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Antonievan Leeuwenhoek 66: 151-164, 1994. @ 1994KluwerAcademicPublishers. Printedin the Netherlands. 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. 152 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. 153 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 154 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. 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