Project description:H. seropedicae wild-type or ntrC mutant were grown on three different nitrogen conditions: nitrogen limiting, ammonium shock and nitrate shock.
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: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:bra-inra09-02_bioen_nitrogen - nitrate induction - nlp mutants - Short term nitrate induction kinetics in wildtype, nlp7-1, nlp7-3 and nlp6nlp7 - 10 days old seedling grown in liquid culture on 3mM nitrate wer starvec for N for 3 days and the kinetic for the resupply of nitrate was studied during a short kinetic (0,5, 10,20 minutes).
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: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:ngs2020_19_arimnet-barley responses to nitrate limitation-What are the molecular mechanisms taking place in barley under nitrate limitation?-Barley were grown on sand under 0.5 mM nitrate (Low nitrate= LN) or 5 mM nitrate (high nitrate = HN)
Project description:BW25113 wild type cells grown to OD = 0.8 in LB, add 2 ug/mL nalidixic acid or 10 ug/mL azlocillin for 90 min. Control was without any antibiotic.
Project description:Herbaspirillum seropedicae are β-proteobacteria that establish as endophytes in various plants. They are able to consume diverse carbon sources, including hexoses and pentoses like D-xylose. D-xylose catabolism pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that D-xylose is the second most abundant sugar in nature. Furthermore, it is found in some potential raw materials such as lignocellulosic biomass. In this work we present a study of D-xylose catabolism pathways in H. seropedicae strain Z69, using RNA-seq analysis and the subsequent study of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD+- dependent D-xylose dehydrogenase. Mutant Z69∆xylB was still able to grow on D-xylose, although at a reduced rate. This is due to expression of an L-arabinose dehydrogenase encoded by G5B88_05250 gene, and was thus able to use D-xylose as substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize D-xylose. The lower portion of metabolism involves co-expression of two routes: Weimberg pathway that produces α-ketoglutarate and a novel pathway recently described that produces pyruvate and glycolate. This novel pathway seems to be essential since a mutant in the last step of this pathway, Z69∆G5B88_06410, was unable to grow on D‑xylose.