Dataset Information

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

ABSTRACT: Herbaspirillum seropedicae is an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth. H. seropedicae produces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association of H. seropedicae with plants, we inoculated roots of Setaria viridis with H. seropedicae strain SmR1 and mutants defective in PHB production (?phaP1, ?phaP1 ?phaP2, ?phaC1, and ?phaR) or mobilization (?phaZ1 ?phaZ2). The strains producing large amounts of PHB colonized roots, significantly increasing root area and the number of lateral roots compared to those of PHB-negative strains. H. seropedicae grows under microaerobic conditions, which can be found in the rhizosphere. When grown under low-oxygen conditions, only the parental strain and ?phaP2 mutant exhibited normal growth. The lack of normal growth under low oxygen correlated with the inability to stimulate plant growth, although there was no effect on the level of root colonization. The data suggest that PHB is produced in the root rhizosphere and plays a role in maintaining normal metabolism under microaerobic conditions. To confirm this, we screened for green fluorescent protein (GFP) expression under the control of the H. seropedicae promoters of the PHA synthase and PHA depolymerase genes in the rhizosphere. PHB synthesis is active on the root surface and later PHB depolymerase expression is activated.IMPORTANCE The application of bacteria as plant growth promoters is a sustainable alternative to mitigate the use of chemical fertilization in agriculture, reducing negative economic and environmental impacts. Several plant growth-promoting bacteria synthesize and accumulate the intracellular polymer polyhydroxybutyrate (PHB). However, the role of PHB in plant-bacterium interactions is poorly understood. In this study, applying the C4 model grass Setaria viridis and several mutants in the PHB metabolism of the endophyte Herbaspirillum seropedicae yielded new findings on the importance of PHB for bacterial colonization of S. viridis roots. Taken together, the results show that deletion of genes involved in the synthesis and degradation of PHB reduced the ability of the bacteria to enhance plant growth but with little effect on overall root colonization. The data suggest that PHB metabolism likely plays an important role in supporting specific metabolic routes utilized by the bacteria to stimulate plant growth.

SUBMITTER: Silveira Alves LP

PROVIDER: S-EPMC6414382 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

Silveira Alves Luis Paulo LP Plucani do Amaral Fernanda F Kim Daewon D Todo Bom Maritza M Piñero Gavídia Manuel M Silvano Teixeira Cícero C Holthman Fernanda F de Oliveira Pedrosa Fabio F Maltempi de Souza Emanuel E Chubatsu Leda Satie LS Müller-Santos Marcelo M Stacey Gary G

Applied and environmental microbiology 20190306 6

Herbaspirillum seropedicae is an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth. H. seropedicae produces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association of H. seropedicae ...[more]

PMID: 30610076

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Project description:Under conditions of carbon starvation or thermal, osmotic, or oxidative shock, mutants affected in the synthesis or mobilization of poly-3-hydroxybutyrate (PHB) are known to survive less well. It is still unclear if the synthesis and accumulation of PHB are sufficient to protect bacteria against stress conditions or if the stored PHB has to be mobilized. Here, we demonstrated that mobilization of PHB in Herbaspirillum seropedicae SmR1 was heat-shock activated at 45°C. In situ proton (1H) nuclear magnetic resonance spectroscopy (i.e., 1H-nuclear magnetic resonance) showed that heat shock increased amounts of 3-hydroxybutyrate (3HB) only in H. seropedicae strains able to synthesize and mobilize PHB. H. seropedicae SmR1 mutants unable to synthesize or mobilize PHB were more susceptible to heat shock and survived less well than the parental strain. When 100 mM 3-hydroxybutyrate was added to the medium, the ΔphaC1 strain (an H. seropedicae mutant unable to synthesize PHB) and the double mutant with deletion of both phaZ1 and phaZ2 (i.e., ΔphaZ1.2) (unable to mobilize PHB) showed partial rescue of heat adaptability (from 0% survival without 3HB to 40% of the initial viable population). Addition of 200 mM 3HB before the imposition of heat shock reduced protein aggregation to 15% in the ΔphaC1 mutant and 12% in the ΔphaZ1.2 mutant. We conclude that H. seropedicae SmR1 is naturally protected by 3HB released by PHB mobilization, while mutants unable to generate large amounts of 3HB under heat shock conditions are less able to cope with heat damage.IMPORTANCE Bacteria are subject to abrupt changes in environmental conditions affecting their growth, requiring rapid adaptation. Increasing the concentration of some metabolites can protect bacteria from hostile conditions that lead to protein denaturation and precipitation, as well as damage to plasma membranes. In this work, we demonstrated that under thermal shock, the bacterium Herbaspirillum seropedicae depolymerized its intracellular stock polymer known as poly-3-hydroxybutyrate (PHB), rapidly increasing the concentration of 3-hydroxybutyrate (3HB) and decreasing protein precipitation by thermal denaturation. Mutant H. seropedicae strains unable to produce or depolymerize PHB suffered irreparable damage during thermal shock, resulting in fast death when incubated at 45°C. Our results will contribute to the development of bacteria better adapted to high temperatures found either in natural conditions or in industrial processes. In the case of H. seropedicae and other bacteria that interact beneficially with plants, the understanding of PHB metabolism can be decisive for the development of more-competitive strains and their application as biofertilizers in agriculture.

Project description:Purpose: The global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi conditions, based on differential intracellular polyP levels Methods: Three independent samples (biological replicates) of H. seropedicae SmR1 cells grown in NFb5 or NFb50 media after 9 h of growth were used to construct 6 sequencing libraries.The libraries were amplified and enriched using the Ion PI Hi-QTM OT2 200 Kit, and sequenced with the Ion PI Hi-QTM Sequencing 200 kit on an Ion PITM Chip Kit v3. Obtained sequences were trimmed, mapped and analyzed using CLC Genomics Workbench 7.5.1 (Qiagen) against the H. seropedicae SmR1 genome (NC_014323). Differential gene expression was accessed using the DESeq2 package. qRT–PCR validation was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and quantified in triplicate using the Power SYBR-Green PCR MasterMix on a Step-One Plus Real Time-PCR System (Applied Biosystems). Results: After sequencing, 26 and 35 total million reads were obtained for NFb50 and NFb5 conditions, respectively, and from those, 12 million and 16 million reads were uniquely mapped to the H. seropedicae SmR1 genome. Biological replicates showed a very high level of correlation (r2 > 0.98). Among the 4805 genes of the H. seropedicae SmR1 genome, 3976 genes were expressed considering a read coverage equal or higher than three-fold. 1330 genes show expression levels statistically different (p-value ≤ 0.05) from which 670 had fold changes of 2 or higher and were considered differentially expressed genes (DEG) in NFb50 vs NFb5 transcriptome, being 385 down-regulated and 285 up-regulated. To confirm the differential expression observed by the RNA-seq data, the regulation of some genes was confirmed by RT-qPCR. Molecular and physiological analyses revealed that features related to Pi metabolism, bacterial flagella biosynthesis and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the mentioned condition, while aspects involved in adhesion, and stress response were repressed. Since environmental conditions can influence the effectiveness of the PGPB, enhancement of bacterial robustness to withstand different conditions is an interesting challenge Conclusions: The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae bacterial traits related to survival and other important physiological characteristics. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients, but also as a base to design strategies to improve different bacterial features focusing in biotechnological and/or agricultural purposes.

Project description:Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

Dataset Information

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

Publications

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

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