Ursula Gerhardt

Mycoplasma membrane potentials determined by potential-sensitive fluorescent dyes

The membrane potentials of mycoplasmas were investigated by using potential-sensitive cyanine dyes. The fluorescence response results from a potential-dependent partition of the dyes between the cells and the extracellular medium. Cell hyperpolarization (inside more negative), e.g., by the addition of valinomycin, results in uptake of the dyes into the cells and, by formation of dye aggregates, in quenching of the fluorescence intensity. The magnitude of the fluorescence change upon addition of valinomycin depended on the external K+ concentration. At a defined external K+ concentration, no change in fluorescence occurred. The intracellular K+ concentration was determined by atomic absorption spectroscopy. Mycoplasma membrane potentials were calculated according to the Nernst equation. The membrane potential of bothMycoplasma mycoides subsp.capri andMycoplasma gallisepticum was −48 mV±10%; the membrane potential ofAcholeplasma laidlawii was −28 mV±20%.

A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of acholeplasmas, mycoplasmas, streptococci and erythrocytes☆

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.

Rheumatoid factor-negative arthritis, especially ankylosing spondylitis, and infections of the male urogenital tract.

146 men with rheumatoid factor-negative (sero-negative) arthritis, i.e., 97 patients with ankylosing spondylitis, 36 patients with Reiters syndrome, and 13 patients with reactive arthritis, were examined for infections of the urogenital tract by following recently established criteria. 74 patients (50.7%) had infections of the male adnexes: 3 patients suffered from balanitis, 14 patients from urethritis, 49 patients from prostatitis, 1 patient from epididymitis, and 7 patients from urinary tract infection. Balanitis and urethritis were almost exclusively associated with Reiters syndrome. In 37 of 97 patients with ankylosing spondylitis, a urogenital tract infection, mainly a prostatitis (31 patients), was detected. The microorganisms isolated most frequently from patients suffering from urethritis and prostatitis, were Chlamydia trachomatis and Ureaplasma urealyticum.

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beEM=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,EM=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,EM=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,EM=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (EM=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.

Cytochemical localization of surface carbohydrates on mycoplasma membranes

Surface carbohydrate structures, containinga-D-glucosyl or sterically closely related residues, were visualized on mycoplasma membranes by a cytochemical staining procedure with concanavalin A and iron-dextran complexes.

The proton gradient across mycoplasma membranes

The proton gradient across mycoplasma membranes was determined by using different probes which distribute between the intracellular space and the suspension medium in response to a transmembrane proton gradient. The intracellular pH of intact glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: pHext=7 and pHint=7.4; hence, ΔpH=0.4. The size of the proton gradient depended upon the extracellular pH. Without nutrient substrate, the mycoplasmas were unable to maintain a transmembrane proton gradient, i.e., ΔpH approximated O.N, N′-dicyclohexylcarbodiimide, an inhibitor of membrane-bound ATPase, carbonyl cyanide-m-chlorophenyl hydrazone, a proton conductor, and gramicidin, an antibiotic forming cation conduction channels across membranes, strongly affected and even abolished the proton gradient across mycoplasma membranes. These substances also impaired the metabolic activity and viability of mycoplasmas.