Activation and inhibition of ATP synthesis in cell envelope vesicles of Halobacterium halobium

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.     

Phosphate transport in Halobacterium halobium depends on cellular ATP levels.

Freshly harvested Halobacterium halobium cells grown in the presence of 0.5 mM Pi took up phosphate with a low apparent Km. Import depended on intracellular ATP levels; sodium and proton (electro)chemical gradients alone were not competent to drive Pi uptake. Although most of the phosphate accumulated as Pi in the cells, efflux of Pi was difficult to achieve.     

Genetic variability in Halobacterium halobium.

Halobacterium halobium exhibits an extraordinary degree of spontaneous variability. Mutants which are defective in the formation of gas vacuoles (vac) arise at a frequency of 10(-2). Other easily detectable phenotypes, like the synthesis of bacterioruberin (Rub) or the synthesis of retinal (Ret) and bacterio-opsin (Ops), the two components which form the purple membrane (Pum) of H. halobium, are lost at a frequency of about 10(-4). With the same frequency a mutant type appears which exhibits an extremely high variability in these phenotypes. With the exception of the ret mutants, all spontaneously arising mutants show alterations, i.e., insertions, rearrangements, or deletions, in the plasmid pHH1. It appears that the introduction of one insertion into pHH1 triggers further insertions, which makes the identification of relationships between phenotypic and genotypic alterations rather difficult. From the analysis of a large number of spontaneous vac mutants and their vac+ revertants it can be concluded that the formation of the gas vacuoles is determined or controlled by plasmid genes. No such conclusion is yet possible for the rub mutants, although all mutants of this type so far analyzed exhibit a defined insertion. pum mutants which have lost the capability of forming bacterio-opsin carry insertions in the plasmid which are distributed over a rather large region of the plasmid. No strains of H. halobium could be obtained which had lost plasmid pHH1 completely.     

Bacteriorhodopsin-mediated photophosphorylation in Halobacterium halobium.

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 +/- 5 photons per ATP molecule synthesized. According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions.     

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.     

Dynamic plasmid populations in Halobacterium halobium.

Deletion events occurring in the major 150-kilobase-pair (kb) plasmid pHH1 of the archaebacterium Halobacterium halobium were investigated. We found four deletion derivatives of pHH1 in gas-vacuole-negative mutants, two of which (pHH23) [65 kb] and pHH4 [36 kb]) we analyzed. Both plasmids incurred more than one deletion, leading to the fusion of noncontiguous pHH1 sequences. pHH23 and pHH4 overlapped by only 4 kb of DNA sequence. A DNA fragment derived from this region was used to monitor the production of further deletion variants of pHH4. A total of 25 single colonies were characterized, 23 of which contained various smaller pHH4 derivatives. Of the 25 colonies investigated, 2 had lost pHH4 entirely and contained only large (greater than or equal to 100-kb) minor covalently closed circular DNAs. One colony contained the 17-kb deletion derivative pHH6 without any residual pHH4. The sizes of the pHH4 deletion derivatives, produced during the development of a single colony, ranged from 5 to 20 kb. In five colonies, pHH4 was altered by the integration of an additional insertion element. These insertions, as well as copies of the various insertion elements already present in pHH4, presumably serve as hot spots for recombination events which result in deletions. A second enrichment procedure led to the identification of colonies containing either a 16-kb (pHH7) or a 5-kb (pHH8) deletion derivative of pHH4 as the major plasmid. pHH8, the smallest plasmid found, contained the 4 kb of unique DNA sequence shared by pHH23 and pHH4, as well as some flanking pHH4 sequences. This result indicates that the 4-kb region contains the necessary sequences for plasmid maintenance and replication.     

ATP synthesis in cell envelope vesicles of Halobacterium halobium driven by membrane potential and/or base-acid transition

Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about -100 mV relative to the external medium, i.e., inside-negative. ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units. The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about -100 mV. Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N,N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 microM/2 mg protein/ml. These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.     

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Methyl-accepting taxis proteins in Halobacterium halobium.

Methyl-accepting taxis proteins were identified and characterized in Halobacterium halobium, an archaebacterial species that is both chemotactic and phototactic. The data suggest direct involvement of methylation and demethylation in mechanisms of both chemotaxis and phototaxis and identify adaptation as the sensory process in which those reactions are likely to be involved. Analysis by electrophoresis and fluorography revealed methyl-accepting species, of apparent Mr between 90,000 and 135,000, that exhibited characteristics of sensory components. Those methyl-3H-labeled species were absent in a mutant blocked in taxis. Methylation of specific bands increased after positive chemostimuli and decreased after negative stimuli. Other methyl-3H-labeled bands, from 17 to 29 kd, exhibited features of biosynthetic intermediates, not of sensory components. Assay of rates of demethylation by measuring release of volatile forms of radiolabeled methyl groups revealed transient changes following chemo- or photostimuli that persisted for periods roughly equivalent to adaptation times. Negative chemostimuli induced increased rates of demethylation, as expected from fluorographic analysis, but positive chemostimuli also resulted in an increase. Photostimuli of either sign were followed by increases in rates of demethylation of shorter duration and lesser magnitude than chemostimuli-induced increases, a relationship that corresponded to differences in adaptation time.     

Primary and secondary chloride transport in Halobacterium halobium.

Chloride uptake in intact cells of Halobacterium halobium was characterized by rates of influx and efflux of 36Cl- under conditions of light, respiration, or both. Halobacterial mutant strains with and without retinal transport proteins allowed study of the effects of halorhodopsin and bacteriorhodopsin under illumination. Two structurally independent chloride transport systems could be distinguished: halorhodopsin, the already known light-driven chloride pump, and a newly described secondary uptake system, which was energized by respiration or by light via bacteriorhodopsin.     

Integration of photosensory signals in Halobacterium halobium.

Stimulation of Halobacterium halobium through its sensory photosystems, PS 370 and PS 565, leads either to a prolonged or to a shortened interval between two reversals of the swimming direction of the cell, the attractant or repellent response. Stimuli are integrated to yield the same response regardless through which photosystem they are given. Simultaneously elicited attractant and repellent signals cancel each other at any time during a reversal interval, even in the period of refractoriness shortly after a reversal, when the cell is insensitive to repellent stimuli. Successively applied stimuli are less completely integrated. The net response depends on the moment of stimulation during the interval, on the sequence of stimuli, and on the delay between them. Integration of successively applied effective stimuli (after refractoriness) is to a great extent explained in terms of a cellular oscillator (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985) which is changed in opposite directions by attractant and repellent signals. Some conclusions on the shape of the oscillator after its disturbance by a stimulus can be made. Integration of signals during refractoriness leads us to postulate an additional step before the oscillator in the sensory pathway. Cancelling of simultaneous opposite signals is thought to proceed at this integrator. It also takes part in the integration of successively evoked signals. At this step signals rapidly decline within 10 ms, and the total life time (at least of repellent signals) does not exceed 1.2 s.     

Photosensory retinal pigments in Halobacterium halobium

In Halobacterium halobium, nicotine is known to block the synthesis of retinal. Cells grown in the presence of nicotine do not show any photophobic response. Addition of retinal₁ or retinal₂ restored the photophobic responses to light-increase in the UV and to light-decrease in the green-yellow part of the spectrum. The action spectra of the two retinal₂-photosystems were red-shifted by 15–20 nm, compared with the corresponding retinal₁ systems. We conclude that each of the two photosystems, PS 370 and PS 565, has its own photosensory pigment with retinal as the chromophoric group.     

An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium

The mechanism by which bacteriorhodopsin (BR) transports protons across the cell membrane of Halobacterium halobium is actively studied in many laboratories. Currently available systems for the synthesis of mutant proteins obtained by site-directed mutagenesis of the gene encoding BR (bop) require reconstitution of the denatured polypeptide after its synthesis Escherichia coli or yeast; this approach is technically difficult and labor intensive, and raises questions about possible differences between in vivo and in vitro folding. Using a newly described transformation system and a halobacterial plasmid vector, we show that it is possible to reintroduce the bop gene into BR- strains of H. halobium. The bop-carrying plasmid expresses native BR in amounts similar to those obtained in several wild type strains. This system allows facile site-directed mutagenesis in halophilic archaebacteria.     

Excitation signal processing times in Halobacterium halobium phototaxis.

Phototaxis responses of Halobacterium halobium were monitored with a computerized cell-tracking system coupled to an electronic shutter controlling delivery of photostimuli. Automated analysis of rates of change in direction and linear speeds provided detection of swimming reversals with 67 ms resolution, permitting measurement of distinct phases of the responses to attractant and repellent stimuli. After stimulation, there was a latency period in which the population reversal frequency was unchanged, followed by an excitation phase in which reversal frequency increased, and a slower adaptation phase in which reversal frequency returned to its prestimulus value. A step-decrease in illumination of the attractant receptor slow-cycling or sensory rhodopsin (SR) (lambda max, 587 nm) was interpreted by the cells as an unfavorable stimulus and, after a minimum latency of 0.70 +/- 0.14 s, induced swimming reversals with the peak response occurring 1.34 +/- 0.07 s after onset of the stimulus. Two distinct repellent responses in the near UV/blue were observed. One was a reversal response to 400 nm light, which was dependent on orange-red background illumination as expected for the photointermediate repellent form of SR (lambda max, 373 nm). The minimum latency of this response was approximately the same as that of the SR attractant system. The second was a reversal response with shorter minimum latency (0.40 +/- 0.07 s) to light of longer wavelength (450 nm) than absorbed by the known SR repellent form. This result confirms recent findings of an additional repellent photosystem in this spectral range. Further, the longer wavelength repellent response is independent of orange-red background illumination, indicating that the photoreceptor mediating this response is not a photointermediate of SR.     

Light energy conservation processes in Halobacterium halobium cells.

In Halobacterium halobium, proton pumping driven by light or by respiration generates an electrochemical potential difference across the membrane. Energy storage in this form is only transient. Cellular energy transducers competing with proton leaks stabilize this free energy as high energy phosphate bonds, electrochemical potential of other ions, and chemical potential of amino acids and possibly other chemical species. The pH changes induced by light or by respiration in cell suspensions are complicated by proton flows associated with the functioning of the cellular energy transducers. Dominant is the proton inflow coupled to the synthesis of ATP, which has been kinetically resolved. A proton-per-ATP ratio of about 3 is calculated from simultaneous measurements of photophosphorylation and the proton inflow. This value is compatible with the chemiosmotic coupling hypothesis. The time course of the light-induced changes in membrane potential indicates that light-driven pumping increases a dark preexisting potential of about 130 mV only by a small amount (20-30 mV). The complex kinetic features of the membrane potential changes do not closely follow those of the pH changes, indicating that flows of ions other than protons are involved. A qualitative model consistent with the available data is presented. A salient feature of this model is a sudden relaxation of the protonmotive force by a proton inflow through the ATPase when the preexisting protonmotive force is increased by light or respiration and reaches a critical value. The trigger could be either the proton-motive force, the pH gradient, or possibly the internal pH.     

Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.