Uridine and uracil transport in Escherichia coli and transport-deficient mutants.

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterize the transport mechanism of uracil and uridine. Mutant U-, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR-, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U-UR- isolated from a culture of mutant U-, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR-R- was isolated from a culture of mutant UR-, and is resistant to a high concentration of showdomycin. This mutant is defective for transport of intact uridine and addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U- and UR-, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these processes.

Mechanism of energy coupling for transport of deoxycytidine, uridine, uracil, adenine and hypoxanthine in Escherichia coli.

The transport processes for uridine, deoxycytidine, uracil, adenine and hypoxanthine require an energy source and are active under anaerobic or aerobic conditions. Inhibitory effects of cyanide, arsenate, carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol and N,N'-dicyclohexylcarbodiimide on the transport of uridine and deoxycytidine differ from the corresponding effects on the transport of uracil, adenine and hypoxanthine. The nature of these inhibitory effects supports the conclusion that uridine and deoxycytidine transport is energized either by electron transport or by ATP hydrolysis via (Ca2+ + Mg2+)-ATPase. The transport or uracil, adenine and hypoxanthine is dependent upon ATP or some high energy phosphate derivative of ATP, but is independent of (Ca2+ + Mg+)-ATPase and electron transport. Uptake of the ribose moiety of uridine by a mutant of Escherichia coli B, which lacks the transport system for uracil and intact uridine, is neither stimulated by energy sources nor inhibited by various inhibitors of energy metabolism under either aerobic or anaerobic conditions.

Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants.

Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.

Transport of purines and deoxyadenosine in Escherichia coli.

The characteristics of adenine, guanine, hypoxanthine, xanthine, and uracil uptake in Escherichia coli B show that each base is transported by a specific system. The data support the concept that the transport of guanine, hypoxanthine, xanthine, and uracil function without direct involvement of the respective purine or pyrimidine phosphoribosyltransferase enzymes. Uracil phosphoribosyltransferase is not demonstrable in E. coli B, and large differences are observed in the inhibitory effects of heterologous purines on the uptake of guanine, hypoxanthine, and xanthine as compared to the corresponding inhibitory effects reported for the soluble purine phosphoribosyltransferase enzymes of E. coli B. Additional evidence is provided by the low Km values determined for the transport of adenine, guanine, hypoxanthine, and xanthine relative to the corresponding Km values for the phosphoribosyltransferase enzymes. Data are presented indicating that adenine may be transported without participation of adenine phosphoribosyltransferase. The stimulatory effect of glucose, the inhibitory effect of KCN, and the high intracellular to extracellular concentration gradients of the bases produced in the presence of glucose provide evidence that the transport processes are energy-dependent. The Km values for transport of the purines and uracil range from 10(-7) M to 5 X 10(-7) M. Characteristics of adenine and uracil uptake are similar in E. coli B, E. coli K-12, and a showdomycin-resistant mutant of E. coli B. Adenosine and deoxyadenosine are transported in E. coli B by independent transport systems. Adenine or hypoxanthine does not share the adenosine or deoxyadenosine transport systems as evidence by the mutual lack of competition of free bases and nucleosides on transport. The transport systems for deoxyadenosine and adenosine are defective in the mutant.

Deoxycytidine transport in the presence of a cytidine deaminase inhibitor and the transport of uracil in Escherichia coli B.

Tetrahydrouridine, a cytidine deaminase inhibitor, prevents periplasmic degradation of deoxycytidine by Escherichia coli B. It does not inhibit deoxycytidine transport and therefore allows an accurate determination of deoxycytidine transport. Data obtained using tetrahydrouridine show that deoxycytidine is transported in E. coli B as the intact nucleoside by an active transport process, with a K-m of 6 times 10-minus 6 M. Cytidine and deoxyadenosine inhibit transport competitively, whereas guanosine has no effect on transport. Arsenate or KCN greatly reduces transport. In a mutant resistant to the nucleoside antibiotic, showdomycin, the active transport of deoxycytidine is lost, and residual slow uptake occurs by passive diffusion. Uracil is accumulated in E. coli B by an active transport process with a K-m of 5 times 10-minus 7 M.