RNA-Seq Provides New Insights into the Gene Expression Changes in Azoarcus olearius BH72 under Nitrogen-Deficient and Replete Conditions beyond the Nitrogen Fixation Process

Abstract

Azoarcus olearius BH72 is an endophyte capable of biological nitrogen fixation (BNF) and of supplying nitrogen to its host plant. Our previous microarray approach provided insights into the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated the induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially expressed 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 the induction of genes involved in carbon metabolism and flagellar and twitching motility. This may support N2-fixation by increasing energy production and by finding suitable microaerobic niches. On the other hand, energy expenditures were reduced by suppressing translation and vitamin biosynthesis. Nonetheless, strain BH72 does not appear to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids; a strong gene induction of machineries for their uptake and assimilation was detected. RNA-Seq has thus provided a better understanding of a lifestyle under limiting nitrogen sources by elucidating hitherto unknown regulated processes.

Deep‐learning‐based removal of autofluorescence and fluorescence quantification in plant‐colonizing bacteria in vivo

“…Its function in plant–endophytic bacteria associations is unknown. Previous RNA‐Seq data (Solaiyappan Mani & Reinhold‐Hurek, 2021) ranked it at a low expression level suitable for CNN learning (60–80 RPKM in comparison to repressed nitrogenase genes nifHDK at 10–50 RPKM).…”

“…Nitrogen fixation appears to be an important feature for the endophytic lifestyle of A. olearius (Hurek et al ., 1987; Egener et al ., 1999; Sarkar & Reinhold‐Hurek, 2014; Solaiyappan Mani & Reinhold‐Hurek, 2021). The nifH gene encodes the iron protein of the nitrogenase complex mediating BNF.…”

“…Common methods to quantify gene expression in bacteria such as quantitative reverse‐transcription PCR (Shidore et al ., 2012), transcriptome studies applying microarrays (Sarkar et al ., 2017) or RNA‐Seq (Solaiyappan Mani & Reinhold‐Hurek, 2021), and transcriptional fusions with reporter genes using enzymatic read‐outs (Egener et al ., 1999), extrapolate over a large number of cells. However, genetically identical bacterial cells are individuals: they may have different phenotypic characteristics even when grow under homogeneous environmental conditions in liquid cultures (Gasperotti et al ., 2020).…”

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq

“…Type IV pili are important for endophytic colonization, enabling effective attachment to and motility by twitching inside the host ( 8 – 10 ). Recent RNA-Seq studies further emphasized the need of diazotrophs to be mobile by demonstrating upregulation of the type IV pilus machinery during nitrogen fixation ( 17 ). Tn-Seq and RNA-Seq revealed a striking overlap of genes found to be significant ( pilY1A , pilW , azo2178 , pilOPQ , and pilAB ).…”

“…The endophytic lifestyle of strain BH72 appears to be interconnected to microaerobiosis—typical for waterlogged roots—and nitrogen fixation. In field-grown roots ( 6 ), pot experiments with field soil ( 14 ), and gnotobiotic cultures ( 15 ), nitrogenase genes are expressed in roots; moreover, several proven or suspected interaction-related genes are upregulated under microaerobiosis or nitrogen fixation conditions according to transcriptome studies ( 13 , 16 , 17 ). Therefore, it is of great interest to analyze the contribution of A. olearius genes under these culture conditions in more detail by a global approach.…”