A collection of hybrid plasmids carrying either the wild-type or mutated
glpT
gene was generated in vitro and used to characterize the
glpT
-dependent active transport system for
sn
-glycerol-3-phosphate in
Escherichia coli
K-12. Restriction endonuclease analysis and recloning of DNA fragments localized
glpT
to a 3-kilobase pair
Pst
I-
Hpa
I segment of DNA. Comparison of DNA carrying
glpT-lacZ
fusions with DNA carrying intact
glpT
allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in
glpT
, it was shown that
glpT
is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of
glpT
, the
sn
-glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50°C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50°C; subsequent treatment at 95°C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in
glpT
were negatively dominant over wild-type
glpT
, indicating that the active form of the permease is multimeric. A gene (named
glpQ
) promoter distal to
glpT
codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the
glpT
gene. The present report demonstrates that it is not the gene product of
glpT
and is not required for active transport of
sn
-glycerol-3-phosphate.
ISOLATION AND REACTIVATION OF THE AXOSTYLE
