Transcriptome analysis of nitrogen assimilation preferences in Burkholderia sp. M6-3 and Arthrobacter sp. M7-15

Abstract

Introduction: Ammonium (NH₄ ⁺) and nitrate (NO₃ ^-) are the two main forms of inorganic nitrogen (N) that exist in soil and both can be absorbed and utilized by plants. As a vast and crucial biome, soil microorganisms are responsible for mediating the inorganic N assimilation process and enhancing nitrogen use efficiency. Understanding how these microorganisms assimilate different forms of inorganic nitrogen is crucial. There are a handful of microorganisms that play a dominant role in the process of soil inorganic nitrogen assimilation and have a significant advantage in abundance. However, microbial preferences for ammonium or nitrate, as well as differences in their metabolic pathways under co-existing ammonium and nitrate conditions, remain unclear.

Methods: In this study, two microbial strains with nitrogen assimilation advantages, Burkholderia sp. M6-3 and Arthrobacter sp. M7-15 were isolated from an acidic Chinese soil and then incubated by different sources of inorganic N to investigate their N preferences. Furthermore, RNA sequencing-based transcriptome analysis was used to map the metabolic pathways of the two strains and explore their explanatory potential for N preferences.

Results: The results showed that strain M6-3 preferred to utilize NH₄ ⁺ while strain M7-15 preferred to utilize NO₃ ^-. Although both strains shared similar nitrogen metabolic pathways, the differential expression of the glutamine synthetase-coding gene glnA played a crucial role in regulating their inorganic N preferences. This inconsistency in glnA expression may be attributed to GlnR, a global regulator of nitrogen utilization.

Discussion: This research strengthens the theoretical basis for exploring the underlying causes of differential preferences for inorganic N forms and provided key clues for screening functional microorganisms to ultimately enhance inorganic nitrogen use efficiency.

Keywords: RNA-seq analysis; bacterial strains; inorganic nitrogen assimilation; nitrogen metabolic pathway; nitrogen preference.

Figure 1

Morphology and genome information of Burkholderia sp. M6-3 (A,C) and Arthrobacter sp. M7-15 (B,D). The circular genome map is displayed from the outer circle to innermost, as follows: scale marks of the genome; protein-coding genes on the forward strand; protein-coding genes on the reverse strand; tRNA (black) and rRNA (red) genes on the forward strand; tRNA (black) and rRNA (red) genes on the reverse strand; GC content; GC skew. Protein-coding genes are color coded according to their COG categories.

Figure 2

Growth of Burkholderia sp. M6-3 (A) and Arthrobacter sp. M7-15 (B) under different inorganic nitrogen application. Linear graphs indicate the growth curve of the bacterial strains in terms of the change in OD₆₀₀ value. The time axis indicates the different time periods of bacterial strain growth. The different periods of strains growth were classified according to whether there was a significant difference between their OD₆₀₀ values (p < 0.05, n = 3, Student’s T-test). Control refers nitrogen-free treatment. Bars indicate the standard deviation (n = 3).

Figure 3

Changes in inorganic nitrogen contents during 72 h of incubation with Burkholderia sp. M6-3 (A–C) and Arthrobacter sp. M7-15 (D–F). “0–24 h,” “0–60 h” and “0–72 h” indicate the changes of inorganic nitrogen content from 0 h to 24 h, from 0 h to 60 h and from 0 h to 72 h, respectively. Bars indicate the standard deviation, different letters indicate significant differences (p < 0.05, n = 3, Duncan’s test).

Figure 4

The intersections of differentially expressed genes (DEGs) of Burkholderia sp. M6-3 (A,B) and Arthrobacter sp. M7-15 (C,D) under different inorganic nitrogen treatments.

Figure 5

Differential transcript expression of functional genes for nitrogen metabolism in ammonium nitrogen, nitrate nitrogen and mixed nitrogen sources by Burkholderia sp. M6-3 (A,C) and Arthrobacter sp. M7-15 (B,D). The significance cutoff was set at 0.01 (−sc 0.01), and a |GFOLD value| > 1 was also required for 2-fold change or greater. Red borders indicate genes significantly up-regulated (Gfold >1); blue borders indicate genes significantly down-regulated (Gfold < −1), and black borders indicate no significant difference. Gray ovals indicate the nitrogen regulatory factors associate with the two-component regulatory system.

Figure 6

The expression levels of major nitrogen metabolism genes in Burkholderia sp. M6-3 (A) and Arthrobacter sp. M7-15 (B) under different inorganic nitrogen treatment.