The P(r) to P(fr) transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Delta2 is accompanied by net acidification in unbuffered solution. The extent of this net photoreversible proton release was measured with a conventional pH electrode and increased from less than 0.1 proton released per P(fr) formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Delta2) H(+)/P(fr) at pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transient absorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constants of approximately 4 and approximately 20 ms that were also observed in parallel measurements of the photocycle (tau(3) and tau(4)). The number of transiently released protons per P(fr) formed is about one. This H(+) release phase is followed by a proton uptake phase of a smaller amplitude that has a time constant of approximately 270 ms (tau(5)) and is synchronous with the formation of P(fr). The acidification observed in the P(r) to P(fr) transition with pH electrodes is the net effect of these two sequential protonation changes. Flash-induced transient absorption measurements were carried out with Cph1 and Cph1Delta2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (tau(1)-tau(5): 5 and 300 micros and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B(1), B(3), and B(5)) showed significant pH dependence. Measurements of the P(r)/P(fr) photoequilibrium indicated that it is pH independent in the range of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested that the phycocyanobilin chromophore deprotonates at alkaline pH in both P(r) and P(fr) with an approximate pK(a) of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both P(r) and P(fr). The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pK(a) changes in the protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome.

Various methods of manipulation of the intracellular pH in Streptococcus lactis result in a unique relationship between the rate of glutamate and glutamine transport and the cytoplasmic pH. The initial rate of glutamate uptake by S. lactis cells increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4. A further increase of the cytoplasmic pH to 8.0 was without effect on transport. The different levels of inhibition of glutamate and glutamine transport at various external pH values by uncouplers and ionophores, which dissipate the proton motive force, can be explained by the effects exerted on the intracellular pH. The dependence of glutamate transport on the accumulation of potassium ions in potassium-ifiled and -depleted cells is caused by the regulation of intracellular pH by potassium movement.

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We determined the wavelength dependence of the specific growth rate of Rhodopseudomonas sphaeroides (the action spectrum of growth). A half-maximal (light-limited) growth rate was obtained when the culture vessel was illuminated with photon intensities between 0.8 x 1014 and 3.5 x 1014 photons cm-2 S-1 in the wavelength region between 400 and 950 nm. In the action spectrum, measured at 1.25 x 1014 photons cm-2 s-l, distinct peaks could be observed at 480, 580, 800, and 870 nm, and minima could be found at 420, 540, 640 to 730, 830, and 940 nm. Both the pigment content and pigment composition of R. sphaeroides varied, depending on the wavelengths of the actinic light used for growth. This demonstrates that chromatic adaptation occurs in this bacterium.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (A1) only. At pH 7.9 the PMF was composed of a high A* of -98 mV and was partially compensated by a reversed pH gradient (ApH) of +37 mV. ATPase According to the chemiosmotic hypothesis, the proton motive force (PMF) across the cytoplasmic membrane plays a key role in biological energy transduction. The PMF is the sum of the transmembrane electrical potential (A*) and the transmembrane pH gradient (ApH) (22). The phototrophic bacterium Rhodobacter sphaeroides can grow either aerobically in the dark or anaerobically in the light. In the absence of electron transfer R. sphaeroides maintains a significant A4s. The origin of this anaerobic-dark potential is unknown.Despite the presence of this anaerobic-dark potential in R. sphaeroides, no PMF-driven transport occurs. However, upon initiation of linear or cyclic electron transfer, PMFdriven solute transport takes place that is accompanied by a depolarization of the A+. These and other observations indicated that the electron transfer chain not only functions as a PMF-generating system but also plays a direct role in the regulation of secondary transport systems (7,8

A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 μM plastocyanin included), using 100 nM ferredoxin:NADP+ reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP+ photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP+, and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233−16241], do not significantly influence the rate of linear electron transport to NADP+. Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 ,uM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 ,uM and maximal rates of choline uptake (Vm..) of 5.4 and 4.2 nmol of choline/min mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCi, betaine transport occurred with a K. of 15.1 ,uM and a Vmax of 3.2 nmol/min mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 ,uM and 9.2 nmol of betaine/min -mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline or betaine to the growth medium did not result in the induction of additional transport systems.Adaptation of bacteria to fluctuations in the osmolarity of their surroundings is crucial for their survival. In order to grow, cells must maintain positive turgor, which is an outward pressure resulting from a cytoplasmic osmolarity which exceeds that of the extracellular milieu (9, 13). In Escherichia coli osmoregulation and -adaptation have been studied in detail (9, 12). When E. coli is exposed to a sudden increase in external osmolarity, dehydration of the cells occurs, which results in an inhibition of growth (2, 19). The bacteria can respond, however, to this osmotic shock by accumulating osmotically active solutes and resume growth when the internal osmotic pressure is appropriately raised (9, 13). The primary response to an increase in osmolarity is the uptake of K+ ions via the constitutive Trk system, the osmotically regulated Kdp system, or both (6). Although turgor pressure can be restored, growth rates usually remain low because of the inhibition of intracellular enzymes by the high cytoplasmic ionic strength (6, 9). If possible, E. coli therefore accumulates so-called compatible solutes to replace K+. A compatible solute is functionally defined as a compound...

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986 Extensive studies with Rhodobacter sphaeroides suggested that the activities of secondary solute transport systems depend directly on the rate of electron transfer (8, 9, 11). Also, transport of lactose in R. sphaeroides L39, which is equipped with the lactose carrier from Escherichia coli, was found to be strictly dependent on linear or cyclic electron transfer, and under anaerobic-dark conditions in the presence of a large A/, no uptake of lactose was observed (10). In a previous paper, we have shown that this anaerobicdark potential in R. sphaeroides is largely counterbalanced by a reversed pH (inside acidic) and therefore the proton motive force (PMF) is low (1). with Na+ via the major proline permease (PP-1, putP) in E. coli and Salmonella typhimurium is stimulated by K+ in cells preloaded with Na+ (6,33). This system is apparently inhibited by intracellular Na+, and stimulation of proline transport by K+ is best explained by K+-Na+ exchange which restores the Na+ gradient necessary for high rates of proline transport. Also, phosphate bond-driven glutamateglutamine and phosphate transport in Lactococcus lactis depends on the accumulation of K+ ions. This is caused by regulation of the intracellular pH by K+ movement. Glutamate-glutamine and phosphate transport is strictly regulated by the intracellular pH, with pKa values of 7.0 and 7.3, respectively (28,29).Lactose transport can be studied in membrane vesicles from R. sphaeroides 4P1 grown aerobically in the dark, in which a PMF is generated by electron transfer in the linear electron transfer chain (2). Under these conditions, L-alanine is not taken up by the membrane vesicles, which suggests that the transport mechanism of L-alanine is different from the H+/lactose symport mechanism. To determine the actual mechanism of L-alanine transport, the properties of the lactose and L-alanine transport systems were compared in cells under various assay conditions. Lactose uptake could be driven by artificial gradients under anaerobic conditions in the dark, indicating that the lactose carrier was active under these conditions. The mechanism of L-alanine transport appeared to be totally different. Transport of the nonmetabolizable L-alanine analog 2-a-aminoisobutyric acid (AIB) was found to be unidirectional and shock sensitive. L-Alanine-binding activity was detected in concentrated 5148 JOURNAL