TY - JOUR
T1 - Cloning and characterization of the aerobic sn-glycerol-3-phosphate dehydrogenase structural gene glpD of Escherichia coli K-12
AU - Schweizer, H.
AU - Larson, T. J.
PY - 1987
Y1 - 1987
N2 - The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a λ glpD transducing phage. The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes. The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment. Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene. The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments. The glpD gene product, a polypeptide of M(r) 55,000, was detected in a maxicell system. Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates. The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site. Determination of the apparent M(r) of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region. The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded β-galactosidase activity. The glpD3 mutation was mapped within the glpD gene, providing additonal evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.
AB - The glpD gene encoding aerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12 was cloned into pACYC177 from a λ glpD transducing phage. The recombinant plasmid, designated pSH55, carried a 7.4-kilobase-pair HindIII fragment containing the glpD and glpR genes. The glpD gene was subcloned into pACYC177 on a 4.4-kilobase-pair BamHI-HindIII fragment. Expression of the cloned glpD gene was regulated in the manner previously described for the chromosomal glpD gene. The position of glpD on this plasmid was determined by Tn1000 insertional inactivation experiments. The glpD gene product, a polypeptide of M(r) 55,000, was detected in a maxicell system. Truncated polypeptides replaced the 55,000-molecular-weight polypeptide when plasmid derivatives harboring Tn1000 insertions that inactivate glpD were used as templates. The sizes of these polypeptides confirmed the previously determined direction of transcription and allowed estimation of the translation start site. Determination of the apparent M(r) of a hybrid protein encoded by a glpD'-'lacZ fusion provided additional evidence for the position of the glpD control region. The amino-terminal 30 to 60 amino acids of this hybrid protein (provided by glpD) were sufficient for efficient membrane localization of glpD'-'lacZ-encoded β-galactosidase activity. The glpD3 mutation was mapped within the glpD gene, providing additonal evidence that glpD is the structural gene for aerobic sn-glycerol-3-phosphate dehydrogenase.
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U2 - 10.1128/jb.169.2.507-513.1987
DO - 10.1128/jb.169.2.507-513.1987
M3 - Article
C2 - 3027031
AN - SCOPUS:0023141781
SN - 0021-9193
VL - 169
SP - 507
EP - 513
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 2
ER -