KINETICS OF UREA DEGRADATION BY GENETICALLY MODIFIED E. COLI

Abstract

Malchesky first reported the degradation of urea by wild type cells grown in media containing an uremic molecule (Trans Am Soc Artif Int Organs 7977 23:726). Chang subsequently described the metabolism of urea by E. Coli cells modified to contain the gene for urease and proposed a therapy format based upon oral ingestion of encapsulated cells (Nat. Med. 1996 2:883). In order to further develop this intriguing technology, we have begun to characterize the kinetics and mechanism of bacterial urea degradation. All studies were conducted with E. Coli DH 5 cells (kindly provided by S. Mulrooney, Mich. State U.) containing the urease plasmid pkAU17 from Klebsiella aerogens along with an ampicillin resistant gene. Cells were passaged in Luria-Bertani (LB) media containing urea and ampicillin. The rate of urea degradation from a 100 mg/dl starting concentration of urea varied in dose dependent fashion on the concentration of modified cells in a challenge solution. Small aliquots of these cells in LB media grew to a concentration of −1 million cells/ml in 48 hours. At this concentration, cells were found to completely degrade urea at a starting concentration of 100 mg/dl in less than one hour. Supernatant from cultures of modified cells had no capacity to degrade urea, suggesting that the degradation process is intracellular. The mass efficiency of urea degradation by genetically modified cells (approximately three mg urea removed per μg of cells per hour) was greater than that of constructs prepared from purified urease suspended in alginate and appeared adequate for therapeutic consideration. In our expereincee at least, it was not possible to condition unmodified control E. Coli cells to degrade urea by prolonged passaging in urea-containing culture media.