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Photomovements of
Microorganisms:
An Introduction
Giovanni Checcucci
Istituto di Biofisica CNR
Antonella Sgarbossa
Istituto di Biofisica CNR
Francesco Lenci
Istituto di Biofisica CNR
120.1 Introduction................................................................. 120-1
120.2 Photobehavioral Responses of Microorganisms ....... 120-1
120.3 Photosensory Transduction Chains ........................... 120-4
Chromophores • Primary Reactions and Signaling
States • Photoreceptor Organelles • Dark Steps
120.4 Concluding Remarks ................................................... 120-8
120.1 Introduction
There is no need to emphasize that without light, life on our planet as we know it would not exist. Solar
radiation, in fact, is a fundamental source of energy for all photosynthetic organisms and microorganisms,
and photosynthesis is one of the most important biological processes on earth, which, by consuming
carbon dioxide and liberating oxygen, has made the world into the livable place we know today. Light is
also a sensory stimulus that provides vital information on the environment to all living beings, terrestrial
and aquatic, diurnal and nocturnal, prey and predators, to creatures provided with “eyes” and nervous
systems, as well as to aneural life forms like plants, fungi, and unicellular microorganisms, such as bacteria,
algae, and protozoa.
Many freely motile microorganisms are provided with a photoreceptor apparatus able to perceive the
quantity and the quality of light (propagation direction, fluence rate, spectral composition, polarization)
in the environment and to transform the absorption of a photon into a biophysical/biochemical signal
that can be recognized, elaborated, and transduced to the motor apparatus. Light constitutes, therefore,
an information signal that controls their movement and eventually brings the cells to accumulate into
settings in which the illumination conditions are best for their growth, survival, and development.1
In this chapter, we will focus our attention on the general features of photomovements of freely motile
microorganisms, referring to the chapters of other authors for deeper insight into some of the most
important case studies (see Chapters 116 and 121–124).
120.2 Photobehavioral Responses of Microorganisms
The main photobehavioral responses are, according to the terminology of Diehn et al.,2 photophobic
reactions, phototaxis, and photokinesis.
In photophobic responses, the sensory stimulus consists of a sudden change in light intensity, which
elicits a transient variation in the motor activity of the microorganisms. Step-up and step-down photophobic responses are caused by a step-wise increase or, respectively, decrease in photon flux. Usually,
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FIGURE 120.1 The step-up photophobic response exhibited by a cell (namely, in this case, the ciliate Blepharisma
japonicum) upon crossing a dark(gray)-light(white) border.
both photophobic responses consist of a brief cessation of forward movement (stop response) followed
by a random change of the direction of movement or, mainly in bacteria, by a variation of the frequency
of tumbling/movement inversion. The response pattern depends on the morphology of the microorganism and is independent of light direction. A photophobic response can typically last for several seconds
or even a few minutes, after which the microorganism can become adapted to the new illumination
conditions. When a swimming cell crosses a dark-light (light-dark) border, it can experience a sudden
change in light intensity that triggers a step-up (step-down) photophobic response. The final outcome
of these sensory reactions is an avoidance of the lighted (shady) region and accumulation in shaded
(lightened) areas (photodispersal or, respectively, photoaccumulation). Usually step-up photophobic
reactions occur with a time lag with respect to the stimulus application. This delay decreases with
increasing photon flux density and depends on the stimulating wavelength. Similarly, in the case of a
step-down photophobic response, the stimulus, now an abrupt decrease in light intensity, finally brings
the cells to escape from shadowed areas and to accumulate in lighted regions (photoaccumulation). In
Figure 120.1, a schematic reconstruction of a step-up photophobic response is presented.
Phototaxis results from the detection of the direction of light propagation and is a directional response.
It is defined as positive or negative according to whether the oriented movement is toward or away from
the source. Such a response implies the existence of a sophisticated photoreceptor apparatus. To perceive
the position of the light source, in fact, an asymmetry in the photoreceptor apparatus, which allows the
cell to sense the vectorial characteristics of the light signal, is required. This can be accomplished, for
instance, by means of a single photosensing unit coupled to a screening device that periodically shades
the photoreceptor proper.
In photokinesis, light intensity affects the absolute value of the microorganism velocity. If the velocity
increases (decreases) when the organism is exposed to light, photokinesis is positive (negative) and causes
photodispersal (photoaccumulation).
It is worth noting that the same organism may be able to exhibit more than one photoresponse, which,
in some cases, causes the same result (e.g., photodispersal induced by negative phototaxis and step-up
photophobic responses) and, in other cases, depends on the environmental illumination condition (e.g.,
positive or negative phototaxis elicited by low- or high-light intensities).
Consequently, photoaccumulation and photodispersal of cell populations can be the result of phobic,
kinetic, and tactic responses, and drawing conclusions on behavioral strategies can be difficult.
Reported in Figure 120.2 is a schematic block diagram of a typical experimental apparatus for measuring photomotile responses in microorganisms. Monitoring IR radiation, which is not perceived by
the microorganisms, allows the operator to observe the cells without “disturbing” them. To quantitatively
measure photoresponses, population methods and single-cell track (usually computer-assisted3) analyses
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Video recorder
Monitor
Computer
Monitoring light
Infrared
video camera
Infrared filters
Cut-off filter or
interference filter
Sample
Stimulating light
Anti-heat filter
Stimulating light
Microscope
Neutral density filter
FIGURE 120.2 A typical experimental apparatus for studying photobehavioral responses in microorganisms.
are currently used. Both have advantages and drawbacks, and here, we only want to point out that these
measurements deserve an accurate choice of meaningful parameters with which to reliably discriminate
among different reactions. As explained above, photodispersal (photoaccumulation) may result not only
from true negative (positive) phototaxis but also from a series of step-up (step-down) photophobic
reactions, as well as from positive (negative) photokinesis. A still useful, even if “old-fashioned,” experimental setup to discriminate between, for example, step-up photophobic responses and negative phototaxis, was used in the case of ciliate Stentor coeruleus4,5 and is reported in Figure 120.3.
Photomotile responses allow photosynthetic bacteria and microalgae to gather in environments in
which light is bright enough to efficiently drive the photosynthetic process but, at the same time, not
too intense to lead to photoinhibition or photobleaching. However, even for microorganisms that do not
harvest and convert light energy directly for their metabolism, like ciliates, for example, light can be an
environmental cue to accumulate into habitats that can be favorable for reproduction or propitious for
their prey and, in general, for food.6 In microorganisms containing endogenous photosensitizers, used
as defensive pigments against predators (e.g., Blepharisma japonicum and Stentor coeruleus, see Chapter
122), even relatively dim light can cause severe damage. Their ability to escape lighted spots is directly
linked to their survival.7
Some microorganisms, finally, have been shown to be able to perceive and transduce short-wavelength
ultraviolet radiation (UV-B = 280 – 315 nm). As in aquatic ecosystems and, in particular, in microorganisms, UV-B has been shown to impair photosynthetic activity, curb growth and metabolic rates,
damage DNA, spoil photo- and gravi-orientation in the water column, and hurt cell viability and motility
(see Chapter 116). This ability of cells to elaborate UV-B photons as environmental sensory stimuli can
directly lead the cells into sheltered areas, thus avoiding harmful UV irradiation.8,9
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Light intensity
Light direction
Step-up Photophobic Response
Negative Phototaxis
hv
hv
hv
hv
FIGURE 120.3 Cell distribution in the light field produced by a convex lens in the case of negative phototaxis and
step-up photophobic responses.
120.3 Photosensory Transduction Chains
In Figure 120.4, the block diagram of the photosensory transduction chain is reported: following absorption of a photon, the photosensing chromophore undergoes molecular modifications leading to the
formation of the signaling state that triggers the transduction chain, eventually acting on the motor
apparatus.
In what follows, we will go through the different blocks, trying to highlight the main and most
promising experimental approaches to the different problems. It is worth noting that because of their
intrinsic multidisciplinary character, most key problems encountered in these kinds of studies can best
be faced using different experimental techniques and methodologies.
To identify the chromophore responsible for the absorption of the photon triggering the whole
photosensory process, action spectroscopy10 is an unmatched, effective nondestructive technique (see
Chapter 115). If not affected by artifacts (screening and reflecting organelles, energy transfer processes,
multiple sensing chromophores, etc.) and not made unreliable by wrongly choosing the behavioral
parameters describing photoresponsiveness, the structure of an action spectrum is proportional to that
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Chromophere identification
Action spectroscopy
In vivo absorption and fluorescence microspectroscopy
Isolation of the photoreceptor
Selection of pigment lacking mutants
Photoreceptor localization
and characterization
Excited
chromophore
TEM and SEM
Confocal microscopy
In vivo absorption and fluorescence microspectroscopy
Biochemistry and molecular biology (genetic engineering)
Molecular
Pocket
Molecular
Pocket
Signaling state
Primary molecular events in the photosensing unit
The photosignalling state
In vivo and in vivo time-resolved optical spectroscopes
Time-resolved X-ray cristallography
Biochemistry and molecular biology
Studies on model systems
Transduction chain
Transduction
Chemical surgery with inhibitors, uncouplers
(warning: reliability, meaningfulness)
Electrophysiology
Selection of mutants
Photobehavioral response
Motor apparatus
Automatic cell tracking
(warning: choice and elaboration of motion parameters;
photoaxis: definition vs. measurement)
Operator-assisted analysis of single cell tracks
(warning: operator errors)
FIGURE 120.4 The photosensory transduction chain in photomotile microorganisms.
of the chromophore absorption spectrum. Action spectra, moreover, relate photoresponsiveness to the
wavelength of the stimulating light, thus directly linking the functional absorption of photons by the
presumed photoreceptor pigment to the observed physiological reaction. Even if it has some limitations
and can be painstakingly time-consuming, nonetheless, action spectroscopy provides direct information
about the photoreceptor pigment, is noninvasive, usually requires simple and nonexpensive instrumentation, and is one of the most, and once the only one, used techniques for studying photoresponses in
microorganisms.
Whenever it is possible to extract the chromophore, or better, to isolate the integral photoreceptor
unit, all chemical, biochemical, and physicochemical assays can be used to carefully characterize the
structural and functional properties of the chromophore. The chromophore, for instance, can be purified
by HPLC, and its molecular weight and structure can be determined by mass, IR, and NMR spectroscopy.11,12 Information on the apoprotein–chromophore complex, as another example, can be obtained
by means of column chromatographies, mono- and bidimensional gel electrophoresis, and enzymatic
assays. When reliable hypotheses are available on the chemical nature of the sensing chromophore, specific
inhibitors of its biosynthesis can advantageously be employed.13
Mutants lacking one or more photopigments can allow discrimination, which is responsible for the
photobehavioral reaction. In mutants with a photosensing unit deprived of the light-detecting chromophore (blind mutants), exogenous addition of chromophores restoring the photoresponse can bring
identification of the nature of the molecule with the role of perception and transduction of light stimuli.14
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Electron microscopy and in vivo absorption and emission microspectroscopy and confocal microscopy
allow detailed morphological information on the photoreceptor unit to be obtained and the spectroscopic
characteristics of the candidate sensing chromophores in their physiological molecular environment to
be determined. In vivo absorption and emission microspectroscopy and confocal microscopy also permit
the photosensing units to be localized within the cell and, in some cases, the maps of their spatial and
spectral distributions to be determined.
Time-resolved optical spectroscopies in vitro, x-ray crystallography, and microspectroscopies in vivo
can provide thorough information on the kind and the time-scale of primary molecular reactions
occurring in the photoreceptor unit, up to some tiny detail of molecular rearrangements following the
absorption of the photon.15
These studies on biological structures and substructures can profitably be complemented with studies
on model systems. Artificial models of natural photoreceptors have, in fact, been widely utilized, with
the aims of elucidating the molecular mechanisms of light-driven biological processes16 and of devising
synthetic tools able to mimic the performance of biological light detectors and transducers and suitable
for technological applications.17 The study of model photosensing and phototransducing systems not
only helps clarify the relationships between light-induced modifications in the photopigment and subsequent biophysical/biochemical signal transduction steps but can also yield impressive advancements
in natural pigments-based photonic devices for applications in holography, neural network optical
computing, and optical memories18,19 (see Chapters 134, 135, and 139).
Mutants deficient in one or more biochemical pathways, or engineered organisms, can supply data
for understanding the molecular machinery operating in the transduction process. Similar figures can
be achieved by means of chemical surgery with metabolic drugs, such as, for example, inhibitors and
uncouplers. Of course, any drug can severely affect not only the specific pathway presumed to be involved
in the sensory process but also many others, and cell viability in general, so that extreme care has to be
taken to avoid unreliable results and misleading conclusions.
Electrophysiological techniques, in particular, patch-clamp, may be difficult to use with microorganisms, the dimensions of which vary from a few hundreds to a few µm. They are the only techniques (with
the exception of some special fluorescent probes, like ion-specific fluorescent indicators), however, that
allow photoelectric effects, like light-induced membrane depolarizations, action potentials, and photic
receptor potentials, to be measured. Then, these data can be related to light-induced modification of the
motor (flagella and cilia) activity.20
Electrophysiological studies are also possible on intact cells, by performing photoelectric measurements
in cell suspensions.21
Chromophores
As photomotile microorganisms belong to almost every phyletic group that includes unicellular organisms, it is not unexpected that a multiplicity of chromophores with completely different molecular
structures and spectroscopic features can act as the prosthetic group of the photoreceptor. To whatever
class the organisms belong, in order to be able to detect light over a large range of the solar spectrum,
their chromophores usually have broad absorption bands with high molecular extinction coefficients. In
some cases, the spectral sensitivity can be widened, if different chromophores, absorbing in contiguous
spectral ranges, are spatially assembled in molecular frameworks to maximize the collective interaction
among them.
The presently known chromophores are 4-hydroxy-cinnamic acid (e.g., in Ectothiorhodospira halophila,
now reclassified as Halorhodospira halophila see Chapter 123), carotenoids (e.g., retinal in Halobacterium
salinarum, see Chapter 124), pterins and flavins (e.g., in Euglena gracilis, see Chapter 115), and dianthronic molecules (e.g., stentorin in S. coeruleus and blepharismins in B. japonicum, see Chapter 122).
These chemically different chromophores undergo different, early, light-induced molecular transformations: photoinduced charge transfer (flavins, stentorin, and blepharismins), cis-trans photoisomerization (retinals, 4-hydroxy-cinnamic acid), and energy transfer (pterins–flavins).
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Primary Reactions and Signaling States
A chromophore acts as a photosensing-phototransducing biological device, because it is not isolated but
rather embedded in and interacting with a molecular framework (an apoprotein, in most cases) that
“senses” the molecular modifications induced by light in the chromophore and, in its turn, gives rise to
the signaling state.22 The intermolecular interactions between the chromophore and its surrounding
microenvironment (its “molecular pocket”) can severely affect its photophysical and photochemical
properties, the probability of radiationless and radiative transitions from the first excited singlet and
triplet states, and the yields of energy and charge transfer processes from excited and metastable states.
Study of them can, therefore, lead to the formulation of hypotheses on the primary mechanisms operating
in the photosensing-phototransducing process.
Photoreceptor Organelles
A chromophore coupled to a transducing protein without any further “superstructure” is a simple
photosensory system seldom found in microorganisms. Instead, to efficiently harvest and transduce the
information carried by light, in most microorganisms, the photosensors are organized in ordered structures, with complexity that amazingly varies between prokaryotes and eukaryotes and within eukaryotes.
This wide variety of ordered structures is due not only to their different origins, generally regarded as
polyphyletic even within the same class, but also to their functional requirements. The photoreceptor
organelles must usually operate over a wide range of light incidence angles and intensities and discriminate between different wavelengths to reduce the environmental noise. Above all, it must match the cell
morphology and locomotion pattern, especially if detection of the direction of light is required. In
phototactic (able to perceive the direction of light) flagellated microalgae, the essential components of a
photoreceptor apparatus are the stigma (or eyespot) and the photoreceptor proper. The main role of the
stigma is, thanks to the helical movement of the cell, to periodically shade the photosensing structure,
so that the stigma–photosensor system constitutes a highly directional apparatus. The modulation of the
photic stimulus on the photosensor allows the cell to track the light source and correct its trajectory,
finally pointing toward (positive phototaxis) or away from (negative phototaxis) the light source.23
In ciliates, the capability of responding to directional light stimuli depends on the structural properties
of the photoreceptor (see Chapter 122), and different subcellular organelles have been suggested to be
decisive factors in photomovements of the cells: from pigment granules to stigmas, from watchglass
(Lieberkuehn) to composed crystalline organelles.24
In bacteria, finally, the photoreceptor molecules can be embedded into the cell membrane (as is the
case of sensory rhodopsins in H. salinarum, see Chapter 124) or can simply be located in the cytoplasm
(as is the case of PYP in H. halophila, see Chapter 123).
Dark Steps
As shown in the block diagram of Figure 120.4, the light stimulus is converted by the photoreceptor,
through its primary molecular reactions, into an intracellular signal that must travel up to the motor
apparatus in order to alter the motile behavior of the cell. The intracellular events of the signal transduction chain triggered but not directly driven by the photic stimulus are called “dark steps.” In general,
they encompass processes for signal amplification without adding noise and feedback mechanisms, in
order to extend dynamic ranges, improve accuracy, prevent overloads, and prevent signal reduction and
uncontrolled fluctuations.25
The large variety of photoreceptor systems of unicellular microorganisms entails a wide diversity of
possible mechanisms of signal transduction. Moreover, notwithstanding their apparent “simplicity,”
microorganisms show a significant richness and complexity of sensory pathways originated by the need
to balance multiple environmental signals (oxygen, carbon and nitrogen sources, pH, light, etc.), to move
toward or maintain themselves in the optimum environment for growth.
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Among bacteria, the halophilic archaebacterium H. salinarum has been thoroughly studied, and many
of the molecular processes at the basis of its phototransducing mechanisms were clarified (see Chapter
124). In this cell, the light stimulus is transduced through mechanisms similar to those discovered in
bacterial chemotaxis. The sensory rhodopsins I and II play their roles of photosensors, thanks to their
complexation with the corresponding transducer proteins, HtrI and HtrII, respectively, all of them
inserted into the membrane. The transducer proteins modulate the kinase activity that, in turn, controls,
through a cytoplasmic phosphoregulator, probably a fumarate molecule, the flagellar motor switching
which is placed in the membrane.26
In the case of the purple-sulfur bacterium H. halophila, the molecular mechanisms responsible for
signal generation were comprehensively studied and clarified (see Chapter 123), but the signal transduction pathway from PYP to the flagella still needs to be identified.
As far as microalgae are concerned, the structural components and the signaling cascade initiated by
the photoreceptor excitation have not yet been definitely ascertained (see Chapter 121). Electrophysiology
measurements can provide crucial complementary information on the light-initiated and light-dependent
processes in green flagellated algae. Unfortunately, algal cells do not usually exceed 10 to 20 µm in
diameter, which makes microelectrode recording problematic. In several cases, however, thanks to the
asymmetric localization of the signal sources within the cell, the photoinduced electrical signals involved
in phototaxis and photophobic response in green flagellates could be measured extracellularly from
individual cells and from cell suspensions.21
In C. reinhardtii, different lines of evidence point to the presence of heterotrimeric G-proteins for
coupling between the rhodopsin and the ion channels. In analogy to the invertebrate visual system, IP3
may be a good candidate for a messenger, mediating activation of the ion channels in the plasma
membrane.27
Recently, in the photoreceptor organelle of Euglena gracilis, a new type of blue-light receptor flavoprotein, photoactivated adenylyl cyclase (PAC) (see Chapters 115 and 121), was discovered and biochemically
characterized, indicating cAMP as the second messenger for step-up photophobic response of this green
alga. It constitutes a unique case of a protein performing two different functions simultaneously: photoreception and transduction (cAMP synthesis catalysis).28
Among ciliates, particularly interesting are the cases of B. japonicum and S. coeruleus, for the richness
of the data available and because of the difficulty in depicting a unitary framework. In fact, two main
hypotheses are, at present, available. The first hypothesis was that, following light stimulation, an intracellular increase of proton concentration could lead to the opening of calcium channels, indirectly by
depolarizing the membrane or directly by altering the conductance of specific calcium channels.29 More
recent results cast doubt on this simple model, suggesting the involvement of amplification and transduction steps similar to those operating in the visual process of metazoans. The fact that in B. japonicum
and S. coeruleus the light-induced ciliary beating stop occurs with a delay up to 1 sec (significantly long
in comparison with the millisecond lag-time of mechanoresponses in the same organisms) has been
considered indicative of specific time-limiting biochemical processes involving G-proteins and second
messengers as cGMP or IP3.30–32 Among the most important questions to clarify in this framework is the
mechanism of G-protein activation in B. japonicum and S. coeruleus. The presently available experimental
results bring us to hypothesize that the G-protein could be activated by the intracellular pH variation
following the release of protons from the photoreceptor, but a direct interaction between the excited
photoreceptor and the G-protein cannot be discarded, even though its nature is currently unknown (see
Chapter 122). It is, finally, worth noting that a substantial number of data allow us to set up a satisfactory
picture of the electrophysiological basis of photosensory transduction in B. japonicum and S. coeruleus.20
120.4 Concluding Remarks
This introductory chapter should provide basic knowledge for better understanding and enjoying the
following chapters devoted to the photobehaviors of some characteristic unicells. In those chapters,
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further details about the richness of the chromophore molecular structures and pockets and of the “dark
steps” involved in microorganism photosensory perception and transduction will be presented.
In nature, of course, light is not the only environmental signal affecting the behavior of microorganisms, and their motile responses result from the integration of “internal needs” (such as, for instance,
metabolism and cell cycle) with a diversity of external stimuli (chemical, mechanical, gravitational, photic,
etc.). Integrated investigations of their motile responses to different environmental signals (chemical and
photic, for example) might, therefore, offer clues for a deeper understanding of sensory processes in
microorganisms.
Finally, we would like to retake a general consideration on the lack of an unitary description of the
mechanisms at the basis of photomovements of microorganisms.33 At present, it looks like (as it does for
almost every single unicell) an ad hoc model holds: specific photosensing chromophores embedded in
and interacting with particular molecular pockets, distinctive signaling states triggering unique and
different molecular cascades. The question then becomes, is the cause of such a multiplicity of interpretations a consequence of our low level of knowledge and understanding or is it an intrinsic feature of
these natural phenomena?
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