Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli

Abstract

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secrete sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra-and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVSl1O) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. col(pVS100). Sucrose uptake was inhibitied by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 ,uM and 6 nmol/mg of protein per min and 275 FIM, respectively.Vibrio alginolyticus is an aerobic, halotolerant, gramnegative bacterium, which has been isolated from marine environments (20) and from damaged salted hides (16,29,30). The ability of V. alginolyticus to utilize sucrose has been used to distinguish it from Vibrio parahaemolyticus, which causes food-borne gastroenteritis (20). Kakinuma and Unemoto (5) investigated the initial step of the sucrose catabolic pathway in a marine V. alginolyticus strain and showed that sucrose is actively transported by the Na+ electrochemical potential and then hydrolyzed intracellularly to glucose and fructose by sucrase.The pathway of sucrose metabolism in the gram-negative marine V. alginolyticus strain differs from that in grampositive Bacilus subtilis (6), Streptococcus lactis (26), and Streptococcus mutans (21, 24), for which sucrose is accumulated in the cell by the phosphoenolpyruvate-dependent phosphotransferase system. Sucrose 6-phosphate, which is the intracellular product of this transport system, is then hydrolyzed to glucose 6-phosphate and fructose by sucrose-6-phosphate hydrolase.We investigated the sucrose metabolism in the collagenolytic V. alginolyticus strain and report here the cloning, expression, and regulation of the V. alginolyticus sucrose utilization system in Escherichia coli. Evidence is presented for the active transport of sucrose by a Na+-independent process in E. coli, as well as for the appearance of extra-and intracellular sucrase enzyme activities. MATERIALS AND METHODSPlasmids, bacterial strains, and growth conditions. V.alginolyticus (16,29,30) was used as the source of chromosomal DNA. E. coli JA221 recAl leuB6 trpE5 (11) was used as the recipient strain. Plasmid pEcoR251, a gift from M. Zabeau, Plant Genetic Systems, Ghent, Belgium, is a positive selection vector containing the E. coli EcoRI endonuclease gene under the control of the bacteriophage X rightward promoter, the ampicillin resistance gene, and...

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

“…Previous localization experiments with E. coli(pBS100) indicated that the B. fragilis BF-1 fructanase was localized primarily in the periplasm (43). Cellular localization experiments with B. fragilis BF-1 also indicated that most of the cell-associated fructanase activity was located in the periplasm (Table 2).…”

“…Plasmid pBS100 containing the B. fragilis BF-1 sucrose utilization system cloned into the positive selection vector pEcoR251 has been described previously (43). The vectors pBluescript SK and pBluescript KS (Stratagene, San Diego, Calif.) were used for cloning and exonuclease III-shortening experiments.…”

“…The gram-negative obligate anaerobe Bacteroides fragilis BF-1 is able to grow on sucrose as the sole source of carbon; sucrose metabolism was shown to involve a sucrose transport protein and a sucrase enzyme (43). The B. fragilis BF-1 sucrose utilization system was cloned and expressed in Escherichia coli on plasmid pBS100 (43).…”

“…The B. fragilis BF-1 sucrose utilization system was cloned and expressed in Escherichia coli on plasmid pBS100 (43). E. coli(pBS100) cells utilized sucrose as the sole source of carbon because of the presence of sucrase and sucrose transport activities.…”

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes

“…Pro A, glutamine synthetase (Bender et al, 1977) and /?-lactamase (Sykes & Nordstrom, 1972) were assayed in E. coli cell-free culture supernatants, cytoplasmic and periplasmic cell fractions. Pro A and sucrase (Scholle et al, 1987) were assayed in B. subtilis cell-free culture supernatants and cytoplasmic fractions.…”