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

Zhendong Li,Jin Wang,Guoping Zhao,Yinhua Lu,Ying Wang,Jingzhi Wang,Xinqiang Liu,Guosong Zheng

doi:10.3389/fmicb.2018.00264

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.

Highlights

Soil-dwelling actinomycetes produce a large number of bioactive secondary metabolites, including antibiotics, immunosuppressants, and antitumor agents (Berdy, 2005)
Three GlnR boxes were found in the promoter of amtB operon, each consisting of one a site and one b site (a3-b3, a1-b1, and a2-b2; Figure 1A)
This three-GlnR-box model was conserved in amtB promoter among Streptomyces, which was distinct from most other GlnR targets, where usually two GlnR boxes were identified (Supplementary Figure S2)

Summary

INTRODUCTION

Soil-dwelling actinomycetes produce a large number of bioactive secondary metabolites, including antibiotics, immunosuppressants, and antitumor agents (Berdy, 2005). Mechanism of amtB Transcriptional Regulation coelicolor, a model actinomycete, the global nitrogen metabolism is stringently regulated by the OmpR-type regulator, GlnR (Fink et al, 2002; Tiffert et al, 2008, 2011). With more and more GlnR targets characterized, more complicated GlnR-binding sequences are found, which may consist of either triple 22-bp GlnR boxes in the amtB promoter (Wang et al, 2012) or merely two a sites, separated by variable length in the promoters of nasA (Wang and Zhao, 2009) and SCO5163 (Lewis et al, 2011) in S. coelicolor. Information-based models were later established to describe GlnR box as consisting of two 11-nt direct repeats (Sola-Landa et al, 2013), the new models were still unable to explain the GlnR-binding cis-elements observed in nasA and SCO5163 promoters in S. coelicolor. We here systematically studied the GlnR binding activities against the three GlnR boxes in amtB promoter and investigated the roles of the three GlnR boxes in the transcriptional regulation of amtB

MATERIALS AND METHODS

RESULTS

DISCUSSION