Insight into the Molecular Mechanism of the Transcriptional Regulation of amtB Operon in Streptomyces coelicolor

Abstract

In Streptomyces coelicolor, amtB transcription is promptly regulated by the global nitrogen regulator GlnR. Although the GlnR binding cis-element has been characterized in amtB promoter, consisting of three GlnR boxes of a3-b3, a1-b1, and a2-b2, its role in GlnR-mediated transcriptional regulation remains unclear. Here, we showed that GlnR had different binding affinity against each pair of GlnR binding sites in amtB promoter (i.e., a3-b3, a1-b1, and a2-b2 sites), and GlnR was able to bind a3-b3 and a1-b1, respectively, but not a2-b2 alone. Consistently, a2 was not a typical GlnR binding site and further experiments showed that a2 was non-essential for GlnR-mediated binding in vitro and transcriptional regulation in vivo. To uncover the physiological role of the three GlnR boxes, we then mutated the wild-type amtB promoter to a typical GlnR-binding motif containing two GlnR boxes (a3-b3–a2-b2), and found although the transcription of the mutated promoter could still be activated by GlnR, its increasing rate was less than that of the wild-type. Based on these findings, one could conclude that the three GlnR boxes assisted GlnR in more promptly activating amtB transcription in response to nitrogen limitation, facilitating bacterial growth under nitrogen stresses.

The developmental regulator MtrA binds GlnR boxes and represses nitrogen metabolism genes inStreptomyces coelicolor

“…Of the three GlnR sites of amtB , a2b2 displays the fewest conserved nt, and this lack of conservation may be more tolerated by MtrA than by GlnR. Our data showing binding of a1b1 and a3b3, but not a2b2, by GlnR is consistent with a previous report (Li et al , ). Collectively, our data indicated that MtrA recognizes all three GlnR boxes upstream of amtB , whereas GlnR recognizes only two of three.…”

“…Shifting of the a3b3 probe was also detected with GlnR (Fig. D and E), consistent with a previous report (Li et al , ) and indicating that a3b3 is a genuine GlnR box. To validate the role of the conserved nucleotides in a3b3, we performed EMSAs using oligonucleotides with point mutations in the repeat motifs of a3b3; such mutated fragments can serve as negative controls and demonstrate the specificity of MtrA binding.…”

“…GlnR has been identified as the major regulator of nitrogen metabolism in Streptomyces (Fink et al , ; Tiffert et al , ), and its consensus recognition sequence, named the GlnR box, was characterized as a 22‐nt sequence (gTnAc‐n6‐GaAAc‐n6) consisting of two direct repeats of five nt (Tiffert et al , ; Sola‐Landa et al , ). When the two GlnR boxes of amtB , glnA , glnII , nirB , ureA and sco2195 , excluding the last six nonspecific nt, were aligned (Tiffert et al , ; Li et al , ), a 16‐nt consensus recognition sequence for GlnR was obtained (Fig. ).…”

“…Altogether, three GlnR boxes were predicted upstream of amtB and were named a1b1, a2b2 and a3b3 (Tiffert et al , ; Wang et al , ; Li et al , ). In our previous study, we predicted one MtrA site upstream of amtB , and this completely overlapped the a3b3 site; we further showed that MtrA bound a probe containing this site (Zhang et al , ).…”

The Balance Metabolism Safety Net: Integration of Stress Signals by Interacting Transcriptional Factors in Streptomyces and Related Actinobacteria

“…Recently, it was experimentally demonstrated that MtrA binds to one of the three putative GlnR binding sequences upstream of the amtB-glnK-glnD cluster (Zhu et al, 2019). Previously, three putative GlnR binding sites (named a1-b1, a2-b2, and a3-b3), each consisting in two binding repeats had been identified in the amtB promoter region (Wang et al, 2012;Li et al, 2018). More recently, Zhu et al (2019) showed that the developmental regulator MtrA recognizes the a3-b3 binding sequence which also binds GlnR efficiently.…”