Evidence for chemiosmotic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei

Desulfomonile tiedjei (strain DCB-1) was previously shown to conserve energy for growth from reductive dechlorination of 3-chlorobenzoate coupled to formate oxidation. We tested the hypothesis that a chemiosmotic mechanism couples reductive dechlorination and ATP synthesis in D. tiedjei. Dechlorination resulted in an increase in the ATP pool of cells. Uncouplers and ionophores decreased both the dechlorination rate and the ATP pool. However, at low concentrations the inhibitors had relatively greater effects on the ATP pool, and in some cases, even appeared to stimulate dechlorination. Those agents could not completely inhibit ATP synthesis while allowing dechlorination activity. The proton-driven ATPase inhibitor, N,N-dicyclohexylcarbodiimide (DCCD), had similar effects. An imposed pH gradient also resulted in an increase in the ATP pool of cells, and this increase was partially inhibited by DCCD. Addition of 3-chlorobenzoate to cell suspensions caused proton translocation by the cells. Proton translocation was stimulated by the permeant thiocyanate anion and inhibited by uncouplers. A maximum H⁺/3-chlorobenzoate ratio of greater than two was observed. These findings suggest that dechlorination supports formation of a proton-motive force which in turn supports ATP synthesis via a proton-driven ATPase.Abbreviations 3CB 3-chlorobenzoate - CCCP m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - P proton-motive force - PCP pentachlorophenol     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pK_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

Specific removal of chlorine from the ortho-position of halogenated benzoic acids by reductive dechlorination in anaerobic enrichment cultures

Anaerobic enrichment cultures catalysing the reductive dechlorination of chlorinated benzoic acids were obtained from three fresh-water sediments collected from seven different locations. Sub-cultures from these enrichments specifically removed ortho-substituted chlorine from 2,3,6-, 2,3,5- and 2,4,6-trichlorobenzoic acid, yielding chloride and 2,5-, 3,5-, and 2,4-dichlorobenzoic acids, respectively. These reductive dehalogenations were stimulated by the addition of benzoate and/or volatile organic acids. In one of these enrichments dehalogenation of ortho- and/or para-chlorine substituents was also observed from 2,3-, 2,4-, 2,5-, and 3,4-dichlorobenzoic acid, yielding 3- and 4-chlorobenzoate. Removal of meta-chlorines was not observed in any of the enrichments.