Twocomponent systems in Archaea

1.2.1 Chemotaxis in H. salinarum

The rod-like H. salinarum are up to 6 ^m long and 0.5 ¬°im in diameter (see Figure 1) [42]. The bacterium is propelled by polarly inserted motor-driven right-handed helical flagella. The forward swimming direction is reversed by switching the motor from a clockwise to a counter clockwise rotation, which is under the control of cytoplasmic factors. The swimming pattern of halobac-teria without stimulus is like a random walk. Forward swimming periods are interrupted by a short stop and a reversal of direction. Angular changes are thereby caused by Brownian motion or by mechanical obstacles in the path of the cells.

The cells respond to various chemicals, e.g. arginine, leucine, or dipeptides such as Met-Val, as attractants and also to phenol, indole or benzoate as repellents [43,44], From more than eighty compounds tested six amino acids and seven peptides turned out to be attractants whereas three substances were shown to induce phobic responses [44], Recently, the genome of Halobacterium species NRC-1 has been sequenced [45]. In this project, at least 17 homologous methyl-accepting taxis transducers (halobacterial transducer proteins, Htps [46]) have been recognised whereas, for comparison, E. coli contains only 5 taxis receptors. Originally, in a screening with oligo nucleotides comprising consensus sequences of the signal domain of eubacterial methyl-accepting proteins 13 genes encoding Htps were identified [47]. The primary amino acid sequences clearly showed that the group of Htps includes not only transmembrane receptors but also soluble cytoplasmic proteins. In a couple of instances the proteins could be functionally assigned [37,41,44,48-50], For example, the membrane-bound transducer HtrVIII is an oxygen sensor and involved in the aerotaxis of the cells [51]. An interesting cytoplasmic arginine sensor has been shown to be physiologically coupled to an arginine:ornithine antiporter [44]. Further evidence was provided that branched chain amino acids like leucine or valine are sensed by BasT [50]. It should be noted that the phototactic transducer Htrll displays a dual function as a photophobic as well as a serine receptor. The latter property is conferred by an extracellular domain inserted between the two transmembrane helices [52].

The signal transduction chain consists of proteins of the two-component signalling chain. Genome analysis of Halobacterium species NRC-1 revealed the complete set of Bacillus subtilis che gene homologues with the exception of CheZ [45], The adaptor protein CheW, the histidine kinase CheA, the response regulators CheY, and CheB, as well as CheJ had been described earlier by Oesterhelt and Rudolph [53,54], The picture emerging from these data indicate a similar signal transduction chain to that described for enteric bacteria (see Figure 3). As in E. coli an adapter molecule (CheW) is attached to the cytoplasmic domain of the transducer. An attractant or repellent stimulus deactivates or activates the histidine kinase CheA [54] which is bound in an unknown fashion to the signalling domain of the transducer. CheA phosphorylates

Figure 3. A model of the two-component system in archaea with NpSRII as an example (the structures were taken from [87,124], not drawn to scale). Excitation of NpSRII by light activates the histidine kinase CheA which becomes phosphorylated. Subsequently, CheA transfers ~P onto the response regulators CheY or CheB. CheY~P is the switch factor for the flaggellar motor. Thereby the periods between reversals are shortened, resulting in a photophobic response of the bacteria. Fumarate was recognised as a second switch factor. If CheB is phosphorylated, CheB~P catalyses the demethylation of glutamic acid residues (small balls), thus enabling the cells to adapt to constant input of stimuli. An intrinsic phosphatase activity of CheY and CheB

deactivates the two proteins.

Figure 3. A model of the two-component system in archaea with NpSRII as an example (the structures were taken from [87,124], not drawn to scale). Excitation of NpSRII by light activates the histidine kinase CheA which becomes phosphorylated. Subsequently, CheA transfers ~P onto the response regulators CheY or CheB. CheY~P is the switch factor for the flaggellar motor. Thereby the periods between reversals are shortened, resulting in a photophobic response of the bacteria. Fumarate was recognised as a second switch factor. If CheB is phosphorylated, CheB~P catalyses the demethylation of glutamic acid residues (small balls), thus enabling the cells to adapt to constant input of stimuli. An intrinsic phosphatase activity of CheY and CheB

deactivates the two proteins.

CheY the switch factor of the flagellar motor, thereby reversing the rotational direction of the motor. Low concentrations of CheY~P prolong the swimming period between reversals. CheA can not only activate CheY but also CheB, which functions as a methylesterase of methylated Asp or Glu residues that flank the signalling domain. CheR, a constitutively active methyltransferase, re-methylates the carboxylates. Thereby, depending on the input of attractant and repellent impulses the methylation level is altered. These methylation/ demethylation reactions are involved in the adaptation of the bacteria to constant stimuli and have been studied in great detail for enteric bacteria (see Figure 3, for a model of the signal transduction chain). It should be noted that an overall chemo- and phototactic signal integration occurs in H. salinarum [25,55]. For more information on the two-component signalling cascade in enteric bacteria and the adaptation processes the reader is referred to recent reviews (e.g. [56,57]).

A second sensing system, triggered by the activated transducer, has been discovered that relies on fumarate [58,59]. This metabolite operates as a second messenger and acts together with CheY at the flagellar motor. This principle is not unique to archaea, it has also been demonstrated in eubacteria [60,61],

Several H. salinarum mutants, defective in taxis, displayed distinct pheno-types corresponding to a mutant missing photoreceptors, a mutant defective in CheR, and mutants on the level of the methylesterase and intracellular signalling [62,63]. Especially, the phototaxis mutant Pho81 proved to be useful for the homologous and heterologous expression of sensory rhodopsins (e.g. [64-67]).

1.2.2 Phototaxis of Halobacteria

A gradient of an external stimulus such as light alters the regular intervals of forward swimming, stopping, and backward swimming by prolonging or reducing the swimming period [22,25,68]. For example, cells swimming up a gradient of light (>500 nm) will increase the period between reversals. If they move against this gradient the interval between reversals will be shortened. Both behavioural responses result in a net movement towards the source of light. Gradients of light at wavelengths below 500 nm produce the opposite effect, consequently the bacteria move away from this kind of irradiation. The responsible pigments are SRI, which guides the halobacteria towards favourable light conditions and, in a two-photon process, away from UV-light [33], as well as HsSRII which enables the bacteria to seek the dark when the oxygen supply is plentiful, thus avoiding photooxidative stress [21].

The phototactic behaviour of halobacteria has been studied by visual tracking of single cells through a microscope (e.g. [22,69]) or subsequently by computer tracking and motion analysis techniques (e.g. [70]). In the 1980s, Stoeckenius and co-workers developed a rapid population method to determine action spectra [71] which the authors successfully used for identifying sensory rhodopsin II [35]. The first paper to describe negative phototaxis other than the SRI-mediated blue light repellent response was published by Takahashi et al. [72], who described a mutant that displayed only negative phototaxis with a maximum of the action spectrum at about 475 nm. In subsequent work the same group isolated the responsible pigment, which they named phoborhodopsin [34], Other groups also described a fourth rhodopsin in H. salinarum [73,74],

Once halobacteria reach areas of constant stimulus influx, the cells adapt to these conditions and resume spontaneous switching. The apparatus behind this adaptation process includes those proteins involved in the methylation and demethylation of Glu or Asp residues located in the cytoplasmic domain of the transducer. These reactions were first described by Schimz [28,29] who discovered that a stepwise increase of orange light results in the liberation of methanol, the product of the demethylation reaction. Conversely, a decrease in light intensity or an increase of UV-light reduces the methanol release. The author also provided evidence that membrane proteins are the target of the methylation reaction. In further work the reactions were analysed in more detail [43,75-77]. Perazzona and Spudich identified the methylation sites in Htrl and Htrll by mutagenic substitutions of Glu residues selected from consensus sequences [77]. Indeed, replacing the Glu265-Glu266 pair in Htrl and the homologous Glu513-Glu514 couple in Htrll by alanine eliminated the methylation of these transducers. The physiology of methylation/demethylation was further investigated by Marwan et al. who proposed a kinetic model of photosensory adaptation that relies on receptor deactivation [78]. It was suggested, in accordance with other publications, that a reversible methylation of Htrl - itself a membrane protein - is the chemical basis for the sensory adaptation.

Halobacterial colour sensing, such as the visual perception of higher eukaryots, can adjust to intensity changes in incoming light. The sensitivity of this process, however, is not yet known. Conversely, measurements of stimulus response curves revealed that H. salinarum can detect a single photon [79].

The cell synthesises sensory rhodopsin II constitutively. In contrast, the biosynthesis of SRI was shown - like BR and HR - to be induced by decreasing oxygen tension in a cell culture [73]. This repertoire of light-sensing pigments, which includes SRI with its dual function (photo-attractant response with maximum at 587 nm and photo-repellent answer at 373 nm) and the photophobic receptor HsSRII (Amax = 490 nm), enables the bacteria to seek, at low oxygen concentrations, optimal light conditions for the functioning of the two ion pumps BR and HR. With ample oxygen supply H. salinarum solely relies on oxidative phosphorylation. Possible photo-oxidative damage can be avoided because HsSRII with an absorption maximum matching that of sun light directs the cells towards the dark [21,80],

1.2.3 Receptor I transducer complexes

The incoming extracellular signal, which can be of either chemical or physical nature, has to reach the cytoplasm to activate the two-component system. The interface between the transmembrane signalling complex and the cellular chemotactic proteins is provided by the cytoplasmic domain of the receptors, and in the case of phototaxis by their cognate transducers. A comparison of the primary sequences of 29 proteins from 16 different species, which also included archaeal Htr's [38,41] (excluding Htrll, whose sequence had not yet been published [37]) revealed a consensus secondary structure consisting mostly of a-helices. A seven-residue repeat (a-b-c-d-e-f-g) with hydrophobic residues in positions a and d indicated a coiled/coil arrangement of the helices [81]. This domain structure was also recognised for Htrl using a sequence alignment and crosslinking studies of single Cys substitutions into selected sites of the membrane domain of Htrl [82], In this latter work it has also been shown that Htrl forms a dimer whose interface is sensitive to receptor photoactivation. A dimer structure of Htrll from the alkalophilic archaea N. pharaonis has been deduced from electron paramagnetic resonance (EPR) investigations [83,84]. The interaction of archaeal transducers with their cognate sensory rhodopsins has been analysed [64,85] and it could be proven that specificity is determined by their transmembrane helices [86].

Since the sequence homology between the archaeal transducers and the bacterial receptors leads to similar secondary structure predictions, the tertiary structure of the cytoplasmic domain of the serine chemotaxis receptor provided by Kim and co-workers [87] can also be taken as a model for the phototaxis transducers. By analogy one can deduce that the cytoplasmic part of a transducer dimer is a distinct four-helix bundle formed by the association of two helical hairpins. This rod extends about 200 A into the cytoplasm with three different functional sections recognisable (Figure 3). At the membranedistal end, a kinase-interaction region is responsible for the interaction with CheW and CheA. Approaching the cytoplasmic membrane a methylation region follows which is involved with the adaptation processes and probably binds CheB and CheR. A linker element connecting the methylation region with the transmembrane domain is, so far, structurally not very well characterised. An alignment of various linker sequences suggests two amphipathic helices [88]. A structural characterisation of this part of the transducer will certainly be the key to understanding the transmembrane signal transduction and activation of the cytoplasmic two-component system. The Htrs region might also harbour the recognition site for their cognate photoreceptors SRI and HsSRII [89-91],

The functionality of a complex between NpSRII with a truncated transducer devoid of most of its cytoplasmic domain is unimpaired, as shown in studies involving the binding of the transducer to the receptor using blue native gel electrophoresis and isothermal calorimetry experiments [83]. Additional information about the functionality of the complex comes from electrophysiological measurements [92], Previous work demonstrated that the innate capability of NpSRII to pump protons on light excitation is blocked by the binding of its cognate transducer [93,94], In his thesis Schmies has demonstrated that a truncated transducer consisting of the N-terminal amino acid sequence from 1 to 113 does indeed block the proton transfer in NpSRII, indicating a functional complex [92],

Chemoreceptors form heterogeneous clusters primarily at the poles of the bacterial cell [95], For the archaeal phototaxis transducers and chemoreceptors no such information is available. It would be important to know whether such complex structures are also established in Archaea and, if they are, whether the components are recruited from the chemotaxis as well as from the phototaxis branch.

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