Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation

Rottem, Shlomo; Linker, C; Wilson, Thérèse

doi:10.1128/jb.145.3.1299-1304.1981

Cited by 48 publications

(27 citation statements)

References 24 publications

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“…Previous studies with M. gallisepticum (12,17) showed that the swelling of energy depleted cells was prevented by the addition of nondiffusable solutes such as sucrose or MgSO4 to the external medium. In the presence of glucose (with its associated high ATP level), no swelling occurs.…”

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confidence: 99%

“…This is due to the inward diffusion of NaCl and water as a result of colloidosmotic and Donnan forces caused by intracellular nondiffusable macromolecules (11,14,22). The two bacteria without cell walls in which this phenomenon has been documented are Acholeplasma laidlawii (8) and Mycoplasma gallisepticum (12,17). Both species are known to be able to maintain their volume in the presence of glucose by an energy-dependent extrusion of NaCl and water from the cell.…”

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confidence: 99%

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Sodium and proton transport in Mycoplasma gallisepticum

Linker¹,

Wilson²

1985

J Bacteriol

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When washed cells of Mycoplasma gallisepticum were incubated at 37°C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 2Na' from the cells. Glucose stimulated 22Na' extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-atninoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+-H+ exchange reaction.

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Sodium and proton transport in Mycoplasma gallisepticum

Linker¹,

Wilson²

1985

J Bacteriol

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“…ApH and Aq~ were calculated as previously described [12] from the partition of radioactive markers between the intra-and extracellular water. For ApH, either 14C-labeled benzoic acid or 140 labeled methylamine were used, whereas for A+, [3H]-tetraphenylphosphonium (TPP +) plus unlabeled TPP + (8 #M) were utilized.…”

Section: Determination Of Intraeellular Volume Aph and A ~mentioning

confidence: 99%

A cation/proton antiport activity in Acholeplasma laidlawii

Lelong

Shirvan

Rottem

1989

FEMS Microbiology Letters

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Sealed membrane vesicles of Acholeplasma laidlawii were obtained by controlled lysis of carotenoid‐rich intact cells. An imposed ΔpH was created by loading membrane vesicles or intact Acholeplasma laidlawii cells with 0.25 M NH4Cl and diluting them into 0.25 M choline chloride. The passive efflux of NH3 from the membrane vesicles or cells resulted in the creation of a ΔpH (inside acid) that could be visualized by the quenching of the fluorescence of the weak base acridine orange. Whereas with isolated membrane vesicles, the fluorescence was dequenched by the addition of Na+, with intact cells, K+ín addition to Na+ was required. These results strongly suggest a Na+/H+ exchange activity that in intact Acholeplasma laidlawii cells is K+‐dependent. The possible role of the Na+/H+ exchange activity in pH homeostasis at the more alkaline pH range, as well as in the extrusion of excess Na+ from the cells is discussed.

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“…At various time intervals, samples (20 ,ul each) were withdrawn, and the DNase I activity was terminated by adding 80 ,ul of A buffer containing 20 mM EDTA. The LUV-encapsulated plasmid was separated from the untrapped, digested plasmid by passing 100-,u samples through a Sephadex G-50 column in a 1-ml tuberculin syringe, as previously described (24). The amount of residual DNA in the LUV preparations was estimated by counting the radioactivity or determining total DNA and comparing with controls containing nonencapsulated plasmids.…”

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“…Samples, in duplicate, were layered onto the surface of 0.3 ml of silicone oil (550:556 grade; Dow Coming Corp.) in 1.5-ml plastic microcentrifuge tubes and centrifuged at 13,000 x g for 2 min. Under these conditions, the cells passed through the oil, forming a pellet at the bottom of the tube (24). The cell pellet was resuspended, and the amounts of residual radioactivity and fluorescence in the cells were determined.…”

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Fusion-mediated transfer of plasmids into Spiroplasma floricola cells

Tarshis

Rottem

1992

J Bacteriol

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We have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells.

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Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation

Cited by 48 publications

References 24 publications

Sodium and proton transport in Mycoplasma gallisepticum

Sodium and proton transport in Mycoplasma gallisepticum

A cation/proton antiport activity in Acholeplasma laidlawii

Fusion-mediated transfer of plasmids into Spiroplasma floricola cells

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