5′-coding sequence of thenasAgene ofAzotobacter vinelandiiis required for efficient expression

Abstract

The operon nasACBH in Azotobacter vinelandii encodes nitrate and nitrite reductases that sequentially reduce nitrate to nitrite and to ammonium for nitrogen assimilation into organic molecules. Our previous analyses showed that nasACBH expression is subject to antitermination regulation that occurs upstream of the nasA gene in response to the availability of nitrate and nitrite. In this study, we continued expression analyses of the nasA gene and observed that the nasA 5'-coding sequence plays an important role in gene expression, as demonstrated by the fact that deletions caused over sixfold reduction in the expression of the lacZ reporter gene. Further analysis suggests that the nasA 5'-coding sequence promotes gene expression in a way that is not associated with weakened transcript folding around the translational initiation region or codon usage bias. The findings from this study imply that there exists potential to improve gene expression in A. vinelandii by optimizing 5'-coding sequences.

Translational coupling ofnasSTexpression inAzotobacter vinelandiiprevents overexpression of thenasTgene

“…However, the expression pattern of nasA′‐′lacZ in AVW600 was dramatically changed when the glucose in the media was replaced with sucrose (Fig. d); the bacterium exhibited relatively constant and much higher levels of NasA – LacZ expression under all tested nitrogen conditions, resembling the expression pattern of the nasA′‐′lacZ deletion mutant that lacks the sequences involved in termination/antitermination in our previous analyses (Wang et al ., , ) . This observation suggests that regulation of nasA′‐′lacZ expression in AVW600 was disrupted when the P scrX‐nasT construct was induced by sucrose, highlighting the genetic significance of preventing overexpression of nasT .…”

“…The activity of P nasA in AVW552 remains relatively constant, regardless of the presence of ammonium, a genetic feature that distinguishes it from NtrC‐regulated promoters in other bacteria (Wang et al ., ). Variations of NasA – LacZ expression under different nitrogen conditions solely reflect the regulatory activity of the NasS/NasT system (Wang et al ., , ). As shown in Fig.…”

“…AVW552 has the intact nasST operon and contains a nasA′‐′lacZ translational fusion at the vnf locus of the genome (Wang et al ., ). nasA′‐′lacZ has been used as an expression reporter for studying the transcriptional regulation of the nasACBH operon (Wang et al ., , ). The nasA′ in the fusion is a 613‐nt sequence containing the P nasA promoter extended on to nasA codon 54.…”

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

“…6 ). The positive effect seen here could be due to more efficient translation initiation related to codon usage (Additional file 2 : Figure S2) [ 17 , 64 ]. On the other hand, it has been shown in bacteria that mRNA structure but not the codon usage at the beginning of genes is the primary factor for efficient translation [ 65 ].…”