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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

ABSTRACT: NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a ?-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

SUBMITTER: Aquino B

PROVIDER: S-EPMC4541686 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Aquino B B Stefanello A A AA Oliveira M A S MA Pedrosa F O FO Souza E M EM Monteiro R A RA Chubatsu L S LS

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 20150710 8

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-bi ...[more]

PMID: 26176311

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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.

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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

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