Effect of Dicyclohexylcarbodiimide on Growth and Membrane-Mediated Processes in Wild Type and Heptose-Deficient Mutants of Escherichia coli K-12

A. P. Singh, P. D. Bragg
1974 Journal of Bacteriology  
The effects of N, N'-dicyclohexylcarbodiimide (DCCD) on the growth of Streptococcus faecalis, and on the growth, 0-galactosidase synthesis, and various membrane-mediated processes, were studied in wild-type Escherichia coli JE1011 and its lipopolysaccharide-defective mutant NS1. DCCD (0.1 mM) completely inhibited the growth of S. faecalis and E. coli NS1 but had little effect on strain JE1011. The same amount of DCCD with E. coli NS1, but not with E. coli JE1011, inhibited the induction of
more » ... e induction of s-galactosidase, increased the permeability of the cells to o-nitrophenyl-fl-D-galactoside without causing extensive cell lysis or release of ultraviolet-absorbing materials, and inhibited the oxidation of certain intermediates of the tricarboxylic acid cycle. Inhibition of the oxidation of malate, fumarate, and a-ketoglutarate by DCCD appeared to be at the level of the transport system for these compounds. Inhibition of the membrane-bound adenosine triphosphatase by DCCD was not entirely responsible for these effects, since oxidation of these substances, and transport of ["4C ]succinate and [14C]fumarate, was inhibited by DCCD in a mutant, N144, which lacked adenosine triphosphatase activity. It is concluded that lipopolysaccharide forms a barrier to DCCD in wild-type E. coli, and that DCCD can inhibit several processes in the cell. N, N'-dicyclohexylcarbodiimide (DCCD) has been shown to have effects on gram-positive bacteria. Harold et al. (11, 12) found that low concentrations of this substance inhibited growth, transport of monovalent cations, amino acids, and phosphate in Streptococcus faecalis. DCCD also prevented the degradation of the adenosine 5'-triphosphate (ATP) pool which occurred if glycolysis was inhibited in these cells. These effects of DCCD were attributed to inhibition of the membrane-bound adenosine triphosphatase (ATPase), which had been shown to be sensitive to this agent (1, 11, 12), and which is believed to have a role in transport (11, 12) . This hypothesis was strengthened when mutants of S. faecalis were isolated which had DCCD-resistant ATPase, and K+ and cycloleucine transport (2). Glutamine transport in intact cells of Escherichia coli was found to be resistant to the inhibitor (28). After treatment with ethylenediaminetetraacetic acid (EDTA), transport of glutamine and proline was inhibited by DCCD (16, 28) . Since the ATPase of E. coli could be inhibited by this substance (7, 8), its effects on transport were postulated to be due to inhibition of this enzyme. In agreement with this hypothesis, DCCD prevented the fall in the ATP pool induced by colicin El (9). The evidence from the action of DCCD on intact cells seems to be consistent, and suggests that DCCD could be used as a specific probe for the involvement of the membrane-bound ATPase in processes within the cell. It has been used in this manner by Prezioso et al. (22) and by Postma et al. (23). In the former case DCCD was used to show that ATP was not the direct energy source for the transport of galactosides and amino acids into membrane vesicles of E. coli driven by oxidation of ascorbate or D-lactate. Postma et al. (23) employed DCCD to show that ATP was directly used for the transport of Krebs-cycle intermediates into intact cells of Azotobacter vinelandii. Further, the studies by Klein and Boyer (16) on the energization of active transport in E. coli were dependent to some extent on the assumption that DCCD specifically reacted with the ATPase. However, DCCD is such a highly reactive compound (15) that its site of action would not 129 on May 7, 2020 by guest
doi:10.1128/jb.119.1.129-137.1974 fatcat:yephzhhxuvglva7gakfi2ikqoe