Project description:Herbaspirillum seropedicae SmR1 was grown in NFbHPN medium until OD600nm of 0.6, when 5mM phenol or 2,5mM benzoic acid was added to the medium. After 30 minutes of growth, the cells were collected by centrifugation and total RNA was extracted with RiboPureBacteria. Also, a derivative strain from Herbaspirillum seropedicae SmR1 resistant to 1mM phenol (named strain RP) was grown in NFbHPN medium containing 1mM phenol until OD600nm of 0.6, then 5mM phenol was added to the medium. After 30 minutes of growth, the cells were collected by centrifugation and total RNA was extracted with RiboPureBacteria. All samples were depleted for ribossomal RNA e sequenced with Solid plataform.
Project description:Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, promoting plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1.
Project description:Herbaspirillum seropedicae is a diazotrophic bacterium which associates endophytically with economically important gramineae. Flavonoids such as naringenin, have been shown to have an effect on the interaction between H. seropedicae and its host plants. We used a high-throughput sequencing based method (RNA-Seq) to access the influence of naringenin on the whole transcriptome profile of H. seropedicae.
Project description:H. seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize and promote plant growth wheat seedlings growing hydroponically in Hoaglandâs medium were inoculated with H. seropedicae the bacteria and incubated for 3 days. mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of bacteria attached to the root and planktonic revealed an extensive metabolic adaptation to the epiphytic life style.
Project description:In this study, we have investigated the physiological consequences of PHB (poly(3-hydroxybutyrate)) synthesis in H. seropedicae by characterising the trancriptional changes in a mutant strain lacking phaC1 gene which codes for the PHA synthase enzyme essential for the last step in the synthesis of PHB. To do this experiment both wild type (SmR1) and phaC1 mutant were cultured on Nfb-Malate-HP media suplemented with 20 mM of ammonium chloride under 30 Celsius degrees and 120 rpm shaking rate until the late log-phase (O.D600 = 1.0), when the peak of PHB production is observed. The cultures obtained as above were harvested for RNA extraction and transcriptome analysis.
Project description:To determine the direct promoter targets of Fnr proteins, we correlated transcriptional changes observed in single fnr1 and fnr3 mutants (E-MTAB-5741) with ChIP-Seq analysis, taking advantage of C-terminally 3xFlag fnr alleles engineered into the H. seropedicae genome. ChIP-seq data were obtained from cultures grown under limited oxygen availability. Using this approach, DNA-binding targets for the H. seropedicae Fnr1 and Fnr3 proteins were unambiguously revealed and correlated with transcript profiles to determine the specific regulons of each protein.
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.