Archeabacterial phototaxis

Martin Engelhard, Georg Schmies and Ansgar A. Wegener

Table of Contents

Abstract 2
1.1 Introduction 2
1.2 Two-component systems in Archea 6
1.2.1 Chemotaxis in H. salinarum 6
1.2.2 Phototaxis of Halobacteria 8
1.2.3 Receptor/transducer complexes 9
1.3 Properties of sensory rhodopsins and the receptor/transducer complex 10
1.3.1 Primary sequences 11
1.3.2 Absorption spectra 12
1.3.3 Photocycle of sensory rhodopsins 14
1.3.4 Proton transfer reactions of sensory rhodopsins 16
1.3.4.1 Receptors as proton pumps 16
1.3.4.2 Proton transfer reactions in the receptor/transducer complexes 19
1.3.5 Properties of the SR/Htr complex 20
1.4 Molecular mechanism of the signal transfer 22
1.4.1 The receptor-transducer interaction 22
1.4.2 Molecular mechanism of the signal transfer 24
1.5 Outlook 26
Acknowledgements 26
References 26

Abstract

Phototaxis in Archaea is regulated by the two receptors sensory rhodopsin I and sensory rhodopsin II which are closely related to the two ion pumps halorhodopsin (HR) and bacteriorhodopsin (BR). These seven helix membrane proteins are activated by light which induces an all-trans to 13-cis isomerisation of the retinal chromophore bound via a protonated Schiff base to helix G. The signal invoked by these reactions triggers structural changes in cognate halobacterial transducers of rhodopsin. The cytoplasmic domains of these membrane proteins are homologous to that of eubacterial chemoreceptors which activate proteins of the two-component signalling cascade. The similarities between the phototaxis machinery with the two-component signalling chain on the one hand and between the photoreceptors with the ion pumps BR and HR on the other direct the present review. The first part addresses the physiological response of the H. salinarum towards light and the underlying protein network. The next section focuses on the shared properties of receptors and ion pumps such as structural similarities and common principles of the light activated reactions. Finally, the molecular mechanism of signal transfer from the photoreceptor to the transducer is discussed.

1.1 Introduction

Bacteria and Archaea have survived the most dramatic environmental changes that have occurred since their first appearance, three billion years ago. They have occupied almost every ecological niche available, including extremes such as high temperatures at acidic or alkaline conditions. One reason for their endurance is their ability to respond adequately and precisely to environmental changes either genetically or by a locomotive answer. The information flow from the external input across the plasma membrane to the activation of the physiological signal is based on the so-called two-component signalling system that has been found in all three domains of life (for recent reviews on eukaryotic and prokaryotic two-component system see, e.g. This signalling pathway consists of sensors, which receive and transmit the external stimuli to cytoplasmic proteins, including both a histidine and an aspartate kinase (hence the name) which function as transmitter and receiver, the latter regulating the physiological response on the level of genes, proteins, or the cellular motor. The input signal can be quite diverse, ranging from magnetic fields, gravity, or osmolarity to chemicals, starvation, or photons, to name a few.

The two-component signalling cascade has been thoroughly investigated for the chemosensory system of Escherichia coli, Salmonella typhimurium, and related enteric bacteria. In recent years a similar signalling cascade from the archaeal Halobacterium salinarum has been discovered while analysing the mechanism of phototaxis. These archaea have been of particular interest since the discovery of bacteriorhodopsin (BR), the light-activated proton pump, in the early 1970s. The wealth of available information on the function and structure of BR has been reviewed (e.g.; see also a special issue of Biochem. Biophys. Acta,1460 (2000) with a comprehensive discussion of BR and related pigments). Various three-dimensional structures of the BR ground state and intermediates (reviewed in are now accessible and provide a basis for the understanding of the molecular mechanism of the light-activated proton transfer. Furthermore, this data is important in elucidating signal transduction as exemplified in the sensory rhodopsins.

During these investigations on BR three other retinylidene proteins were discovered. Halorhodopsin (HR), an ion pump like BR (both reviewed e.g. in and, was first described and named by Mukohata and co-workers. In subsequent work, HR has been recognised as an inward directed chloride pump and the amino acid sequence has been determined. Since 2000 the tertiary structure of HR has been available at 1.8 Å resolution. The other two pigments, sensory rhodopsin I (SRI) and sensory rhodopsin II (HsSRII), are responsible for the phototaxis of the bacteria and enable them to seek optimal light conditions for the functioning of the ion pumps HR and BR (SRI) and to avoid photo-oxidative stress (HsSRII) (Figure 1). The earliest report on the phototactic behaviour of H. salinarum was published in 1975 although the involvement of retinal proteins was only recognised in subsequent work. At about the same time it was demonstrated that methylation of membrane proteins is involved in the photosensory and chemosensory behaviour of H. salinarum which suggested that a sensory pathway similar to that in E. coli exists.

Research into halobacterial photosensing made a decisive step forward when Spudich and Spudich isolated HR-deficient mutants. These so-called flux mutants were obtained by exciting HR in cells in which a small proton leak had been introduced with a protonophore. The method selects for mutants which escape cytoplasmic acidification. In such a way isolated mutants lacking BR as well as HR were used for phototaxis studies. The photo-sensory behaviour of these bacteria was unimpaired, demonstrating that neither BR nor HR are involved in phototaxis (however, see below for more recent experiments on BR as photosensor). The authors identified a retinal-containing protein absorbing between 580 and 590 nm. It was named 'slow rhodopsin-like pigment' (later renamed as sensory rhodopsin I; SRI) because of its photocycle turnover of 800 ms, in contrast to that of about 10 ms for BR or HR.

On light excitation SR forms, in analogy to the BR-photocycle, a long-lived intermediate with a fine-structured absorption band with a maximum at 373 nm. This species is also photoactive and has been correlated with the photophobic response of H. salinarum. The notion that the same photoreceptor is responsible for both the repellent as well as the attractant responses has been further elaborated by the same authors. The observations were summarised in a mechanism of colour sensing mediated by a single receptor (SRI). The essence of the model is the discrimination between visible and UV light by one- and two-photon processes, respectively. The absorption of a photon (λ > 500 nm) by SRI triggers the photocycle, which results in the activation of the attractant signal transduction chain. However, in the presence of both visible and UV light the long-lived intermediate (S373) is excited and the repellent signalling cascade is turned on. This proposal of Spudich and Bogomolni was confirmed in later work and is now the accepted explanation for the colour discrimination of H. salinarum.

During further work on the halobacterial phototaxis, another repellent pigment was identified, named phoborhodopsin (pR) or sensory rhodopsin II (HsSRII). HsSRII covers the blue-green region of the spectrum. Its photocycle, like that of SRI, is quite slow and also contains, similar to BR, an M-like intermediate. Contrary to SRI, this pigment induces in H. salinarum only a single answer to light, i.e. a photophobic response. The four archaeal rhodopsins detected in H. salinarum are depicted in Figure 1. The corresponding amino acid sequences are shown in Figure 2.

The amino acid sequences of the two sensory rhodopsins have been determined. Additionally, the primary structures of HsSRII from the archaeal species Natronobacterium pharaonis (NpSRII) and Haloarcula vallismortis are available. The amino acid sequences of the SRs reveal considerable homologies to those of BR and HR. Positions defining the retinal binding site are usually identical; however, the site which in BR is intimately involved in the proton uptake from the cytoplasm (Asp96) is changed to an aromatic amino acid. It has been proposed that this change interferes with an efficient reprotonation of the Schiff base. Indeed, the NpSRII mutant F86D displays an unperturbed M-decay, although the overall turnover is unchanged.

The sequence determinations revealed upstream of the sopI and sopII loci open reading frames correspond to the halobacterial transducer of rhodopsin (Htr). Both sop and their corresponding Htr genes are under the control of the same promoter. Sequence alignments with the chemotactic receptors from enteric bacteria revealed considerable homologies, especially in regions important for signalling and adaptation processes. This observation connects the well-known two-component system with the signalling chain in phototaxis, combining our knowledge of the two separate fields of archaeal phototaxis and eubacterial signal transduction.

1.2 Two-component systems in Archaea

1.2.1 Chemotaxis in H. salinarum

The rod-like H. salinarum are up to 6 pm long and 0.5 pm in diameter (see Figure 1). 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 halobacteria 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. 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. Recently, the genome of Halobacterium species NRC-1 has been sequenced. In this project, at least 17 homologous methyl-accepting taxis transducers (halobacterial transducer proteins, Htps) 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. 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. For example, the membrane-bound transducer HtrVIII is an oxygen sensor and involved in the aerotaxis of the cells. An interesting cytoplasmic arginine sensor has been shown to be physiologically coupled to an arginine:ornithine antiporter. Further evidence was provided that branched chain amino acids like leucine or valine are sensed by BasT. It should be noted that the phototactic transducer HtrII 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.

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. 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. 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 which is bound in an unknown fashion to the signalling domain of the transducer. CheA phosphorylates 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. 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.).

A second sensing system, triggered by the activated transducer, has been discovered that relies on fumarate. 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.

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

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. 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, as well as HsSRII which enables the bacteria to seek the dark when the oxygen supply is plentiful, thus avoiding photooxidative stress.

The phototactic behaviour of halobacteria has been studied by visual tracking of single cells through a microscope (e.g.) or subsequently by computer tracking and motion analysis techniques (e.g.). In the 1980s, Stoeckenius and co-workers developed a rapid population method to determine action spectra which the authors successfully used for identifying sensory rhodopsin II. The first paper to describe negative phototaxis other than the SRI-mediated blue light repellent response was published by Takahashi et al., 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. Other groups also described a fourth rhodopsin in H. salinarum.