduba o. A estabilidade e maior consistência da resposta da pl
Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'
Baldotto, Lílian Estrela Borges;Olivares, Fábio Lopes;Bressan-Smith, Ricardo;
Brazilian Journal of Microbiology , 2011, DOI: 10.1590/S1517-83822011000100015
Abstract: the events involved in the structural interaction between the diazotrophic endophytic bacterium herbaspirillum seropedicae, strain ram10, labeled with green fluorescent protein, and pineapple plantlets 'vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. after 6 hours of inoculation, h. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. moreover, within 1 day after inoculation the bacteria were colonizing the shoots. in this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. the penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. after 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of h. seropedicae.
Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy
Silva, Lúcia Gracinda da;Miguens, Flávio Costa;Olivares, Fabio Lopes;
Brazilian Journal of Microbiology , 2003, DOI: 10.1590/S1517-83822003000500023
Abstract: the interaction between sugar cane plantlets and h. seropedicae was investigated using high pressure freezing followed by freeze substitution. microscopical observation showed consistent differences between this approaches when compared with the conventional preparation, specially related to appearance of the bacteria cell and the endophytic attachment to the host cell wall.
Analysis of the Promoter Region, Motif and CpG Islands in AraC Family Transcriptional Regulator ACP92 Genes of Herbaspirillum seropedicae  [PDF]
Mihret Yirgu, Mulugeta Kebede
Advances in Bioscience and Biotechnology (ABB) , 2019, DOI: 10.4236/abb.2019.106011
Abstract: Identification of promoters and their regulatory elements are the most important phases in bioinformatics. To understand the regulation of gene expression, identification, and analysis of promoters region, motif and CpG islands are the most important steps. The accurate prediction of promoter’s is basic for proper interpretation of gene expression patterns, construction and understanding of genetic regulatory system. Therefore, the objective of this study was to analyze the promoter region, motif such as a transcription factor and CpG islands in AraC family transcriptional regulator ACP92 genes of Herbaspirillum seropedicae. The analysis was carried out by identifying transcription start sites in ACP92 genome sequences taken from the H. seropedicae assembly of NCBI genome browser, and 29 ACP92 genes sequences. Accordingly, transcription start sites (TSS) were identified, and the result indicated that 37.9% had more than one TSS whereas only 62.1% had one TSS. In the analysis, seven motifs were identified from the thought sequences and MV6 was revealed the common promoter motif for all (100%) in H. seropedicae ACP92 gene that serves as binding sites for transcription factors which shared a minimum of 48.27%. Based on a common motif MV6 to find out similar motifs using TOMTOM from the databases of prokaryotes DNA, most of them are transcription factors of fur family. The others are bacterial histone-like protein family, matp and sigma-54 factor family also transcription factor families that are binding candidate to MV6. H. seropedicae ACP92 genes are CpG Island which implies that the regulation of gene expression plays an important role.
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