Bioscience Reports, Vol. 17, No. 1, 1997
REVIEW
Mechanisms of Oxygen Taxis in Bacteria
Ruslan N. Grishanin1 and Sergei I. Bibikov2-3
Received November 1, 1996
Since 1881 when Englemann reported aerotaxis in bacteria, an understanding of the molecular nature
of the signal transduction remains a daring goal for microbiologists. This short review discusses known
facts and recent advances in the field including the discovery of the flavoprotein receptor which drives
Escherichia coli towards oxygen. Possible mechanisms of oxygen sensing in various bacterial species
are considered in connection with the existing, often fragmental, data on phototaxis, redox taxis and
taxis repellent effect of the reactive oxygen species (ROS).
KEY WORDS: Aerotaxis; phototaxis; taxis reppelent effect; signal transduction; bacteria; reactive
oxygen species; E. coli; H. Salinarium.
INTRODUCTION
Bacteria swim by rotating their semirigid helical flagella and using rotary motor,
which is driven by the electrochemical gradient of protons across the cytoplasmic
membrane (A/IH*)- They change swimming direction by change of the direction
of flagellar rotation, that causes reversals (Spirilla, Halobacteria) or brief
tumbling (peritrichiously flagellated bacteria, such as Escherichia coli or
Salmonella typhimurium). Some bacterial species change the direction by a
transient stop of the flagellar movement (Rhizobia, Rhodobacter). Motile bacteria
respond tactically to a range of chemical substances, temperature, pH, oxygen,
light. Bacteria measure gradients in a time scale, because they are too small to
sense a spatial gradient along their body length. An increase in an attractant
concentration and a decrease in a repellent concentration lows the probability of
the next direction-changing event, while a decrease in an attractant concentration
and increase in a repellent concentration cause the opposite effect, increasing the
pace of events. If there are no following changes in the environment then a
direction-changing frequency is adjusted to a prestimulus frequency. These
adjustments in time reflect an intrinsic resetting of the taxis system, that allows
bacteria to measure changes in effector concentration in a temporal manner. As a
On leave to Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
On leave to Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
3 To whom correspondence should be addressed.
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2
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0144-8463/97/0200-0077$12.50/0© 1997 Plenum Publishing Corporation
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Grishanin and Bibikov
result of such a mechanism bacteria alter their direction-changing frequency to a
bias of their overall movement towards an attractant or away from a repellent.
The most detailed model of chemotaxis was developed for E. coli. A special
group of receptors/transducers, methyl-accepting chemotaxis proteins (MCPs),
initially respond to a change in concentration of chemoeffectors and transmit the
signal across the membrane. They are transmembrane proteins, with extracellular
receptory domain and intracellular signalling domain. Cytoplasmic domains of
MCP interact with soluble proteins CheA and CheW. CheA is histidine
autokinase, while CheW plays the role of an adaptor interacting with MCP and
CheA. Excitation of the MCPs either directly by binding a chemoeffector
molecule like aspartate or serine, or indirectly by interacting with periplasmic
binding proteins, causes a change in the level of CheA autophosphorylation. An
attractant stimulus causes the decrease in autophosphorylation while a repellent
stimulus causes the opposite effect. The phosphate group from the histidine
residue of CheA is transferred to CheY. CheY is a relatively small cytoplasmic
protein which upon phosphorylation traverses the cytoplasm and binds to a switch
of the flagellar motor, causing the reversal of rotation. Decrease of the
phosphorylation level of CheY leads to supression of reversals. The level of CheY
phosphorylation is also regulated by protein CheZ with phosphatase activity
directed toward CheY-P. Another target for CheA kinase is methylesterase
CheB, which modifies MCPs by deamidation and demethylation of a certain
glutamic residues. Changes in activity of CheB, together with activity of
methyltransferase CheR regulates the level of MCP methylation, resetting the
transducer into a non-signalling conformation and providing the negative feedback for taxis signal, or adaptation. The organization of the system varies in
details in different species through the basic components are highly conserved [1].
TWO TYPES OF AEROTACTIC RECEPTORS
In spite of great progress in the study of the mechanism of bacterial
chemotaxis the mechanism of the oxygen reception was for a long time a matter
for wide speculations. The study of aerotaxis in bacteria has a very long history.
Aerotaxis has been described first by Engelmann, Beyerinck and Pfeffer in the
19th century as tightly related to the type of metabolism. It was demonstrated
that obligate aerobes had a strong positive aerotaxis, microaerophiles were
attracted by low oxygen concentration but repelled by high oxygen and
anaerobiosis, whereas obligate anaerobes were strictly repulsed by oxygen.
Facultative anaerobes E. coli and S. typhimurium in response to a gradient of
oxygen generated by respiration, accumulated close to the water/air interface
when grown aerobically but were repelled by very high oxygen concentration.
Growth under anaerobic conditions decreases the preferable concentration of
oxygen. The behavior of bacteria suggested that oxygen can function as both an
attractant and a repellent. This dual aerotactic behavior is not surprising. On one
hand oxygen supports highly efficient aerobic metabolism, but on the other hand
superoxide radicals generated during the respiration are highly toxic for cells.
Oxygen Taxis in Bacteria
79
Aerotaxis enables bacteria to move toward the concentrations of oxygen that are
optimal for respiration (for aerobes) while avoiding the harmful high concentrations of oxygen. Bacteria could find an optimal concentration of oxygen behaving
in respect to an integral signal from both positive and negative systems of the
oxygen reception. Putative receptors may have different affinity for oxygen, with
high affinity for positive and low affinity for negative receptor. Consistent to that
KQ.S (stimulus intensity producing 50% photoresponse) calculated for a positive
aerotactic response in E. coli was found to be in a micromolar range, whereas KO.S
for a negative response was 1 mM [2],
ROLE OF ELECTRON TRANSPORT IN POSITIVE AEROTAXIS
Data obtained primarily in Barry Taylor's Laboratory in Loma-Linda suggest
that the respiratory electron transport is essential for reception of oxygen in
positive aerotaxis in different bacteria. This fact outlines a singificant difference
between aerotaxis and the other chemotactic responses where the change of the
occupancy of the highly specialized receptors induces the signal. The data imply
that the terminal oxidases of the electron transport system, such as cytochrome o
and d in E. coli and S. typhimurium, and cytochrome aa3 in Bacillus cereus serve
as the primary receptors for oxygen in aerotaxis [3,4]. The inhibitory effect of
cyanide on aerotaxis is consistent with this implication. Moreover, an addition of
cyanide was shown to induce a repellent response in S. typhimurium, possibly
mimicking the effect of a decrease in concentration of oxygen (both are resulted
in a decrease in respiratory electron transport) [5]. The inhibition of the
respiration in B. cereus by 2-heptyl-4-hydroxyquinoline N-oxide which blocks
electron transport at cytochrome b but not at terminal oxidase aa3 also
suppressed the aerotactic response [4]. The alternative electron acceptors for
electron transport system, like nitrate, fumarate for anaerobic E. coli, DMSO and
TMAO for anaerobic Rb. sphaeroides were shown to attract bacteria [6,7]. The
involvement of photosynthetic electron transport in phototaxis of purple nonsulphur bacteria also suggests the coupling of the electron transport to taxis [8].
IDEA OF PROTON-MOTIVE FORCE SENSING
Bacteria void of all the receptors tend to move randomly but accumulate in
the regions where the speed of the cells is low. Because the speed of the cells is
proportional to the level of A/I H », bacteria would accumulate in the regions
where pmf is low. Thus a mechanism should exist to prevent this potentially fatal
situation. In 1980 Khan and Macnab described the loss of the reversal ability in £.
coli at low energy levels [9] and this mechanism was confirmed for several other
species [10]. The existence of the A/IH--receptor or protometer as a possible
solution for aerotaxis was suggested in many publictions [6, 11, 12]. By measuring
the A^H, generated as a result of respiration the protometer should be able to
drive the cell directly to the regions where the oxygen is available from the
80
Grishanin and Bibikov
anaerobic regions where the speed of pmf-generation is low. Though the
mechanisms underlying the protometer and electron transport reception hypothesis look pretty similar, there is an important detail which is used to distinguish
between those mechanisms. According to protometer hypothesis putative receptor should be able to measure both components of the proton-motive force—
electric potential (AW) and pH difference on the membrane (ApH). The difficulty
is the mutual interdependence of electron flow through the respiratory chain and
proton flow across the membrane.
It is known that the proton ionophores collapsing A/Z H - induce the repellent
response in some bacteria [12, 13, 14]. The direct evidence that the change in
A/ZH+ may induce behavioral response was obtained in Halobacterium salinarium. The transformation of the blind strain devoid of all retinal proteins with a
plasmid encoding light-driven proton pump bacteriorhodopsin restored a photosensitivity to the mutant [10]. The response however was mediated by the
light-driven changes in AW rather than ApH [15]. The respiratory chain remained
intact in this mutant and we cannot exclude the possibility that generation of
A/ZH+ by bacteriorhodopsin was, in fact, stimulating the reverse electron flow
through the respiratory chain, which was actually sensed. The data on the
aerotactic and phototactic behavior of Rhodobacter sphaeroides also suggested
that the responses were directly coupled to respiratory or photosynthetic electron
transport, rather than mediated by A£ H » sensing [7,8]. It is interesting to note
that the coupling of one of the transcriptional regulators to the respiratory system
has been recently reported. FnrL protein with an Fe-S cluster is controlled by
activity of the ebb-type cytochrome oxidase system [16].
DISCOVERY OF THE AEROTACTIC RECEPTOR IN E. coli
Progress in the genome sequencing lead to the identification of a new open
reading frame in E. coli, which exhibited several hallmarks of an aerosensing
function. New gene dubbed aer (for aerotaxis) had sequence homologies to the
conserved part of the signal transducer MCPs and to a group of proteins like
NifL, FixL and others, involved in triggering the regulatory responses to a change
in the oxygen levels. The in-frame deletion of the gene [17] as well as the
insertional deletion [Rebbapragada et al, personal communication] resulted in
the loss of the positive aerotaxis responses. Moreover, as shown in [17] the loss of
the positive aerotaxis produced a strong air-avoidance response, with cells
accumulated in the center of the drop of a medium surrounded by air. The loss of
the function in the deletion mutants was complemented by the plasmids carrying
the aer gene in a dose-dependent manner. When Aer protein was overexpressed
the response to air gradients was even stronger than in the wild type in full
accordance with the idea of existence of two oppositely tuned oxygen receptors.
Though experiments with air bubbles and soft agar plates strongly show that Aer
protein constitute the major receptor for positive aerotaxis, its interaction with
the respiratory chain components and oxygen remains to be discovered.
As was shown recently, Aer protein contains the non-covalently connected
Oxygen Taxis in Bacteria
81
prostetic group which was identified in [17] as flavinadenine dinucleotide (FAD).
The presence of FAD produces immediate constrains on what the possible
interaction of the protein may be with the respiratory transport components.
The Aer protein could detect oxygen directly if a formation of a flavin
hydroperoxide complex takes place like in flavoprotein oxygenases and bacterial
luciferases. However, it seems really unlikely at the moment as the earlier
experiments identified the terminal oxidases as primary receptors. Therefore we
assume that FAD is used for interaction with the respiratory chain components
and upon reduction/oxidation induces a conformational change in the Aer
protein which activates or deactivates respectively the CheA kinase.
Possible link between the FAD reduction/oxidation and conformational
change may be the presence of three cysteine residues in Aer protein. Cysteins
are rarely presnet in MCPs and Aer has two in the transmembrane part of the
protein and one in the cytoplasmic part. The distance between the transmembrane cysteins is 50 amino acid residues or exactly 5 turns of the
transmembrane a-helix. Oxidation of the cystein residues may lead to the
formation of the cystein bridges between the homologous proteins and formation
of the active dimers. Studies of the Aer protein will provide information about
signalling which is especially valuable because of the current difficulties with the
crystallization of the MCPs.
POSSIBLE MECHANISM OF SIGNALLING IN AEROTAXIS
The pathways for aerotaxis and chemotaxis in Entherobacteria converge at
the level of CheA kinase [18]. An intriguing discovery was the independence of
the aerotactic response in E. coli from the methylation/demethylation processes
controlled by enzymes CheR an CheB [2]. The structure of the Aer protein may
provide some clues for this. It appears that methylation domains contain several
glutamines in place of alternate glutamine-glutamate pairs in other receptors
which are normally used by methylesterase CheB as recognition sites. As a result
the glutamines never become deamidated and cannot be used by methylase CheR
for methylation. The Aer protein seems to be locked in the "fully methylated"
neutral form and is incapable of adaptation by means of methylation/
demethylation. It is quite possible that due to the labile form of the signalling
state in the redox receptor, adaptation mechanism was deliberately switched off
by evolution giving place to a mechanism which relies completely on the level of
electron flow through the redox chain and never resets.
It is also possible that the recently discovered property of protein phosphatase CheZ to change the phosphatase activity via CheY-P-mediated polymerization of CheZ provides an additional loop for negative regulation and adaptation
[19]. In contrast to oxygen response in Entherobacteria, aerotaxis in Bacillus
subtilis and H. salinarium seemed to depend on the methyl group turnover [20,
21]. The difference in the adaptation mechanisms for the aerotaxis in different
species reflects a different functional organization of the taxis systems, which is
apparent despite the homology of the components [22, 23].
Grishanin and Bibikov
82
Another unusual finding was the discovery of the inverted positive aerotactic
response in a mutant with deleted methylation/demethylation system ACheR
ACheB. It was shown [2] that additional mutation removing tse gene complements
the defect and restores normal aerotaxis. The mechanism by which the nonmodified Tsr (receptor for serine and repellents in E. coli) can invert the positive
aerotactic response is unknown. The negative aerotactic response was not
modified in that case.
NEGATIVE RESPONSE TO OXYGEN
The repellent response to oxygen is one of many ways of protection from
dangerously high oxygen concentration. There is a hypothesis that the repellent
response can be mediated by the reception of some oxygen by-product. The
hypothesis is backed by the data that strong tumbling response to blue light can
be induced in E. coli mutants accumulating protoporphyrin IX due to the
photoinduced production of reactive oxygen species on the protoporphyrin [24].
Repulsion of E. coli by H2O2 was demonstrated with quite low (1 ^iM)
concentration of H2O2. Also E. coli was shown to be repelled by OC1~ and
N-chlorotaurine, which together with H2O2 are produced by stimulated phagocytic leucocytes in the respiratory burst response. The response to these species
was methylation-independent like the repellent response to high concentration of
oxygen, suggesting the same mechanism [25]. The reception of ROS could play
the role in the switching of aerotaxis in Kb. sphaeroides from positive to negative
under the light: photoexcited components of light harvesting complex and
bacteriochlorophillis of photosynthetic reaction centers can provide a generation
of ROS under aerobiosis. However, there is an alternative explanation implying
that bacteria swim to a prefered redox potential zone and the repellent response
to oxygen may be mediated by a redox reception. In the gradients of
redox-mediators like TMPD (Axospirillum brasiliense) or substituted quinones
(E. coli) bacteria form sharply defined bands resembling those of aerotactic bands
[26,27].
DsrA—the protein from the anaerobic bacterium Desulfovibrio vulgaris
bearing homology to MCPs from other bacteria is believed to be the oxygen
repellent receptor. It contains the c-type heme and it was suggested that the
oxidation of the heme generates a repellent signal [28].
CONCLUSION
In spite of 100 years of history, the studies on oxygen taxis in bacteria are still
in progress and the mechanism of aerotaxis is not known yet. Recent discovery of
the sensory flavoprotein in E. coli may provide the necessary instrument for
future studies. It appears that redox interaction may be part of the signal
transduction pathway from oxygen to flagella and can be considered as an
interesting link of the systems of energy and signal transduction.
Oxygen Taxis in Bacteria
83
REFERENCES
l.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Eisenback, M. (1996) Mol. Microbiol. 20:903-910.
Shioi, J., Dand, C. V. and Taylor, B. L. (1987) J. Bacterial. 169:3118-3123.
Shioi, J., Tribhuwan, R. C, Berg, S. T. and Taylor, B. L. (1988) J. Bacterial. 170:5507-5511.
Laszlo, D. J., Niwano, M., Goral, W. W. and Taylor, B. L. (1984) J. Bacteriol. 159:820-824.
Shioi, J. and Taylor, B. L. (1984) J. Biol. Ckem. 259:10938-10988.
Taylor, B. L. (1983) Trends Biochem. Sci. 8:438-441.
Gauden, D. E. and Armitage, J. P. (1995) J. Bacteriol. 177:5853-5859.
Grishanin, R. N., Gauden, D. E. and Armitage, J. P. (1997) J. Bacteriol. 179:24-30.
Khan, S. and Macnab, R. (1980) J. Mol. Biol. 138:563-597.
Bibikov, S. I., Grishanin, R. N., Kaulen, A. D., Marwan, W., Oesterhelt, D. and Skulachev, V. P.
(1993) Proc. Natl. Acad. Sci. USA. 90:9446-9450.
Glagolev, A. (1980)7. Theor. Biology. 82:171-185.
Miller, J. B. and Koshland, D. E. Jr. (1977) Proc. Natl. Acad. Sci. USA. 74:4752-4756.
Sherman, M. Yu., Timkina, E. O. and Glagolev, A. N. (1981) FEMS Microbiol. Lett. 12:121-124.
Barishev, V. A., Glagolev, A. N. and Skulachev, V. P. (1981) Nature. 292:338-340.
Grishanin, R. N., Bibikov, S. I., Altschuler, I. M., Kaulen, A. D., Armitage, J. P. and Skulachev,
V. P. (1996) 7. Bacterial. 178:3008-3014.
Zeistlra-Ryalls, J. H. and Kaplan, S. (1996) J. Bacterial. 174:985-993.
Bibikov, S. 1., Brian, R., Rudd, K. E. and Parkinson, J. S. (1996) (in preparation).
Rowsell, E. H., Smith, J. M., Wolfe, A. and Taylor, B. L. (1995) J. Bacterial. 177:6011-6014.
Blat, Y. and Eisenbach, M. (19%) J. Biol. Chem. 271:1226-1231.
Wong, L. S., Johnson, M. S., Zhulin, I. B. and Taylor, B. L. (1995) /. Bacterial. 177:3985-3991.
Lindbeck, J. C., Goulboume, E. A., Johnson, M. S., Taylor, B. L. (1995) Microbiology.
141:2945-2953.
Rosario, M. M., Kirby, J. L., Bochar, D. A. and Ordal, G. W. (1995) Biochemistry. 34:3823-3831.
Rudolph, J. and Oesterchelt, D. (1995) EMBO J., 14:667-673.
Yang, H., Inokuchi, M. and Adler, J. (1995) Proc. Natl. Acad. Sci. USA., 92:9332-9336.
Benov, L., Fedorovich, I. (1996) Proc. Natl. Acad. Sci. USA. 93:4999-5002.
Grishanin, R. N., Chalmina, I. I. and Zhulin, 1. B. (1991) J. Gen. Microbiol., 137:2781-2785.
Bespalov, V., Zhulin, I. B. and Taylor, B. L. (1996) Proc. Natl. Acad. Sci. USA (in press).
Fu, R., Wall, J. D. and Voordouw, G. (1994) J. Bacteroi, 176:344-350.