Cloning and characterization of a gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces clavuligerus.
With primers designed for the conserved region of the gamma-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb- SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33-43%; similarity, 51-62%) to known gamma-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR ( S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the beta-lactam producer S. clavuligerus contains a gene for the gamma-butyrolactone autoregulator receptor.
Kim
Hyun Soo
HS
Department of Microbiology, Keimyung University, 1000 Shindang-dong, 704-701, Daegu, South Korea.
Lee
Yong Jik
YJ
Lee
Chang Kwon
CK
Choi
Sun Uk
SU
Yeo
Soo-Hwan
SH
Hwang
Yong Il
YI
Yu
Tae Shick
TS
Kinoshita
Hiroshi
H
Nihira
Takuya
T
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
C037481:4-butyrolactone receptor; D017931:DNA Primers; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D015107:4-Butyrolactone