Identification and characterization of glnA promoter and its corresponding trans-regulatory protein GlnR in the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32.
The genetic requirements for the transcription of glnA, encoding the major glutamine synthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain, U32, were investigated. Primer extension experiments showed that the promoter of U32 glnA (pglnA) was likely to have two transcription initiation sites: P(1) and P(2), located 157 and 45 nucleotides (nt) upstream of the translational start codon, respectively. Gel mobility shift and DNase I footprinting analyses revealed a 30 bp cis-element located at 45 to 75 nt downstream of P1, or 38 to 68 nt upstream of P(2). The sequence of the cis-element displayed high similarity to the corresponding regions of pglnA from Streptomyces coelicolor and S. roseosporus. With xylE as a reporter gene, the expression levels of U32 pglnA and its deletion derivatives under different nitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in S. lividans TK54, S. coelicolor J508 and S. coelicolor FS10 (glnR mutant). These in vivo studies showed that the activation of U32 pglnA in S. coelicolor required GlnR, and its binding to the U32 pglnA was further confirmed by the gel mobility shift assay. Cloning and heterologous expression of the U32 glnR allowed us to detect the in vitro interaction between the U32 GlnR and the corresponding pglnA cis-element. Further evidence shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for the active transcription of glnA in U32.
Yu
Hao
H
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Peng
Wen-Tao
WT
Liu
Yang
Y
Wu
Ting
T
Yao
Yu-Feng
YF
Cui
Ming-Xue
MX
Jiang
Wei-Hong
WH
Zhao
Guo-Ping
GP
D039903:Actinobacteria / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D050548:Catechol 1,2-Dioxygenase / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012294:Rifamycins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C082451:GlnR protein, Streptomyces coelicolor; D012294:Rifamycins; D015534:Trans-Activators; C023808:rifamycin SV; D050548:Catechol 1,2-Dioxygenase; D003850:Deoxyribonuclease I; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase