Journal of General Microbiology (1987), 133, 3495-3504. Printed in Great Britain 3495 Autostimulation of Dihydrostreptomycin Uptake in Baciffussubtifis By FRANZ EMLING AND JOACHIM-VOLKER HoLTJE*t Institut fur Biologie II, LRhrstuhl Mikrobiologie I, Uniuersitat Tiibingen, 7400 Tubingen, FRG (Received 6 July 2987) ~ ~~~ ~ ~ ~ ~~~ In Bacillus subtilis it was shown that the membrane potential (At,$) has to reach a threshold value of - 180 to - 190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of A+ is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N’-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of At,$. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing A$ due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins. INTRODUCTION Efficient killing of bacteria by streptomycin correlates well with the rate of its accumulation inside the cell (Hancock, 1962; Bryan et af.,1976, 1979; Taber & Halfenger, 1976; Miller et al., 1980; Eisenberg et al., 1984). The factors which determine the uptake of streptomycin are thus of particular importance for its therapeutic efficiency. In many aerobic bacterial strains [3H]dihydrostreptomycin uptake is autostimulated resulting in a biphasic uptake process (Anand et al., 1960; Hurwitz & Rosano, 1962; Bryan & Van den Elzen, 1976). An early energy-dependent phase (EDPI) with a low rate of uptake is followed by a second greatly enhanced energy-dependent phase (EDPII) of uptake (Bryan & Van den Elzen, 1977). Self-enhancement of dihydrostreptomycin accumulation, a prerequisite for its antibacterial action (Hancock, 198l), has been suggested to depend on codon-misreading during protein synthesis (Hurwitz & Rosano, 1962; Holtje, 1978; Davis et al., 1986). With Escherichia culi it has been shown that preincubation with streptomycin not only increases dihydro- streptomycin accumulation but also the uptake of the polyamines putrescine and spermidine (Holtje, 1978). However, the molecular basis that triggers streptomycin uptake with increased rate (EDPII) remains obscure. A recent model proposes the creation of membrane channels by misread proteins to be responsible for the increase in uptake (Davis et al., 1986). While the model indeed can account for a number of previously unexplained observations it fails to explain convincingly the energy t Present address: Max-Planck-Institut fur Entwicklungsbiologie, Spemannstrasse 35/11, 7400 Tiibingen, FRG. Abbreviations: CCCP, carbonyl cyanide mchlorophenylhydrazone ; DCCD, N,N’-dicyclohexylcarbodiimide; TPMP+, triphenylmethylphosphonium ion; EDPI, EDPII, energydependent phases I and 11; A+, transmembrane potential difference (‘membrane potential’); ApH, transmembrane pH difference; Ap, transmembrane proton electrochemical difference (‘protonmotive force’). 0001-4284 0 1987 SGM 3496 F. EMLING AND J.-V. HOLTJE dependence of uptake as well as the specificity of stimulated uptake. Furthermore, it cannot explain a number of findings described in the present communication. It is now well established that the uptake of dihydrostreptomycin and other aminoglycoside antibiotics into bacteria is energized by the electrical component (A$) (interior of cell negative) rather than the chemical component (ApH) of the proton electrochemical difference (Ap) across the cytoplasmic membrane (Damper & Epstein, 1981 ; Mates et al., 1982; Bryan 8z Kwan, 1983; Eisenberg et a/., 1984; Arrow & Taber, 1986). It was, therefore, of interest to follow the membrane potential during the course of self-enhancement of dihydrostreptomycin accumu- lation. The method most used to estimate A$ determines the partition of a permeable lipophilic cation, like tetraphenylmethylphosphonium (TPMP+), inside and outside the cell. Being a trivalent cation dihydrostreptomycin interferes with this technique, which therefore cannot be used in this case. Interestingly, among the many protein synthesis inhibitors besides aminoglycosides causing misreading, only puromycin also stimulates streptomycin uptake. The mechanism of puromycin-induced enhancement of uptake may be considered an analogous situation to the autostimulation phenomenon since both antibiotics cause ribosomes to synthesize non- functional proteins. Therefore, an attempt was made to follow A$ during puromycin-stimulated dihydrostreptomycin uptake. Based on the experimental results a model was developed proposing a common basis for the mechanism of autostimulation and puromycin-enhanced dihydrostreptomycin uptake. METHODS Bacterial strains and growth conditions. All strains were grown routinely in 1 % Tryptone broth (Bacto, Difco) with shaking at 37 "C. This medium contained approximately 11.5 mM-Na+, 0.8 mM-K+ and 0-19 mM-Mg*+. Bacillus subtilis 60015 (trpC rnetC) was a gift from Dr E. Freese (NIMH, NIH, Bethesda, Md., USA). The K+- retention mutant 168KL was kindly provided by Dr K. Willecke (Westdeutsches Tumorzentrum, Essen, FRG) and the ribosomally streptomycin-resistant strain SRBIS (trpC lys3 metB strA ) was obtained from Dr G. H. Chambliss (University of Wisconsin, Madison, Wis., USA). Uptake experimenrs. Uptake measurements were started by adding appropriate concentrations of the respective radioactive solute ([3H@ihydr~~treptomycin,6.6 VM; 37 kBq mi-' ; or [14C]spermidine,6-6 g~;4-625 kBq ml-I) to cells growing in the mid-exponential phase of growth at an ODs7*of about 0.2. At the indicated times 0.5 ml samples were withdrawn and collected by filtration through cellulose nitrate membranes (pore size 0.45 pm; Sartorius) which had been pre-soaked in 0.1 M-LiCI. The filters were immediately rinsed twice with 3 ml 0.1 M- LiCI. placed into scintillation vials and dried at 70 "C for 30 min. After the addition of 5 ml scintillation fluid (Quickscint 401 ; W. Zinsser) the radioactivity was determined in a liquid scintillation counter (mark 11; Nuclear Chicago Corp.). The results were corrected for adsorption of radioactive material to membrane filters, non- specific binding to the bacterial cell surface and uptake by passive diffusion. For this, controls were prepared either lacking cells or with cells pre-treated with carbonyl cyanide rn-chlorophenylhydrazone (CCCP). Further control samples were quickly cooled to 4°C before the addition of the respective solute. Growth of the cells throughout the uptake experiment was monitored and the results were corrected for changes in the optical density of the culture. The relationship between ODS78and cell titre was determined using the viable count method; dry weight and intracellular water space were calculated on the basis of the values published by Shioi er al. (1980). Measurernenr o/'A+. This was done by measuring the distribution of the lipophilic cation [I4C]TPMP+at a final concentration of 8.4 p~ (1.48 kBq ml-I) as described by Shioi et al. (1980). The lipophilic anion tetraphenylboron was added to the cell suspension at a final concentration of 2 ~LM0.5 min before TPMP+ addition to enhance TPMP' uptake. Because TPMP+ adsorbs strongly to cellulose nitrate, in these experiments cellulose acetate membrane filters (pore size 0.45 p~;Schleicher and Schiill) were used. The filters were pre-soaked with 8.4 ~LM unlabelled TPMP+ in 0.1 M-LicI solution. After collection of the cells on the filters they were rinsed twice with 3 mi 0.1 M-LICI containing 8.4~~unlabelled TPMP+. A# was calculated from the Nernst equation: A$ = (2.3 RT/F)log((TPMP+],,,/[TPMP+],,). At 37 "C, the value of 2-3 RT/F is about 61.25 mV. TPMP+ uptake was usually complete in less than 1 min and remained constant for about 1 to 6 min. The mean of the values measured within that time range (normally three values per individual experiment) was taken to calculate the momentary A$ of the cells at zero time, i.e. the addition of t3H]TPMP+ to the cell suspension. The results were corrected for adsorption of TPMP+ to the filters. Controls for non-specific binding of TPMP+ to the cells were done as stated for dihydrostreptomycin uptake measurements but with toluenized cells. This procedure showed that there was no substantial non-specific binding of TPMP+ to the cells. Autostimulation of dihydrostreptomycin uptake 3497 0 14t 2 4 6 8 10 Time (min) Fig. 1. Effect of various inhibitors on the uptake of dihydrostreptomycin by B. subtilis 60015. Cells were grown in complex medium at 37 "C to an OD5,* of about 0.2 and pre-incubated for 3 min in the presence of 10 pg actinomycin ml-l u),10 pg bacitracin mi-' (O), 10 pg DCCD ml-l a),8 pg puromycin ml-'(U) or for 5 min in the presence of lOOpg chloramphenicol ml-l (m). One culture received no inhibitor and served as a control (a).[3H]Dihpdrostreptomycin was added at time zero to give a final concentration of 5 pg ml-I (37 kBq ml-l); samples were withdrawn at the indicated times and accumulation of dihydrostreptomycin