Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12

Abstract

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of E. coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a srcB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3',5'-phosphate receptor protein. Sucrose uptake (apparent K(m) = 10 μM) was dependent on the scrA gene product and on the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (K(m) = 0.17 mM), sucrose (K(m) = 60 mM), and raffinose (K(m) = 150 mM). The active enzyme was shown to be a dimer of M(r) 110,000.