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Antonievan Leeuwenhoek 66: 151-164, 1994.
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151
Photosynthetic electron transport and anaerobic metabolism in purple
non-sulfur phototrophic bacteria
Alastair G. McEwan
Department of Microbiology, The University of Queensland, Brisbane QLD. 4072, Australia
Key words: purple non-sulfur bacteria, Rhodobacter, photosynthesis, CO2 fixation, anaerobic respiration, gene
expression
Abstract
Purple non-sulfur phototrophic bacteria, exemplifed by Rhodobacter capsulatus and Rhodobacter sphaeroides,
exhibit a remarkable versatility in their anaerobic metabolism. In these bacteria the photosynthetic apparatus,
enzymes involved in CO2 fixation and pathways of anaerobic respiration are all induced upon a reduction in
oxygen tension. Recently, there have been significant advances in the understanding of molecular properties of the
photosynthetic apparatus and the control of the expression of genes involved in photosynthesis and CO2 fixation.
In addition, anaerobic respiratory pathways have been characterised and their interaction with photosynthetic
electron transport has been described. This review will survey these advances and will discuss the ways in which
photosynthetic electron transport and oxidation-reduction processes are integrated during photoautotrophic and
photoheterotrophic growth.
1. Introduction
The anoxygenic phototrophic bacteria are a diverse
collection of microorganisms which carry out
bacteriochlorophyll-dependent photosynthesis as a
common metabolic process (Nitschke & Rutherford
1991). There are two major groups of anoxygenic phototrophs: the phototrophic green bacteria (Chlorobiaceae and Chloroflexaceae) (Olson et al. 1988) and
the phototrophic purple bacteria which can be further
divided into purple sulfur bacteria (Chromatiaceae and
Ectothiorhodospiraceae) and purple non-sulfur bacteria (Drews & Imhoff 1991). The purple non-sulfur bacteria exhibit a remarkable range of morphological, biochemical and metabolic properties (Imhoff et al. 1984).
They are also diverse taxonomically with genera being
placed in the c~ and /3 subgroups of Proteobacteria
(Stackebrandt et al. 1988). This review will focus on
two species of purple non-sulfur bacteria: Rhodobacter capsulatus and Rhodobacter sphaeroides. Although
these two species do not exhibit all of the nutritional properties of other purple non-sulfur phototrophs
they exhibit metabolic capabilities which are broad-
ly representative. Indeed, R. capsulatus has been
described as the most versatile of prokaryotes (Madigan & Gest 1979) since it can grow photoautotrophically, chemoautotrophically, photoheterotrophically,
and chemoheterotrophically with a variety of electron
acceptors as well as by fermentation of sugars. Confronted with this diversity of metabolic processes it is
not possible in this review to be comprehensive and so
the focus will be photosynthesis and anaerobic respiration. For recent progress in the area of nitrogen fixation
in phototrophic bacteria the reader is referred to papers
by Schneider et al. (1991), Preker et al. (1992) and
Wang G e t al. (1993).
2. Phototrophic metabolism
2.10rganisation and development of the
photosynthetic apparatus
Most purple non-sulfur bacteria are facultative anaerobes and under oxic conditions are able to grow
chemotrophically with oxygen as electron acceptor.
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R. capsulatus, for example, possesses a branched aerobic respiratory chain which is typical of many nonenteric Gram negative bacteria (La Monica & Marrs
1976). However, lowering the oxygen tension below a
species-specific threshold value triggers the formation
of photosynthetic pigments and pigment-binding proteins (Drews & Oelze 1981; Kiley & Kaplan 1988).
The pigment-protein complexes are incorporated into
the cytoplasmic membrane (CM) and through the process of invagination a specialised membrane system
is formed; the intracytoplasmic membrane (ICM). The
ICM is contiguous with the CM but appears to be functionally distinct since it is the site of light-harvesting
and photosynthetic electron transfer. Formation of the
ICM results in remarkable morphological changes in
the cell membrane structure. The ICM of Rhodobacter
and Rhodospirillum are vesicular structures (known as
'chromatophores') but a variety of membrane structures are found in other genera (Imhoffet al. 1984).
In R. sphaeroides and R. capsulatus three distinct
pigment-protein complexes are involved in the transduction of light energy into chemical energy. Two types
of light harvesting (LH) complexes absorb light and
transfer excitation energy to the photochemical reaction centre (RC) which is the site of primary photochemistry and electron transport (Drews 1986; Zuber
1986). LHI and LHII are also known as B875 and
B800-850 antenna complexes, respectively, in order
to identify the wavelength of maximum absorption
of the Bchl pigment but note that this wavelength,
and hence the designation, is often different in other genera. The B875 antenna is composed of two small
membrane-spanning polypeptides ~ and/3 (Mr 50006000) which bind two molecules of Bchl and one or
two carotenoid molecules. The B800-850 antenna is
made up of two subunits c~ (Mr = 7000) and/3 (Mr =
5000) which, like the B875 polypeptides, have a single
central membrane-spanning c~-helix. They bind two or
three molecules of Bchl and one carotenoid.
The RC of Rhodobacter is composed of three subunits H, M and L and has a total Mr of about 100,000
(Drews 1985). In many species, including Rhodopseudomas viridis, a fourth cytochrome subunit is present.
The L and M subunits are integral membrane proteins
and they bind four Bchl molecules, two bacteriophaeophytin molecules, two quinones and one ferrous iron
atom. The pathway and kinetics of electron transfer
in the RC is now understood at the molecular level
as a consequence of spectroscopic studies (reviewed
by Jackson 1988) and the determination of the crystal
structure of the RC from Rps. viridis (Deisenhofer et al.
1985; Deisenhofer & Michel 1989) and R. sphaeroides
(Allen et al. 1987).
Molecular genetic analysis of purple non-sulfur
bacteria has also advanced considerably over the last
decade (Scolnik & Marrs 1987; Donohue & Kaplan
1991). Most of the genes involved in the formation of
the LH and RC pigment-protein complexes are encoded by a 46 kb gene cluster on the chromosome of R.
capsulatus (Taylor et al. 1983) and a similar but not
identical situation exists in R. sphaeroides (Wu et al.
1991). The puf operon is composed of genes encoding LH1 polypeptides (pu3'B, pufA), RC-L and RC-M
(pujS~, pufiVl) and open reading frames pufQ and pu3"X
(Bauer et al. 1988). There is some genetic evidence
that PufQ is involved in bacteriochlorophyll biosynthesis (Bauer & Marrs 1988) while PufX appears to
be required for the correct insertion of the RC into the
ICM (Farchaus et al. 1992). The pufA gene encoding
RC-H is located 39 kb away from the pufoperon in R.
capsulatus (Zsebo & Hearst 1984). The puc operon is
composed of genes encoding LHII polypeptides (pucB,
pucA) (Youvan & Ismail 1985; Kiley & Kaplan 1987)
as well as genes involved in regulation and assembly of LHII (pucCDE) (Lee et al. 1989; Tichy et al.
1991). The organization of the bch and crt genes which
are involved in pigment biosynthesis is now also well
understood. Indeed, the nucleotide sequence of the
entire photosynthetic gene cluster ofR. capsulatus has
been completed (Burke et al. 1991).
In Rhodobacter the core component of the photosynthetic apparatus is an RC surrounded by LHI antennae in a ratio of 1 RC to 20-30 molecules of LHI Bchl
and this photosynthetic unit is constant under different
light regimes. The RC-LHI core complexes are surrounded by LHII antenna complexes and the level of
LHII is inversely related to the light intensity (Aagaard
& Sistrom 1972). However, although light intensity has
a key role in the regulation of the size of the photosynthetic apparatus (Drews & Oelze 1981; Kiley & Kaplan
1988) it has been known for several decades that oxygen is the major controlling factor in the development
of the photosynthetic apparatus (Cohen-Bazire et al.
1957). Upon a decrease in oxygen tension, biosynthesis of Bchl and pigment binding proteins is strictly
coordinated (Chory et al. 1984). Levels ofpuc and puf
mRNA are increased 10-20 fold when oxygen tension
is reduced. Some bch and crt mRNAs may also be elevated although in general they seem to be regulated by
oxygen to a lesser extent.
Regulation of the puf and puc operons involves
both transcriptional and post-transcriptional control.
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Analysis of puf and puc promoters has been carried
out in detail and cis-acting regulatory elements have
been identified (reviewed by Klug 1993a). However,
the situation regarding trans-acting regulators is less
clear. Sganga & Bauer (1992) have identified a gene
regA which encodes a trans-acting regulator responsible for activating expression of puf, puh and puc
operons in response to a decrease in oxygen tension
but is not involved in the oxygen-controlled expression
of nif genes. RegA has extensive sequence homology
in its amino terminal sequence with two component
response regulators (Stock et al. 1989). It would be
expected that in common with other systems, a sensor kinase would phosphorylate RegA in response to
an environmental signal such as a change in oxygen
tension. The identity of the sensor kinase has not yet
been established but Bauer et al. (1993) have indicated that it is probably RegB since the regB gene
predicts a transmembrane protein and regB mutants
have the same phenotype as regA mutants. However,
since RegA does not appear to possess a DNA binding
region in its carboxy-terminal region it may not function directly in transcriptional control. Instead, Sganga
& Bauer (1992) have suggested that RegA may resemble smaller response regulators such as CheY, CheB
and SpoOF which act as intermediaries in more complex phosphoryl transfer regulatory cascades (Bouret et
al. 1991). The same authors have identified additional
loci associated with RegA in the control of photosynthetic gene expression by light and oxygen and exciting
progress in this area is anticipated. Of particular interest will be to find out whether the putative sensor kinase
RegB resembles FixL, the oxygen sensor kinase in Rhizobium meliloti, since the latter is an oxygen-binding
haemoprotein (Gilles-Gonzalez et al. 1991) or whether
it resembles ArcB, which is thought to sense oxygen
tension indirectly (Stock et al. 1989).
Additional factors are certainly important in the
regulation of photosynthetic gene expression. The first
is the 'superoperonal organisation' of genes in which
transcription of operons encoding crt and bch genes
continue into puf and puh operons (Wellington et al.
1992). It appears that this transcriptional organization is important for the coordination of photosynthetic gene expression. A second factor is differential
decay of mRNA as well as decreased mRNA stability of puf and puc transcripts in the presence of oxygen (reviewed by Klug 1993b). The importance of
differential decay of mRNA is most apparent for puf
gene expression where a ratio of 10-15 LHI : 1 RC
is achieved via greater stability ofpufBA mRNA corn-
pared to puj-'BALMX mRNA (Belasco et al. 1985; Klug
1991). Finally, an unresolved question is the nature
of the transcription factors involved in photosynthetic
gene expression. The nature of the transcription factor
involved in the RegA-mediated sensor kinase regulatory system is not established nor is the question of
whether acr factor other than ~r7° is required for expression ofpuf puh andpuc operons (see Bauer et al. 1993
for discussion).
2.2 Photosynthetic electron transport
The purple non-sulfur bacteria normally carry out
photosynthesis under anaerobic conditions. Photosynthetic electron transport is cyclic and involves two
multimeric transmembrane proteins: the RC and the
cytochrome bcl complex (Jackson 1988). Electron
transfer between these two redox complexes is mediated by the mobile electron carriers ubiquinone, located
in the hydrophobic domain of the cytoplasmic membrane, and a c-type cytochrome, usually located in the
periplasmic space in Rhodobacter. The RC catalyses
a light-driven ferrocytochrome c-ubiquinone oxidoreductase activity while the cytochrome bCl complex
catalyses a ubiquinol-ferricytochrome c oxidoreductase activity (Fig. 1). This cycle of electron transfer is
linked to the generation of a proton motive force (z~p)
across the cytoplasmic membrane (Jackson 1988). The
inner membrane of photosynthetic bacteria such as R.
sphaeroides is one of the best-understood energy transducing systems in biology as a result of the molecular
characterisation of the cytochrome bCl complex (Gennis et al. 1993) and the RC (Feher et al. 1989).
A major surprise in the apparently well-defined area
of cyclic electron transport has been the identification of alternative pathways of electron flow between
the cytochrome bcl comples and the oxidised bacteriochlorophyll dimer of the RC which is generated after a
photochemical event (Fig. 1). A large body of biochemical and spectroscopic data which had accumulated up
to the mid- 1980s indicated that in R. capsulatus and R.
sphaeroides cytochrome c2 mediated electron transfer
between cytochrome bcl and the RC. Cytochrome c2
is a periplasmic monohaemoprotein of MT = 13,000
whose interaction with the RC has been investigated
in great detail (Dutton & Prince 1978). It came as
a great surprise when it was found that cytochrome
c2-deficient mutants (cycA mutants) of R. capsulatus, constructed using reverse genetic techniques, were
able to grow phototrophically (Daldal et al. 1986). The
kinetics of electron transfer in these cycA mutants was
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R.
capsulatum
periplasm
/ ~ ~
cytoplasm
R.
sphaeroides
perip!asm
RC
> ua
>
cytoplas=
Fig. 1. Pathwaysof cyclicphotosyntheticelectron transferin R. capsulatus (upperpanel) and R. sphaeroides(lowerpanel). In R. capsulatus electrontransferbetweenthe RC and cytochromebc~ complex
is mediated by periplasmic cytochromec2 and membrane-bound
cytochrome cz which operate in parallel. In R. sphaeroides only
cytochrome c2 is utilised in wild-type cells but in spd mutants
iso-cytochromec2 can replacecytochromec2.
altered and the favoured interpretation was that direct
electron transfer between the cytochrome bcl complex
and the RC could take place (Prince et al. 1986; Prince
& Daldal 1987). This view was challenged by Jackson
and co:workers who identified, using spectroscopic
techniques, a novel high potential c-type cytochrome
(cytochrome cx) which was able to donate electrons to
the RC in a mutant which lacked both cytochrome c2
and a functional cytochrome bcl complex (Jones et al.
1990). Cytochrome c~ was shown to be a membranebound protein with a mid-point redox potential of +
360 mV (Jones et al. 1990). Further work by Jenney
& Daldal (1993) has identified a gene cycY, which
is required for phototrophic growth of cytochrome c 2
deficient mutants ofR. capsulatus. It seems likely that
the c-type cytochrome encoded by cycY is the same
as cytochrome c~ identified by Jones et al. (1990)
although this needs to be confirmed.
In R. sphaeroides a distinct but equally interesting
picture has emerged (Fig. 1). In contrast to the situation in R. capsulatus, cycA mutants of R. sphaeroides
were unable to grow phototrophically (Donohue et al.
1988). This result was in agreement with the 'classical' view of the role of cytochrome c2 in cyclic electron
transfer in Rhodobacter. However, cycA mutants were
found to be able to revert to photosynthetic competence
via mutation at a second site (Rott & Donohue 1990).
These revertants, known as spd (suppressor of photosynthetic deficiency) mutants were found to produce a
periplasmic isocytochrome c2 which had 44% amino
acid sequence identity with R. sphaeroides cytochrome
c2 (Rott et al. 1993).
It does not appear that a membrane-bound
cytochrome equivalent to cytochrome cx of R. capsulatus (Jones et al. 1990) is present in R. sphaeroides
because mutants of the latter species lacking both
cytochrome c2 and isocytochrome c2 were unable
to grow phototrophically (Rott et al. 1993). Isocytochrome c2 is normally present at only 1.0-2.5% of
the level of cytochrome c2 in wild-type phototrophic R.
sphaeroides (Rott et al. 1992) and its physiological role
is still unclear. However, it has been noted by Donohue
and co-workers that an open reading frame adjacent to
the isocytochrome c2 structural gene encodes a zinccontaining alcohol dehydrogenase (Rott et al. 1993).
This has led to the suggestion that expression of isocytochrome c2 may be linked to the metabolism of
alcohols in R. sphaeroides and preliminary evidence
indicates that levels of this cytochrome are elevated
during growth with ethanol (T.J. Donohue, personal
communication). This point will be discussed in Section 3.3.
2.3 Photosynthetic electron transport and
metabolism
The proton motive force (Ap) which is generated during photosynthetic electron transport is used to drive
ATP synthesis, solute transport, and certain redoxlinked reactions (Jackson 1988). However, photosynthetic electron transport is not a closed cycle since it
is in communication with a variety of primary dehydrogenases (Ferguson et al. 1987). This interaction
occurs predominantly at the level of the ubiquinone
155
pool (Q-pool) which is pivotal in connecting electron transport with metabolism during phototrophic growth. The diversity of carbon sources which
can be metabolised by purple non-sulfur bacteria has
been reviewed by Sojka (1978) and by Dutton &
Evans (1978). During photoheterotrophic growth with
dicarboxylic acids as carbon source the major dehydrogenases will be NADH dehydrogenase and succinate dehydrogenase. The ubiquinol generated during succinate oxidation can be oxidised by energylinked reverse electron flow to NAD +. This reaction
is catalysed by the rotenone-sensitive NADH dehydrogenase which catalyses NADH oxidation under aerobic conditions but under phototrophic conditions acts
as ubiquinol-NAD + oxidoreductase. The energetically unfavourable electron transfer from ubiquinol to
NAD + is linked to the consumption of Ap (Klemme
1969).
A variety of reductants which support photoautotrophic growth of Rhodobacter donate electrons directly into the cyclic electron transport pathway. Molecular hydrogen is oxidised by membranebound hydrogenase which functions as a hydrogenubiquinone oxidoreductase (Vignais et al. 1985).
Despite their name, most purple non-sulfur bacteria
are able to use inorganic sulfur molecules as electron
donors. The slow recognition of this capability probably arose because many purple non-sulfur bacteria
have a low tolerance towards sulfide (Hansen & van
Gemerden 1972). However, certain species such as
Rhodobacter sulfidophilus can tolerate up to 3 mM
sulfide which is comparable to the sulfide tolerance of
purple sulfur bacteria such as Chromatium vinosum
(Hansen & Veldkamp 1973). In R. capsulatus, R.
sphaeroides and Rhodospirillum species extracellular
elemental sulfur is the final oxidation product of sulfide oxidation (Hansen & van Gemerden 1972) but in
R. sulfidophilus and a number of other Rhodobacter
and Rhodopseudomonas species, sulfide is oxidised
completely to sulfate (reviewed by Brune 1989). R.
sulfidophilus is interesting because it appears to oxidize sulfide to sulfate without intermediate formation
of elemental sulfur, but instead accumulates sulfite
(Neutzling et al. 1985). Sulfite is oxidised to sulfate
only when sulfide has been exhausted. It seems likely
that the oxidation of sulfur molecules takes place in
the periplasm and involves the c-type cytochromes as
electron acceptors (Brune 1989). However, in R. sulfidophilus, although sulfide oxidation probably occurs
in the periplasm, the enzyme involved may operate as
a sulfide-ubiquinone oxidoreductase (Brune & Truper
1986).
Recently, it has been established that organic sulfur
can also be used as an electron donor in photosynthetic metabolism. Dimethylsulfide has been shown to
support photoautotrophic growth of the purple sulfur
bacterium Thiocapsa roseopersicina (Visscher & van
Gemerden 1991). A previous report had indicated that
a number of species of phototrophic bacteria might also
have this capability (Zeyer et al. 1987) and work in the
author's laboratory has shown that a new isolate of
R. sulfidophilus (strain SH1) is able to grow photoautotrophically with DMS as electron donor (Hanlon et
al. 1994). DMS oxidation was linked to the accumulation of dimethylsulfoxide (DMSO) indicating that
DMS was used purely as an electron donor. In further
work we have identified a periplasmic redox protein
which is able to catalyse electron transfer from DMS
to a variety of redox dyes. This enzyme, DMS: acceptor oxidoreductase, has been purified and has been
shown to contain a pterin molybdenum cofactor and a
cytochrome b562(Hanlon & McEwan, manuscript in
preparation). The physiological electron acceptor for
DMS: acceptor oxidoreductase has not been identified
but it could be ubiquinone or a periplasmic cytochrome
which in turn is able to donate electrons to the RC. The
latter case resembles the situation which must operate
for methylotrophic phototrophs such as Rhodopseudomonas acidophila during the operation of the dyelinked methanol dehydrogenase (Bamforth & Quayle
1978, 1979). Interestingly, R. sulfidophilus strain SH1
is also able to utilise methanol and methylamine as sole
carbon source (Hanlon et al. 1994).
The use of the oxidised bacteriochlorophyll dimer
as an electron sink during the anaerobic oxidation
of electron donors during phototrophic growth is
analogous to the use of cytochrome c oxidase by
chemotrophic bacteria under aerobic conditions. Some
electron donors which are used by chemoautotrophs
(Wood 1988), for example nitrite, have not yet been
shown to support photoautotrophic growth but it seems
likely that new electron donors in phototrophs will be
identified. In this context the recent report of photoantrophic growth of Rhodomicrobium-like isolates
with ferrous iron as electron source is interesting
(Widdel et al. 1993). The oxidation of ferrous iron
at neutral pH has previously only been reported in
chemoautotrophs such as Gallionellaferruginea (see
Wood 1988). During photoautotrophic growth, electrons donated into cyclic electron transport are used
to reduce NAD + via energy-linked reverse electron
156
flow. The NADH generated can be used in variety of
metabolic reactions including CO2 fixation.
2.4 CO,fixation
A major feature of the purple phototrophic bacteria
is their ability to grow photoautotrophically fixing
CO2 via the reductive pentose phosphate cycle (Calvin
cycle) (Tabita 1988). The molecular details of CO2 fixation are best understood in R. sphaeroides. This bacterium possesses two distinct forms of the key enzyme
of the Calvin cycle ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Form I RubisCO has an
Mr = 550,000 and it is composed of a larger catalytic subunit (Mr = 56,000) and a small subunit (Mr =
15,000). The quarternary structure of Form I RubisCO is probably L8S8 (Gibson & Tabita 1977). This
type of RubisCO appears to be widespread amongst
autotrophic bacteria (Tabita 1988) and it resembles the
enzyme in chloroplasts. A second form of RubisCO
which has been found in R. sphaeroides has an Mr =
360,000 (Gibson & Tabita 1985) and it is composed
of identical subunits Mr = 52,000 (Gibson & Tabita
1977). Both forms of RubisCO are present in R. capsulatus (Shiveley et al. 1984) but in general the Form
II enzyme appears not to be present in most species
of phototrophic bacteria (Tabita 1988). It appears that
the two forms of RubisCO are not closely related (Gibson & Tabita 1977, 1985). In R. sphaeroides the genes
encoding Form I RubisCO (cbbLz, cbbSi) and the gene
encoding Form II RubisCO (cbbMH) are components
of two distinct CO2 fixation operons (Gibson & Tabita
1988; Tabita et al. 1992). It is interesting that these
operons each contain structural genes for phosphoribulokinase, the other unique enzyme of the Calvin
cycle (Hallenbeck & Kaplan 1988; Gibson & Tabita
1988). However, the organisation of the two operons
is not identical. For example, cbbGi1 (encoding glyceraldehyde 3-phosphate dehydrogenase) is present on
the Form II CO2 fixation gene cluster only (Gibson &
Tabita 1988).
Expression of Calvin cycle genes is tightly controlled. During aerobic growth there is virtually no
expression of these genes (Jouanneau & Tabita 1986)
while under photosynthetic growth conditions, varying
degrees of expression of the Form I and II operons are
observed. In general, Form II enzyme predominates
during heterotrophic metabolism (Jouanneau & Tabita
1986; Falcone et al. 1988) while Form I enzyme is
predominant during autotrophic growth (Falcone et al.
1988) and is essential for CO2 fixation at low CO2 ten-
sion (Hallenbeck et al. 1990b). The Form I and Form II
gene clusters each constitute an operon (Gibson et al.
1990, 1991) but their regulation is not fully understood.
Although the two operons must be subject to independent regulation (Jouanneau & Tabita 1986) it is also
known that their expression must in some way be coordinated (Falcone et al. 1988; Hallenbeck et al. 1990a,
b). Recently, Gibson & Tabita (1993) have sequenced a
gene, cbbR, which is divergently transcribed upstream
of the Form I operon. This gene is essential for expression of Form ! genes and in a cbbR mutant, expression
of Form II genes was reduced to 30% of their wild-type
level. CbbR shows homology to members of the LysR
family of transcriptional regulatory proteins (Henikoff
et al. 1988) and it is proposed that it acts as an activator of Calvin cycle operon expression. A similar
situation has been described in Alcaligenes eutrophus
(Windhovel & Bowien 1991). The effector(s) which
activates CbbR has not yet been identified. Gibson &
Tabita (1993) have also indicated that in a cbbR mutant
expression of the Form II operon continued to be low
during aerobic growth. This may indicate that an as
yet unidentified transcriptional regulator is involved in
the regulation of Form II operon expression. It may
be the case that this putative regulator is a 'redox sensor', because in addition to oxygen, alternative electron
acceptors such as DMSO decrease the levels of Calvin
cycle enzymes during photoheterotrophic growth (Hallenbeck 1990a). Conversely, higher levels of Calvin
cycle enzymes are observed as carbon sources become
more reduced (Tabita 1988). Over the past decade it
has become recognized that the complex pattern of
CO2 fixation in Rhodobacter arises because this activity is important for photoautotrophic growth and photoheterotrophic growth.
Early investigations into the physiology of photoheterotrophic growth of purple non-sulphur phototrophs had speculated on the importance of electron
sinks for excess reducing power which might be generated during the oxidation of carbon substrates (Muller
1933). Specifically, CO2 fixation via the Calvin cycle
was suggested to have such a role (Lascelles 1960; Ferguson et al. 1987). Support for this view came from
Richardson et al. (1988) who observed that addition of
bicarbonate or alternative electron acceptors (discussed
in Section 3) to media was required for phototrophic
growth of R. capsulatus on the reduced carbon sources
propionate and butyrate but not for growth on more oxidised carbon sources such as malate or succinate. Studies of mutants ofR. sphaeroides have added to the view
that CO2 fixation has a role in maintaining redox bal-
157
2.5 Phototrophic growth and redox homeostasis
NAD*
"
~
~
Q pool ~-
~fumQrQl~
"~ ("
CO2 fixQtion
innte
THAOI OHSO
cyt.cz
h o ""-~ PeTo
~
N zO
Oz
Fig. 2. Interaction between cyclic photosynthetic electron transfer
and redox-balancing pathways in R. capsulatus. NADH generated
during oxidation" of organic carbon can be oxidised via the CO2
fixation or via respiration to a variety of electron acceptors which
branch from cyclic electron transfer at the level of the Q-pool or
cytochrome c2.
ance during photoheterotrophic growth. A double null
mutant in cbbFl and cbbFtz was constructed in which
expression of RubisCO was essentially abolished (Hallenbeck et al. 1990a). This double mutant was unable
to grow photoheterotrophically with succinate as carbon source unless an external electron acceptor such
as dimethylsulfoxide (DMSO) was added. Essentially the same result was obtained when both RubisCO
genes were deleted from R. sphaeroides (Falcone &
Tabita 1991). Taken together these data confirm that
the Calvin cycle has an important role in maintaining
intracellular redox balance during photoheterotrophic
growth even under conditions where addition of bicarbonate to the medium is not required. Tabita and coworkers have recently reported that a revertant of the
RubisCO double deletion mutant ofR. sphaeroides can
be isolated which is able to grow photoheterotrophically on malate but will still not fix CO2 via the Calvin
cycle (Wang X et al. 1993a). This mutant will not grow
photolithoautotrophically with H2 as electron donor
but will grow under these conditions with thiosulfate
as an electron source (Wang X et al. 1993b). These
data indicate that a hitherto unrecognised pathway of
CO2 fixation exists in R. sphaeroides which is distinct
from the Calvin cycle. The nature of this alternative
CO2 fixation pathway is still unclear although there
are several possibilities (see Wang X et al. 1993b for
discussion).
A feature of photoheterotrophy which distinguishes it from chemoheterotrophy is that during photoheterotrophic growth, catabolism of carbon molecules
is not of major importance for energy generation.
Instead, pathways of carbon metabolism are linked
solely to anaplerotic and biosynthetic pathways. The
TCA cycle during photoheterotrophic growth is incomplete since oxoglutarate dehydrogenase activity is
extremely low (Beatty & Gest 1981). An additional problem which has already been alluded to is the
dissipation of reducing power in the form of NADH
which is generated during carbon catabolism. Consider growth on malate; the oxidation of malate to
oxaloacetate has an unfavourable standard free energy and in order that this reaction should proceed in
the oxidative direction, NADH must be consumed.
One sink for NADH is the Calvin cycle described in
the preceding section (Fig. 2). Another consumer of
NADH is pyridine nucleotide transhydrogenase (H +THase). This enzyme catalyses hydride ion transfer
from NADH to NADP + in a reaction which consumes
Ap (Jackson 1991). Although the equilibrium constant
for this reaction is 1 the influence of the ~ p generated during photosynthetic electron transport means
that the cell maintains a high mass action ratio of
[NADPH] [NAD+]/[NADP +] [NADH]. This NADPH
can be used for biosyntheticpurposes while a high ratio
of NAD+/NADH is maintained in order to provide an
electron acceptor for catabolic redox reactions. Jackson and co-workers have found that the light-driven
H +-THase activity in chromatophores ofR. capsulatus
constitutes about 50% of the ATPase activity (Cotton &
Jackson 1988) and thus H+-THase may have a major
role in the redox and energy economy of the cell.
Intracellular redox balancing mechanisms, including fixation of CO2 produced during catabolism,
appear to be sufficient to allow growth of Rhodobacter
on malate or succinate. However, on more reduced carbon sources such as propionate and butyrate external
oxidants must be provided in the form of bicarbonate or as respiratory electron acceptors (Richardson
et al. 1988). It has been demonstrated that the action
of the anaerobic respiratory electron acceptors during
phototrophic growth is to prevent overreduction of the
ubiquinone pool by reduced substrates and maintain
the uhiquinone pool at close to optimal redox state for
cyclic electron transport (McEwan et al. 1985; Jones
et al. 1989) (Fig. 2).
158
3. Anaerobic respiration
A number of purple non-sulfur bacteria are able to grow
anaerobically in the dark by fermentation of hexoses
(Uffen 1978) but R. capsulatus and R. sphaeroides
exhibit very limited fermentative growth (Schulz &
Weaver 1982). The problem in the case of Rhodobacter was identified by Gest and co-workers as an
inability to dissipate reducing power generated during fermentation and it was shown that oxidants such
as trimethylamine-N-oxide (TMAO) could facilitate
growth via sugar oxidation (Madigan & Gest 1978). It
was subsequently shown that in R. capsulatus reduction of TMAO and DMSO (Yen & Marrs 1977) was,
in fact, an anaerobic respiratory process which was
linked to generation of /Xp (McEwan et al. 1983).
Further work has revealed that a variety of energyconserving anaerobic respiratory pathways is present
in purple non-sulfur bacteria (Ferguson et al. 1987;
McEwan et al. 1990) (Fig. 2). However, the distribution
of these pathways is highly species and strain specific. In addition to facilitating slow growth in the dark,
anaerobic respiratory pathways appear to be important in the dissipation of reducing power in the light
(Richardson et al. 1988): This enables photosynthetic
electron transport to operate at close to optimal redox
poise as described in section 2.5 (McEwan et al. 1985;
Jones et al. 1990b).
3.1 Sulfoxide and amine oxide respiration
A widespread property of purple non-sulfur bacteria
is their ability to use DMSO and TMAO as electron
acceptors. A periplasmic enzyme is able to reduce both
DMSO and TMAO (McEwan et al. 1985a, 1987). This
enzyme has been purified from R. capsulatus (McEwan et al. 1991) and R. sphaeroides f. sp. denitrificans
(Satoh & Kurihara 1987) and since its K,~ for DMSO
is much lower than for TMAO it is now described as
DMSO reductase. DMSO reductase is able to reduce a
wide variety of S-oxides and N-oxides as well as chlorate. The purified enzyme is a monomer Mr = 82,000
and it contains a pterin molybdenum cofactor as its only
prosthetic group (Satoh & Kurihara 1987; McEwan et
al. 1991). This makes it very useful for investigation
of the molecular properties of molybdoenzymes (Benson et al. 1992). Recently, we have obtained crystals
of DMSO reductase from R. capsulatus and a native
data set has been collected to 2.5 A resolution (S. Bailey & A.G. McEwan, unpublished data). We have also
cloned the DMSO respiratory genes of R. sphaeroides
by complementation of two DMSO reductase deficient
mutants ofR. sphaeroides (T.C. Bonnett & A.G. McEwan, unpublished data). This 9 kb DMSO respiratory
gene cluster also complements the DMSO respiratory
mutant R. capsulatus strain DK9 described by Kelly et
al. (1988). Sequencing of this DNA is in progress but
we have evidence that the DMSO reductase of R. capsulatus is related to the periplasmic TMAO reductase
ofE. coli (T.C. Bonnett & A.G. McEwan, unpublished
data). Recently, it has been reported that peptides from
R. sphaeroides DMSO reductase have sequence identity with biotin sulfoxide reductase of E. coli and R.
sphaeroides (Pollock & Barber 1993) and so it is anticipated that the periplasmic DMSO/TMAO reductases
and biotin sulfoxide reductases will form a group of
closely-related molybdoenzymes (see Wooton et al.
1991 for a review of this field).
Electron transfer to DMSO reductase in R.
capsulatus proceeds via a membrane-bound b-type
cytochrome and periplasmic cytochrome c556 but
does not involve the cytochrome bcl complex or
cytochromes involved in the pathways of denitrification (McEwan et al. 1989) (Fig. 3). Another ctype cytochrome which was considered to be associated with DMSO respiration in R. capsulatus and
R. sphaeroides was the 44 kDa cytochrome. This
cytochrome is induced during phototrophic growth of
cells in the presence of DMSO (Ward et al. 1983; Zsebo & Hearst 1984). However, it has now been established that the 44 kDa cytochrome is a cytochrome
c peroxidase (Hanlon et ai. 1992) and presumably it
terminates a respiratory pathway which uses hydrogen
peroxide as an electron acceptor. The elevated levels of
cytochrome c peroxidase observed in the presence of
DMSO are probably associated with a stress response
to the presence of this molecule. In this context it is now
well-established that hydroperoxidases are induced in
many bacteria in response to physiological stress (Barr
& Kogoma 1991).
3.2 Pathways of denitrification
There are very few reports of purple non-sulfur bacteria
which are able to catalyse the complete denitrification
of nitrate to dinitrogen gas. The best-known example
is R. sphaeroides f. sp. denitrificans (Satoh et al. 1976)
but many strains of R. capsulatus and R. sphaeroides
have been shown to respire using one or more of the
electron acceptors in the denitrification pathway (Ferguson et al. 1987).
159
N z O ~ N z
[ ;:7, I
""
3-Hz
,.~,~
bL
I~0~
' b
-',
,
,.Ol-lz'A~'". . . . .
•--.
NO?,
.OMSO
lib.
o'dI
I
(llnS
:
,
OHz
,
.....
l(oa~o
anfiA. jOHz
cytoptctsm
Fig. 3. Organisationof anaerobicrespiratorypathwaysof electron transferto nitrous oxide, dimethylsulphoxideand nitrate in R. capsulatus.
The b-type cytochromesinvolvedin electron transfer between ubiquinol and the terminal reductases of nitrate and DMSO respiration are
indicated by dashed lines since their molecularpropertiesare not known.
Nitrate respiration in R. capsulatus and R.
sphaeroides f. sp. denitrificans involves a periplasmic nitrate reductase (McEwan et al. 1984; Sawada & Satoh 1980). The enzyme from R. capsulatus
can be isolated as a single polypeptide M~ = 90,000
and contains a pterin molybdenum cofactor but no
haem centres (McEwan et al. 1987). This enzyme is
relatively insensitive to inhibition by azide and does
not use chlorate as a substrate. A second enzyme
preparation from R. capsulatus has been described
which contains a cytochrome c552 (M~ = 13,000) as
part of a water-soluble nitrate reductase redox complex (Richardson et al. 1990). Electron transfer from
ubiquinol to the periplasmic nitrate reductase complex involves a membrane-associated oxidoreductase
which is inhibited by HOQNO and low concentrations
of cyanide and contains a cytochrome b559 (Richardson
et al. 1990) (Fig. 3). Very similar redox components
have been identified in the nitrate respiratory pathway
of R. sphaeroides f. sp. denitrificans (Yokota et al.
1984). Until recently the periplasmic nitrate respiratory pathway was considered to be restricted to phototrophic bacteria but it is now known that periplasmic
nitrate reductase is quite widely distributed among nonphototrophic bacteria and allows nitrate respiration to
occur under aerobic conditions (Bell et al. 1990). The
respiratory nitrate reductases of E. coli and Paracoccus denitrificans are quite distinct from the periplasmic enzyme described above. However, this nitrate
reductase has been identified in R. sphaeroides f. sp.
denitrificans (Byrne & Nicholas 1987) and R. capsulatus strain BK5 (Ballard et al. 1990). Nitrite does not
appear to be used by many strains of photosynthetic
bacteria but a copper-containing nitrite reductase has
been purified from the periplasm of R. sphaeroides f.
sp. denitrificans (Sawada et al. 1978). In contrast it
appears probable that the use of nitrous oxide (McEwan et al. 1985) and nitric oxide (Bell et al. 1992)
as electron acceptors is a widely distributed property
of purple non-sulfur bacteria. A periplasmic nitrous
oxide reductase has been purified from R. capsulatus
(McEwan et al. 1985) and R. sphaeroides f. sp. denitrificans (Michelski et al. 1986) and it resembles the
multiple Cu-containing enzyme of non-phototrophic
bacteria. Similarly, it is expected that the NO reductase of photosynthetic bacteria will be a membranebound cytochrome bc complex as described by Zumft
(1993). Cytochrome c2 is essential for nitrous oxide
respiration in R. capsulatus (Richardson et al. 1991)
(Fig. 3) and nitric oxide reductase (Bell et al. 1992).
The cytochrome bcl complex is a major component
of the ubiquinol-cytochrome ca oxidoreductase activity which is linked to electron transfer to nitrous oxide
(Fig. 3), nitrite and nitric oxide but the situation is
rather more complicated.
160
3.3 Evidence for a ubiquinol-cytochrome c
oxidoreductase activity in Rhodobacter which is
independent of the cytochrome bcl complex
The cytochrome bcl complex is an integral component
of the cyclic electron transport system of Rhodobacter
and sincefbc mutants are unable to grow phototrophically (Prince & Daldal 1987) then it can be regarded as essential. However, a light-induced membrane
potential was generated in an fbc mutant of R. capsulatus and was maintained during continuous illumination (Richardson et al. 1989). This indicates that
cyclic electron transfer can occur in R. capsulatus
in the absence of a functional cytochrome bcl complex. However, the Ap generated is rather small (lower than that observed for N20 respiration) and is of
insufficient magnitude to support phototrophic growth
(Richardson et al. 1989). It has been estimated that,
at concentrations of myxothiazol which are sufficient
to completely block turnover of the cytochrome bcl
complex, residual cyclic electron transfer activity can
be observed which constitutes about 3% of the uninhibited rate (Myatt et al. 1987). This cytochrome bclindependent pathway makes a small contribution to
cyclic electron transport but its presence is revealed
when pathways of lower flux such as nitric oxide respiration and nitric oxide respiration were investigated
in R. capsulatus. The rate of turnover of this alternative
ubiquinol-cytochrome c oxidoreductase was sufficient
to support about 30% of the rate of N20 reduction
in myxothiazol=inhibited cells compared to uninhibited cells (Richardson et al. 1989). Since nitrous oxide
respiration is absolutely dependent upon cytochrome
c2 in R. capsulatus (Richardson et al. 1991) it seems
probable that this cytochrome accepts electrons from
the alternative ubiquinol-cytochrome c oxidoreductase
(Fig. 3). The situation regarding nitric oxide respiration is less clear because this respiratory pathway
can operate in mutants whick lack cytochrome bcl and
cytochrome c2. One possibility is that cytochrome c,
which is suggested to operate in cyclic electron transport in parallel with cytochrome c2 (Jones et al. 1990)
(Fig. 1) can also function in nitric oxide respiration
as a mediator of electron transfer between the alternative ubiquinol-cytochrome c oxidoreductase and nitric
oxide reductase.
The observations described above raise the question
of the nature of this alternative ubiquinol-cytochrome
c oxidoreductase and its physiological function. Since
there is no evidence for an alternative to the cytochrome
bci complex in non-photosynthetic denitrifiers such
as Paracoccus denitrificans (Parsonage et al. 1986) it
seems likely that the alternative ubiquinol-cytochrome
c oxidoreductase is specifically associated with photosynthetic electron transport. Spectroscopic studies of
intact cells of R. capsulatus have shown that the alternative ubiquinol-cytochrome c oxidoreductase contains a b-type cytochrome (c~ma~ = 557,560 nm) and
electron transfer via this cytochrome is sensitive to
HOQNO (Richardson et al. 1989). These properties
are almost identical to those of the cytochrome bcontaining oxidoreductase which operates in nitrate
respiration (Richardson et al. 1991). Taken together, these data suggest that a single type of alternative ubiquinol-cytochrome c oxidoreductase functions
in these pathways of anaerobic respiration (Fig. 3).
Furthermore, the link between nitrate respiration and
photosynthesis is clearer following the observation of
Rott et al. (1992) who showed that antibodies raised
against isocytochrome c2 from R. sphaeroides crossreacted with cytochrome c552 from R. sphaeroides f.
sp. denitrificans. This leads to the view that the ctype cytochrome which functions in electron transfer
to nitrate reductase in R. sphaeroides f. sp. denitrificans
is isocytochrome c2. The above data suggest that in R.
capsulatus and R. sphaeroides a ubiquinol-cytochrome
c oxidoreductase may be present which can operate as
an alternative to the cytochrome bcl complex in photosynthesis and in anaerobic respiratory pathways other
than DMSO respiration.
Why should an enzyme with such a low activity
compared to cytochrome bcl complex be involved in
photosynthetic electron transport? The answer may lie
in the need to prevent inhibition of the turnover of
the cytochrome bCl complex which might occur under
phototrophic conditions at high values of Ap and when
the Q-pool was highly reduced. This could lead to an
attenuation of the Q-cycle. The presence of an alternative pathway of ubiquinol oxidation, probably involving a linear pathway of electron flow, would enable
the cell to avoid such a situation. The requirement for
an alternative ubiquinol-cytochrome c oxidoreductase
activity would be most acute during growth on highly
reduced carbon sources such as alcohols and aliphatic
acids.
4. Concluding remarks
The molecular characterisation of the protein complexes involved in photosynthetic electron transfer and CO2
fixation is now at an advanced stage. Recent progress in
161
the understanding of the expression of photosynthesis
and CO2 fixation genes should lead to a more complete understanding of how adaptation to phototrophic conditions is coordinated. Although there is less
detailed information concerning the molecular properties of anaerobic respiratory pathways their importance
in the regulation of phototrophic growth is now established. This work has revealed that during phototrophic
growth Rhodobacter uses a variety of pathways including CO2 fixation and anaerobic respiration to achieve
redox homeostasis. Finally, it is clear that although
the metabolic capabilities of purple non-sulfur bacteria appear to be quite well documented new capabilities
continue to be found. In this context it is particularly
important that species other than R. sphaeroides and R.
capsulatus continue to be investigated.
Acknowledgement
I am grateful to David Richardson and Tim Donohue
for discussion. Work in the author's laboratory was
supported by grants from SERC (UK) and The Royal
Society.
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