Michelle Z. Tadra-Sfeir

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.

Agaricus bisporus and Agaricus brasiliensis (1 → 6)-β-d-glucans show immunostimulatory activity on human THP-1 derived macrophages

The (1→6)-β-D-glucans from Agaricus bisporus and Agaricus brasiliensis were purified to evaluate their effects on the innate immune system. THP-1 macrophages were used to investigate the induction of the expression of TNF-α, IL1β, and COX-2 by RT-PCR. The purification of the polysaccharides gave rise to fractions containing 96-98% of glucose. The samples were analyzed by GC-MS, HPSEC and (13)C NMR, which confirmed the presence of homogeneous (1→6)-β-D-glucans. The β-glucans were incubated with THP-1 derived macrophages, for 3 h and 6 h to evaluate their effects on the expression of pro-inflammatory genes. Both β-glucans stimulated the expression of such genes as much as the pro-inflammatory control (LPS). When the cells were incubated with LPS+β-glucan, a significant inhibition of the expression of IL-1β and COX-2 was observed for both treatments after 3 h of incubation. By the results, we conclude that the (1→6)-β-D-glucans present an immunostimulatory activity when administered to THP-1 derived macrophages.

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.

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.

Defluorination of sodium fluoroacetate by bacteria from soil and plants in Brazil.

The aim of this work was to isolate and identify bacteria able to degrade sodium fluoroacetate from soil and plant samples collected in areas where the fluoroacetate-containing plants Mascagnia rigida and Palicourea aenofusca are found. The samples were cultivated in mineral medium added with 20 mmol L−1 sodium fluoroacetate. Seven isolates were identified by 16S rRNA gene sequencing as Paenibacillus sp. (ECPB01), Burkholderia sp. (ECPB02), Cupriavidus sp. (ECPB03), Staphylococcus sp. (ECPB04), Ancylobacter sp. (ECPB05), Ralstonia sp. (ECPB06), and Stenotrophomonas sp. (ECPB07). All seven isolates degraded sodium-fluoroacetate-containing in the medium, reaching defluorination rate of fluoride ion of 20 mmol L−1. Six of them are reported for the first time as able to degrade sodium fluoroacetate (SF). In the future, some of these microorganisms can be used to establish in the rumen an engineered bacterial population able to degrade sodium fluoroacetate and protect ruminants from the poisoning by this compound.

Isolation and Identification of Sodium Fluoroacetate Degrading Bacteria from Caprine Rumen in Brazil

The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100 μL in mineral medium added with 20 mmol L−1 sodium fluoroacetate (SF), incubated at 39°C in an orbital shaker. Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08) and Ancylobacter dichloromethanicus (ECPB09). These bacteria degraded sodium fluoroacetate, releasing 20 mmol L−1 of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L−1 of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.

Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

Herbaspirillum seropedicae is a diazotrophic ß-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.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere

Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.