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RNA-Seq provides new insights into the nitrogen response of Azoarcus olearius BH72 during N2-fixation beyond the nitrogen fixation process

ABSTRACT: Azoarcus olearius BH72 is a diazotrophic endophyte carrying out biological nitrogen fixation (BNF) and supplying nitrogen to its host plant. Our previous microarray approach provided insights in the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially regulated genes (DEGs). Thus we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24 % of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected induction of genes involved in carbon metabolism and flagellar and twitching motility. On the other hand, genes encoding for proteins involved in translation and vitamin biosynthesis were detected to be suppressed. Nonetheless, strain BH72 appears not to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids: we detected strong induction of machineries for uptake and assimilation.

ORGANISM(S): Azoarcus olearius

PROVIDER: GSE176473 | GEO | 2021/09/06

REPOSITORIES: GEO

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Project description:Purpose: Klebsiella oxytoca M5a1 (previously Klebsiella pneumonia M5a1) is a principle model organism for free-living biological nitrogen fixation. This strain has been used for decades in the characterisation of nitrogenase function and the genetic regulation of nitrogen fixation physiology. Currently it represents a key model for synthetic biology and biotechnology approaches. This project involves a multi-omics approach to modelling nitrogen physiology in Klebsiella oxytoca M5a1, in order to inform rational genetic engineering for improved nitrogen fixation activity. Here we studied the transition from nitrogen replete (high NH4Cl) to nitrogen starved (NH4Cl run-out) conditions, the latter of which induce nif gene expression and synthesis of the nitrogenase enzyme. We mapped RNA-sequencing (Illumina) reads to our assembled M5aI genome in order to compare the transcriptome between nitrogen replete and nitrogen fixing conditions using differential gene expression (DEG) analysis. Methods: Total RNA was extracted from fixed M5a1 cell samples after culturing in nitrogen replete (10 mM NH4Cl; one replicate) or nitrogen starved (3 hours after complete run-out of 0.5 mM NH4Cl; two replicates) growth medium (NFDM). cDNA libraries were prepared for Illumina (NextSeq 500) sequencing, with a read length of 75 nt and a depth of 100 million reads. Following processing and quality control, sequence reads were mapped to a M5a1 genomic template (GenBank WGS accession JAFHKG010000000; BioSample SAMN17288411) and gene count normalisation performed (RPKM). Differential gene expression analysis was performed using the DEseq2 package utilising settings of regularized logarithm (rlog) normalisation of read counts, Wald hypothesis testing and p-value adjustment using a False Discovery Rate threshold of 0.05. Log2 fold changes between conditions were calculated for differentially expressed genes (DEGs) with an adjusted p value of <0.05. Results: Across the three samples, between 22.0-25.1 million reads (81.4-87.7%) were mapped to the genome, including 56.7-68.5% mapped to annotated genes. More than 50% of genes (2694/5279 genes) were differentially expressed (adjusted p-value <0.05) in the nitrogen starved condition with respect to the nitrogen replate condition. 925 genes were upregulated and 1117 genes downregulated by more than 2-fold. Of these, 1515 differentially expressed genes correspond to annotated proteins in the KEGG functional orthology (KO) database. The 20 nitrogen fixation (nif) genes were ranked among the 200 genes with highest calculated expression values (RPKM) in the nitrogen starved condition. Conclusions: Our study provides a detailed insight into the global gene expression profile associated with diazotrophic (nitrogen fixing) growth, revealing a dominant role for nif and other nitrogen regulatory genes in the adaptation to nitrogen stress, in addition to a large array of secondary genes indirectly associated with shifts in metabolism and growth phenotype (e.g. metabolic enzymes, gene expression machinery, transporters).

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RNA-Seq provides new insights into the nitrogen response of Azoarcus olearius BH72 during N2-fixation beyond the nitrogen fixation process

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