Citrate Transport inKlebsiella pneumoniae

Abstract

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also .**GRAPHIC**. and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. .**GRAPHIC**. ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of .**GRAPHIC**. ions in K. pneumoniae grown aerobically on citrate and in Escherichia coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required .**GRAPHIC**. specifically for aerobic growth on citrate indicating that the .**GRAPHIC**. citrate transport system is operating. .**GRAPHIC**. and .**GRAPHIC**. were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also .**GRAPHIC**. dependent and was energized by the protein motive force .**GRAPHIC**. Dissipation of .**GRAPHIC**. or its components .DELTA.pH or .DELTA..psi. by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding .DELTA.pH and by the decarboxylation of oxaloacetate yielding .**GRAPHIC**. and .DELTA..psi.. In citrate-fermenting K. pneumoniae an ATPase which is activated by .**GRAPHIC**. was not found. The cells contain however a proton translocating ATPase and a .**GRAPHIC**. antiporter in their membrane.