Plasmid-mediated sucrose metabolism in Escherichia coli: Identification of a new sucrose porin, ScrC

In most enteric bacteria the ability to ferment sucrose is plasmid-mediated. A sucrose-fermenting clinical isolate of Salmonella typhimurium was found to contain a 72 megadalton plasmid, pUR400, which was easily transferred to Escherichia coli by conjugation. Previous work had shown that the sucrose regulon contained a phosphoenolpyruvate: dependent phosphotransferase system enzyme II$\sp{\rm Scr}$ (scrA), a sucrose-6-phosphate hydrolase (scrB), and a regulatory locus, scrR. The sucrose regulon from pUR400 was cloned in E. coli and mapped to a 5.7-kb SalI fragment of DNA. Analysis of deletion mutants identified a third locus, scrC, involved in sucrose transport. Mutations in this region resulted in an approximately 20-fold decrease in sucrose transport. The scrC gene was found to encode an outer membrane protein having an apparent molecular weight of 68,000 as determined by migration on SDS-PAGE gels. The DNA sequence of the scrC gene was determined to further characterize the gene and its product, ScrC. The open reading frame contained 1515-bp which encoded for a 505 amino acid protein with a molecular weight of 55,408. N-terminal amino acid sequencing of the ScrC protein confirmed that the 68 kDa protein was the product of the scrC gene. The deduced primary structure was used in computer models to predict the secondary structure of ScrC. The ScrC protein was found to have characteristics similar to other E. coli outer membrane proteins. A NBRF protein bank search revealed that ScrC shared 23% identity with the maltose porin, LamB. Liposome swelling experiments provided evidence that the ScrC protein formed diffusion pores. The rate of diffusion of sucrose through the ScrC pore was greater than the rate of diffusion of the disaccharides lactose and maltose, suggesting that the ScrC protein had sucrose specificity. The ScrC protein represents the only known porin protein that is associated with a PTS regulon