Photosynthetic Electron Transport and Anaerobic Metabolism in Purple Non-Sulfur Phototrophic Bacteria

Antonie van Leeuwenhoek 66: 151-164, 1994. 151 @ 1994KluwerAcademic Publishers. Printedin the Netherlands.

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 ly representative. Indeed, R. capsulatus has been described as the most versatile of prokaryotes (Madi- The anoxygenic phototrophic bacteria are a diverse gan & Gest 1979) since it can grow photoautotroph- collection of microorganisms which carry out ically, chemoautotrophically, photoheterotrophically, bacteriochlorophyll-dependent photosynthesis as a and chemoheterotrophically with a variety of electron common metabolic process (Nitschke & Rutherford acceptors as well as by fermentation of sugars. Con- 1991). There are two major groups of anoxygenic pho- fronted with this diversity of metabolic processes it is totrophs: the phototrophic green bacteria (Chlorobi- not possible in this review to be comprehensive and so aceae and Chloroflexaceae) (Olson et al. 1988) and the focus will be photosynthesis and anaerobic respira- the phototrophic purple bacteria which can be further tion. For recent progress in the area of nitrogen fixation divided into purple sulfur bacteria (Chromatiaceae and in phototrophic bacteria the reader is referred to papers Ectothiorhodospiraceae) and purple non-sulfur bacte- by Schneider et al. (1991), Preker et al. (1992) and ria (Drews & Imhoff 1991). The purple non-sulfur bac- Wang Get al. (1993). teria exhibit a remarkable range of morphological, biochemical and metabolic properties (Imhoff et al. 1984). They are also diverse taxonomically with genera being 2. Phototrophic metabolism placed in the c~ and /3 subgroups of Proteobacteria (Stackebrandt et al. 1988). This review will focus on 2.10rganisation and development of the two species of purple non-sulfur bacteria: Rhodobac- photosynthetic apparatus ter capsulatus and Rhodobacter sphaeroides. Although these two species do not exhibit all of the nutrition- Most purple non-sulfur bacteria are facultative anaer- al properties of other purple non-sulfur phototrophs obes and under oxic conditions are able to grow they exhibit metabolic capabilities which are broad- chemotrophically with oxygen as electron acceptor. 152

R. capsulatus, for example, possesses a branched aer- 1985; Deisenhofer & Michel 1989) and R. sphaeroides obic respiratory chain which is typical of many non- (Allen et al. 1987). enteric Gram negative bacteria (La Monica & Marrs Molecular genetic analysis of purple non-sulfur 1976). However, lowering the oxygen tension below a bacteria has also advanced considerably over the last species-specific threshold value triggers the formation decade (Scolnik & Marrs 1987; Donohue & Kaplan of photosynthetic pigments and pigment-binding pro- 1991). Most of the genes involved in the formation of teins (Drews & Oelze 1981; Kiley & Kaplan 1988). the LH and RC pigment-protein complexes are encod- The pigment-protein complexes are incorporated into ed by a 46 kb gene cluster on the chromosome of R. the cytoplasmic membrane (CM) and through the pro- capsulatus (Taylor et al. 1983) and a similar but not cess of invagination a specialised membrane system identical situation exists in R. sphaeroides (Wu et al. is formed; the intracytoplasmic membrane (ICM). The 1991). The puf operon is composed of genes encod- ICM is contiguous with the CM but appears to be func- ing LH1 polypeptides (pu3'B, pufA), RC-L and RC-M tionally distinct since it is the site of light-harvesting (pujS~, pufiVl) and open reading frames pufQ and pu3"X and photosynthetic electron transfer. Formation of the (Bauer et al. 1988). There is some genetic evidence ICM results in remarkable morphological changes in that PufQ is involved in bacteriochlorophyll biosyn- the cell membrane structure. The ICM of Rhodobacter thesis (Bauer & Marrs 1988) while PufX appears to and Rhodospirillum are vesicular structures (known as be required for the correct insertion of the RC into the 'chromatophores') but a variety of membrane struc- ICM (Farchaus et al. 1992). The pufA gene encoding tures are found in other genera (Imhoffet al. 1984). RC-H is located 39 kb away from the pufoperon in R. In R. sphaeroides and R. capsulatus three distinct capsulatus (Zsebo & Hearst 1984). The puc operon is pigment-protein complexes are involved in the trans- composed of genes encoding LHII polypeptides (pucB, duction of light energy into chemical energy. Two types pucA) (Youvan & Ismail 1985; Kiley & Kaplan 1987) of light harvesting (LH) complexes absorb light and as well as genes involved in regulation and assem- transfer excitation energy to the photochemical reac- bly of LHII (pucCDE) (Lee et al. 1989; Tichy et al. tion centre (RC) which is the site of primary photo- 1991). The organization of the bch and crt genes which chemistry and electron transport (Drews 1986; Zuber are involved in pigment biosynthesis is now also well 1986). LHI and LHII are also known as B875 and understood. Indeed, the nucleotide sequence of the B800-850 antenna complexes, respectively, in order entire photosynthetic gene cluster ofR. capsulatus has to identify the wavelength of maximum absorption been completed (Burke et al. 1991). of the Bchl pigment but note that this wavelength, In Rhodobacter the core component of the photo- and hence the designation, is often different in oth- synthetic apparatus is an RC surrounded by LHI anten- er genera. The B875 antenna is composed of two small nae in a ratio of 1 RC to 20-30 molecules of LHI Bchl membrane-spanning polypeptides ~ and/3 (Mr 5000- and this photosynthetic unit is constant under different 6000) which bind two molecules of Bchl and one or light regimes. The RC-LHI core complexes are sur- two carotenoid molecules. The B800-850 antenna is rounded by LHII antenna complexes and the level of made up of two subunits c~ (Mr = 7000) and/3 (Mr = LHII is inversely related to the light intensity (Aagaard 5000) which, like the B875 polypeptides, have a single & Sistrom 1972). However, although light intensity has central membrane-spanning c~-helix. They bind two or a key role in the regulation of the size of the photosyn- three molecules of Bchl and one carotenoid. thetic apparatus (Drews & Oelze 1981; Kiley & Kaplan The RC of Rhodobacter is composed of three sub- 1988) it has been known for several decades that oxy- units H, M and L and has a total Mr of about 100,000 gen is the major controlling factor in the development (Drews 1985). In many species, including Rhodopseu- of the photosynthetic apparatus (Cohen-Bazire et al. domas viridis, a fourth cytochrome subunit is present. 1957). Upon a decrease in oxygen tension, biosyn- The L and M subunits are integral membrane proteins thesis of Bchl and pigment binding proteins is strictly and they bind four Bchl molecules, two bacteriophaeo- coordinated (Chory et al. 1984). Levels ofpuc and puf phytin molecules, two quinones and one ferrous iron mRNA are increased 10-20 fold when oxygen tension atom. The pathway and kinetics of electron transfer is reduced. Some bch and crt mRNAs may also be ele- in the RC is now understood at the molecular level vated although in general they seem to be regulated by as a consequence of spectroscopic studies (reviewed oxygen to a lesser extent. by Jackson 1988) and the determination of the crystal Regulation of the puf and puc operons involves structure of the RC from Rps. viridis (Deisenhofer et al. both transcriptional and post-transcriptional control. 153

Analysis of puf and puc promoters has been carried pared to puj-'BALMX mRNA (Belasco et al. 1985; Klug out in detail and cis-acting regulatory elements have 1991). Finally, an unresolved question is the nature been identified (reviewed by Klug 1993a). However, of the transcription factors involved in photosynthetic the situation regarding trans-acting regulators is less gene expression. The nature of the transcription factor clear. Sganga & Bauer (1992) have identified a gene involved in the RegA-mediated sensor kinase regu- regA which encodes a trans-acting regulator respon- latory system is not established nor is the question of sible for activating expression of puf, puh and puc whether acr factor other than ~r7° is required for expres- operons in response to a decrease in oxygen tension sion ofpuf puh andpuc operons (see Bauer et al. 1993 but is not involved in the oxygen-controlled expression for discussion). of nif genes. RegA has extensive sequence homology in its amino terminal sequence with two component 2.2 Photosynthetic electron transport response regulators (Stock et al. 1989). It would be expected that in common with other systems, a sen- The purple non-sulfur bacteria normally carry out sor kinase would phosphorylate RegA in response to photosynthesis under anaerobic conditions. Photosyn- an environmental signal such as a change in oxygen thetic electron transport is cyclic and involves two tension. The identity of the sensor kinase has not yet multimeric transmembrane proteins: the RC and the been established but Bauer et al. (1993) have indi- cytochrome bcl complex (Jackson 1988). Electron cated that it is probably RegB since the regB gene transfer between these two redox complexes is mediat- predicts a transmembrane protein and regB mutants ed by the mobile electron carriers ubiquinone, located have the same phenotype as regA mutants. However, in the hydrophobic domain of the cytoplasmic mem- since RegA does not appear to possess a DNA binding brane, and a c-type cytochrome, usually located in the region in its carboxy-terminal region it may not func- periplasmic space in Rhodobacter. The RC catalyses tion directly in transcriptional control. Instead, Sganga a light-driven ferrocytochrome c-ubiquinone oxidore- & Bauer (1992) have suggested that RegA may resem- ductase activity while the cytochrome bCl complex ble smaller response regulators such as CheY, CheB catalyses a ubiquinol-ferricytochrome c oxidoreduc- and SpoOF which act as intermediaries in more com- tase activity (Fig. 1). This cycle of electron transfer is plex phosphoryl transfer regulatory cascades (Bouret et linked to the generation of a proton motive force (z~p) al. 1991). The same authors have identified additional across the cytoplasmic membrane (Jackson 1988). The loci associated with RegA in the control of photosyn- inner membrane of photosynthetic bacteria such as R. thetic gene expression by light and oxygen and exciting sphaeroides is one of the best-understood energy trans- progress in this area is anticipated. Of particular inter- ducing systems in biology as a result of the molecular est will be to find out whether the putative sensor kinase characterisation of the cytochrome bCl complex (Gen- RegB resembles FixL, the oxygen sensor kinase in Rhi- nis et al. 1993) and the RC (Feher et al. 1989). zobium meliloti, since the latter is an oxygen-binding A major surprise in the apparently well-defined area haemoprotein (Gilles-Gonzalez et al. 1991) or whether of cyclic electron transport has been the identifica- it resembles ArcB, which is thought to sense oxygen tion of alternative pathways of electron flow between tension indirectly (Stock et al. 1989). the cytochrome bcl comples and the oxidised bacteri- Additional factors are certainly important in the ochlorophyll dimer of the RC which is generated after a regulation of photosynthetic gene expression. The first photochemical event (Fig. 1). A large body of biochem- is the 'superoperonal organisation' of genes in which ical and spectroscopic data which had accumulated up transcription of operons encoding crt and bch genes to the mid- 1980s indicated that in R. capsulatus and R. continue into puf and puh operons (Wellington et al. sphaeroides cytochrome c2 mediated electron transfer 1992). It appears that this transcriptional organiza- between cytochrome bcl and the RC. Cytochrome c2 tion is important for the coordination of photosyn- is a periplasmic monohaemoprotein of MT = 13,000 thetic gene expression. A second factor is differential whose interaction with the RC has been investigated decay of mRNA as well as decreased mRNA stabili- in great detail (Dutton & Prince 1978). It came as ty of puf and puc transcripts in the presence of oxy- a great surprise when it was found that cytochrome gen (reviewed by Klug 1993b). The importance of c2-deficient mutants (cycA mutants) of R. capsula- differential decay of mRNA is most apparent for puf tus, constructed using reverse genetic techniques, were gene expression where a ratio of 10-15 LHI : 1 RC able to grow phototrophically (Daldal et al. 1986). The is achieved via greater stability ofpufBA mRNA corn- kinetics of electron transfer in these cycA mutants was 154

is required for phototrophic growth of cytochrome c 2 R. capsulatum 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. periplasm / ~ ~ 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 'classi- cal' 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 cytoplasm via mutation at a second site (Rott & Donohue 1990). These revertants, known as spd (suppressor of photo-

R. sphaeroides synthetic 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. cap- perip!asm sulatus (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). Isocy- RC tochrome c2 is normally present at only 1.0-2.5% of > ua > the level of cytochrome c2 in wild-type phototrophic R. sphaeroides (Rott et al. 1992) and its physiological role cytoplas= is still unclear. However, it has been noted by Donohue and co-workers that an open reading frame adjacent to Fig. 1. Pathwaysof cyclic photosyntheticelectron transfer in R. cap- the isocytochrome c2 structural gene encodes a zinc- sulatus (upper panel) and R. sphaeroides(lower panel). In R. capsu- containing alcohol dehydrogenase (Rott et al. 1993). latus electron transferbetween the RC and cytochromebc~ complex This has led to the suggestion that expression of iso- is mediated by periplasmic cytochrome c2 and membrane-bound cytochrome cz which operate in parallel. In R. sphaeroides only cytochrome c2 may be linked to the metabolism of cytochrome c2 is utilised in wild-type cells but in spd mutants alcohols in R. sphaeroides and preliminary evidence iso-cytochromec2 can replace cytochromec2. indicates that levels of this cytochrome are elevated during growth with ethanol (T.J. Donohue, personal communication). This point will be discussed in Sec- altered and the favoured interpretation was that direct tion 3.3. electron transfer between the cytochrome bcl complex and the RC could take place (Prince et al. 1986; Prince 2.3 Photosynthetic electron transport and & Daldal 1987). This view was challenged by Jackson metabolism and co:workers who identified, using spectroscopic techniques, a novel high potential c-type cytochrome The proton motive force (Ap) which is generated dur- (cytochrome cx) which was able to donate electrons to ing photosynthetic electron transport is used to drive the RC in a mutant which lacked both cytochrome c2 ATP synthesis, solute transport, and certain redox- and a functional cytochrome bcl complex (Jones et al. linked reactions (Jackson 1988). However, photosyn- 1990). Cytochrome c~ was shown to be a membrane- thetic electron transport is not a closed cycle since it bound protein with a mid-point redox potential of + is in communication with a variety of primary dehy- 360 mV (Jones et al. 1990). Further work by Jenney drogenases (Ferguson et al. 1987). This interaction & Daldal (1993) has identified a gene cycY, which occurs predominantly at the level of the ubiquinone 155

pool (Q-pool) which is pivotal in connecting elec- a sulfide-ubiquinone oxidoreductase (Brune & Truper tron transport with metabolism during phototroph- 1986). ic growth. The diversity of carbon sources which Recently, it has been established that organic sulfur can be metabolised by purple non-sulfur bacteria has can also be used as an electron donor in photosyn- been reviewed by Sojka (1978) and by Dutton & thetic metabolism. Dimethylsulfide has been shown to Evans (1978). During photoheterotrophic growth with support photoautotrophic growth of the purple sulfur dicarboxylic acids as carbon source the major dehy- bacterium Thiocapsa roseopersicina (Visscher & van drogenases will be NADH dehydrogenase and suc- Gemerden 1991). A previous report had indicated that cinate dehydrogenase. The ubiquinol generated dur- a number of species of phototrophic bacteria might also ing succinate oxidation can be oxidised by energy- have this capability (Zeyer et al. 1987) and work in the linked reverse electron flow to NAD +. This reaction author's laboratory has shown that a new isolate of is catalysed by the rotenone-sensitive NADH dehydro- R. sulfidophilus (strain SH1) is able to grow photoau- genase which catalyses NADH oxidation under aero- totrophically with DMS as electron donor (Hanlon et bic conditions but under phototrophic conditions acts al. 1994). DMS oxidation was linked to the accumu- as ubiquinol-NAD + oxidoreductase. The energetical- lation of dimethylsulfoxide (DMSO) indicating that ly unfavourable electron transfer from ubiquinol to DMS was used purely as an electron donor. In further NAD + is linked to the consumption of Ap (Klemme work we have identified a periplasmic redox protein 1969). which is able to catalyse electron transfer from DMS A variety of reductants which support pho- to a variety of redox dyes. This enzyme, DMS: accep- toautotrophic growth of Rhodobacter donate elec- tor oxidoreductase, has been purified and has been trons directly into the cyclic electron transport path- shown to contain a pterin molybdenum cofactor and a way. Molecular hydrogen is oxidised by membrane- cytochrome b562 (Hanlon & McEwan, manuscript in bound hydrogenase which functions as a hydrogen- preparation). The physiological electron acceptor for ubiquinone oxidoreductase (Vignais et al. 1985). DMS: acceptor oxidoreductase has not been identified Despite their name, most purple non-sulfur bacteria but it could be ubiquinone or a periplasmic cytochrome are able to use inorganic sulfur molecules as electron which in turn is able to donate electrons to the RC. The donors. The slow recognition of this capability prob- latter case resembles the situation which must operate ably arose because many purple non-sulfur bacteria for methylotrophic phototrophs such as Rhodopseu- have a low tolerance towards sulfide (Hansen & van domonas acidophila during the operation of the dye- Gemerden 1972). However, certain species such as linked methanol dehydrogenase (Bamforth & Quayle Rhodobacter sulfidophilus can tolerate up to 3 mM 1978, 1979). Interestingly, R. sulfidophilus strain SH1 sulfide which is comparable to the sulfide tolerance of is also able to utilise methanol and methylamine as sole purple sulfur bacteria such as Chromatium vinosum carbon source (Hanlon et al. 1994). (Hansen & Veldkamp 1973). In R. capsulatus, R. The use of the oxidised bacteriochlorophyll dimer sphaeroides and Rhodospirillum species extracellular as an electron sink during the anaerobic oxidation elemental sulfur is the final oxidation product of sul- of electron donors during phototrophic growth is fide oxidation (Hansen & van Gemerden 1972) but in analogous to the use of cytochrome c oxidase by R. sulfidophilus and a number of other Rhodobacter chemotrophic bacteria under aerobic conditions. Some and Rhodopseudomonas species, sulfide is oxidised electron donors which are used by chemoautotrophs completely to sulfate (reviewed by Brune 1989). R. (Wood 1988), for example nitrite, have not yet been sulfidophilus is interesting because it appears to oxi- shown to support photoautotrophic growth but it seems dize sulfide to sulfate without intermediate formation likely that new electron donors in phototrophs will be of elemental sulfur, but instead accumulates sulfite identified. In this context the recent report of pho- (Neutzling et al. 1985). Sulfite is oxidised to sulfate toantrophic growth of Rhodomicrobium-like isolates only when sulfide has been exhausted. It seems likely with ferrous iron as electron source is interesting that the oxidation of sulfur molecules takes place in (Widdel et al. 1993). The oxidation of ferrous iron the periplasm and involves the c-type cytochromes as at neutral pH has previously only been reported in electron acceptors (Brune 1989). However, in R. sul- chemoautotrophs such as Gallionellaferruginea (see fidophilus, although sulfide oxidation probably occurs Wood 1988). During photoautotrophic growth, elec- in the periplasm, the enzyme involved may operate as trons 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 sion (Hallenbeck et al. 1990b). The Form I and Form II metabolic reactions including CO2 fixation. gene clusters each constitute an operon (Gibson et al. 1990, 1991) but their regulation is not fully understood. 2.4 CO,fixation Although the two operons must be subject to independent regulation (Jouanneau & Tabita 1986) it is also A major feature of the purple phototrophic bacteria known that their expression must in some way be coor- is their ability to grow photoautotrophically fixing dinated (Falcone et al. 1988; Hallenbeck et al. 1990a, CO2 via the reductive pentose phosphate cycle (Calvin b). Recently, Gibson & Tabita (1993) have sequenced a cycle) (Tabita 1988). The molecular details of CO2 fix- gene, cbbR, which is divergently transcribed upstream ation are best understood in R. sphaeroides. This bac- of the Form I operon. This gene is essential for expres- terium possesses two distinct forms of the key enzyme sion of Form ! genes and in a cbbR mutant, expression of the Calvin cycle ribulose-1,5-bisphosphate carboxy- of Form II genes was reduced to 30% of their wild-type lase/oxygenase (RubisCO). Form I RubisCO has an level. CbbR shows homology to members of the LysR Mr = 550,000 and it is composed of a larger catalyt- family of transcriptional regulatory proteins (Henikoff ic subunit (Mr = 56,000) and a small subunit (Mr = et al. 1988) and it is proposed that it acts as an acti- 15,000). The quarternary structure of Form I Rubis- vator of Calvin cycle operon expression. A similar CO is probably L8S8 (Gibson & Tabita 1977). This situation has been described in Alcaligenes eutrophus type of RubisCO appears to be widespread amongst (Windhovel & Bowien 1991). The effector(s) which autotrophic bacteria (Tabita 1988) and it resembles the activates CbbR has not yet been identified. Gibson & enzyme in chloroplasts. A second form of RubisCO Tabita (1993) have also indicated that in a cbbR mutant which has been found in R. sphaeroides has an Mr = expression of the Form II operon continued to be low 360,000 (Gibson & Tabita 1985) and it is composed during aerobic growth. This may indicate that an as of identical subunits Mr = 52,000 (Gibson & Tabita yet unidentified transcriptional regulator is involved in 1977). Both forms of RubisCO are present in R. cap- the regulation of Form II operon expression. It may sulatus (Shiveley et al. 1984) but in general the Form be the case that this putative regulator is a 'redox sen- II enzyme appears not to be present in most species sor', because in addition to oxygen, alternative electron of phototrophic bacteria (Tabita 1988). It appears that acceptors such as DMSO decrease the levels of Calvin the two forms of RubisCO are not closely related (Gib- cycle enzymes during photoheterotrophic growth (Hal- son & Tabita 1977, 1985). In R. sphaeroides the genes lenbeck 1990a). Conversely, higher levels of Calvin encoding Form I RubisCO (cbbLz, cbbSi) and the gene cycle enzymes are observed as carbon sources become encoding Form II RubisCO (cbbMH) are components more reduced (Tabita 1988). Over the past decade it of two distinct CO2 fixation operons (Gibson & Tabita has become recognized that the complex pattern of 1988; Tabita et al. 1992). It is interesting that these CO2 fixation in Rhodobacter arises because this activ- operons each contain structural genes for phospho- ity is important for photoautotrophic growth and pho- ribulokinase, the other unique enzyme of the Calvin toheterotrophic growth. cycle (Hallenbeck & Kaplan 1988; Gibson & Tabita Early investigations into the physiology of pho- 1988). However, the organisation of the two operons toheterotrophic growth of purple non-sulphur pho- is not identical. For example, cbbGi1 (encoding glyc- totrophs had speculated on the importance of electron eraldehyde 3-phosphate dehydrogenase) is present on sinks for excess reducing power which might be gener- the Form II CO2 fixation gene cluster only (Gibson & ated during the oxidation of carbon substrates (Muller Tabita 1988). 1933). Specifically, CO2 fixation via the Calvin cycle Expression of Calvin cycle genes is tightly con- was suggested to have such a role (Lascelles 1960; Fer- trolled. During aerobic growth there is virtually no guson et al. 1987). Support for this view came from expression of these genes (Jouanneau & Tabita 1986) Richardson et al. (1988) who observed that addition of while under photosynthetic growth conditions, varying bicarbonate or alternative electron acceptors (discussed degrees of expression of the Form I and II operons are in Section 3) to media was required for phototrophic observed. In general, Form II enzyme predominates growth of R. capsulatus on the reduced carbon sources during heterotrophic metabolism (Jouanneau & Tabita propionate and butyrate but not for growth on more oxi- 1986; Falcone et al. 1988) while Form I enzyme is dised carbon sources such as malate or succinate. Stud- predominant during autotrophic growth (Falcone et al. ies of mutants ofR. sphaeroides have added to the view 1988) and is essential for CO2 fixation at low CO2 ten- that CO2 fixation has a role in maintaining redox bal- 157

2.5 Phototrophic growth and redox homeostasis NAD*

A feature of photoheterotrophy which distinguish- es it from chemoheterotrophy is that during photo- "~ ~ ~fumQrQl~ CO2 fixQtion heterotrophic growth, catabolism of carbon molecules Q pool ~- "~ (" innte 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 incom- THAO I OHSO plete since oxoglutarate dehydrogenase activity is cyt.cz extremely low (Beatty & Gest 1981). An additional problem which has already been alluded to is the h o ""-~ PeTo ~ N zO Oz dissipation of reducing power in the form of NADH which is generated during carbon catabolism. Con- sider growth on malate; the oxidation of malate to Fig. 2. Interaction between cyclic photosynthetic electron transfer and redox-balancing pathways in R. capsulatus. NADH generated oxaloacetate has an unfavourable standard free ener- during oxidation" of organic carbon can be oxidised via the CO2 gy and in order that this reaction should proceed in fixation or via respiration to a variety of electron acceptors which the oxidative direction, NADH must be consumed. branch from cyclic electron transfer at the level of the Q-pool or One sink for NADH is the Calvin cycle described in cytochrome c2. 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 ance during photoheterotrophic growth. A double null Ap (Jackson 1991). Although the equilibrium constant mutant in cbbFl and cbbFtz was constructed in which for this reaction is 1 the influence of the ~p gener- expression of RubisCO was essentially abolished (Hal- ated during photosynthetic electron transport means lenbeck et al. 1990a). This double mutant was unable that the cell maintains a high mass action ratio of to grow photoheterotrophically with succinate as car- [NADPH] [NAD+]/[NADP +] [NADH]. This NADPH bon source unless an external electron acceptor such can be used for biosyntheticpurposes while a high ratio as dimethylsulfoxide (DMSO) was added. Essential- of NAD+/NADH is maintained in order to provide an ly the same result was obtained when both RubisCO electron acceptor for catabolic redox reactions. Jack- genes were deleted from R. sphaeroides (Falcone & son and co-workers have found that the light-driven Tabita 1991). Taken together these data confirm that H +-THase activity in chromatophores ofR. capsulatus the Calvin cycle has an important role in maintaining constitutes about 50% of the ATPase activity (Cotton & intracellular redox balance during photoheterotrophic Jackson 1988) and thus H+-THase may have a major growth even under conditions where addition of bicar- role in the redox and energy economy of the cell. bonate to the medium is not required. Tabita and co- Intracellular redox balancing mechanisms, includ- workers have recently reported that a revertant of the ing fixation of CO2 produced during catabolism, RubisCO double deletion mutant ofR. sphaeroides can appear to be sufficient to allow growth of Rhodobacter be isolated which is able to grow photoheterotrophical- on malate or succinate. However, on more reduced car- ly on malate but will still not fix CO2 via the Calvin bon sources such as propionate and butyrate external cycle (Wang X et al. 1993a). This mutant will not grow oxidants must be provided in the form of bicarbon- photolithoautotrophically with H2 as electron donor ate or as respiratory electron acceptors (Richardson but will grow under these conditions with thiosulfate et al. 1988). It has been demonstrated that the action as an electron source (Wang X et al. 1993b). These of the anaerobic respiratory electron acceptors during data indicate that a hitherto unrecognised pathway of phototrophic growth is to prevent overreduction of the CO2 fixation exists in R. sphaeroides which is distinct ubiquinone pool by reduced substrates and maintain from the Calvin cycle. The nature of this alternative the uhiquinone pool at close to optimal redox state for CO2 fixation pathway is still unclear although there cyclic electron transport (McEwan et al. 1985; Jones are several possibilities (see Wang X et al. 1993b for et al. 1989) (Fig. 2). discussion). 158

3. Anaerobic respiration by complementation of two DMSO reductase deficient mutants ofR. sphaeroides (T.C. Bonnett & A.G. McE- A number of purple non-sulfur bacteria are able to grow wan, unpublished data). This 9 kb DMSO respiratory anaerobically in the dark by fermentation of hexoses gene cluster also complements the DMSO respiratory (Uffen 1978) but R. capsulatus and R. sphaeroides mutant R. capsulatus strain DK9 described by Kelly et exhibit very limited fermentative growth (Schulz & al. (1988). Sequencing of this DNA is in progress but Weaver 1982). The problem in the case of Rhodobac- we have evidence that the DMSO reductase of R. cap- ter was identified by Gest and co-workers as an sulatus is related to the periplasmic TMAO reductase inability to dissipate reducing power generated dur- ofE. coli (T.C. Bonnett & A.G. McEwan, unpublished ing fermentation and it was shown that oxidants such data). Recently, it has been reported that peptides from as trimethylamine-N-oxide (TMAO) could facilitate R. sphaeroides DMSO reductase have sequence iden- growth via sugar oxidation (Madigan & Gest 1978). It tity with biotin sulfoxide reductase of E. coli and R. was subsequently shown that in R. capsulatus reduc- sphaeroides (Pollock & Barber 1993) and so it is antic- tion of TMAO and DMSO (Yen & Marrs 1977) was, ipated that the periplasmic DMSO/TMAO reductases in fact, an anaerobic respiratory process which was and biotin sulfoxide reductases will form a group of linked to generation of /Xp (McEwan et al. 1983). closely-related molybdoenzymes (see Wooton et al. Further work has revealed that a variety of energy- 1991 for a review of this field). conserving anaerobic respiratory pathways is present Electron transfer to DMSO reductase in R. in purple non-sulfur bacteria (Ferguson et al. 1987; capsulatus proceeds via a membrane-bound b-type McEwan et al. 1990) (Fig. 2). However, the distribution cytochrome and periplasmic cytochrome c556 but of these pathways is highly species and strain specif- does not involve the cytochrome bcl complex or ic. In addition to facilitating slow growth in the dark, cytochromes involved in the pathways of denitrifi- anaerobic respiratory pathways appear to be impor- cation (McEwan et al. 1989) (Fig. 3). Another c- tant in the dissipation of reducing power in the light type cytochrome which was considered to be asso- (Richardson et al. 1988): This enables photosynthetic ciated with DMSO respiration in R. capsulatus and electron transport to operate at close to optimal redox R. sphaeroides was the 44 kDa cytochrome. This poise as described in section 2.5 (McEwan et al. 1985; cytochrome is induced during phototrophic growth of Jones et al. 1990b). cells in the presence of DMSO (Ward et al. 1983; Zse- bo & Hearst 1984). However, it has now been estab- 3.1 Sulfoxide and amine oxide respiration lished that the 44 kDa cytochrome is a cytochrome c peroxidase (Hanlon et ai. 1992) and presumably it A widespread property of purple non-sulfur bacteria terminates a respiratory pathway which uses hydrogen is their ability to use DMSO and TMAO as electron peroxide as an electron acceptor. The elevated levels of acceptors. A periplasmic enzyme is able to reduce both cytochrome c peroxidase observed in the presence of DMSO and TMAO (McEwan et al. 1985a, 1987). This DMSO are probably associated with a stress response enzyme has been purified from R. capsulatus (McE- to the presence of this molecule. In this context it is now wan et al. 1991) and R. sphaeroides f. sp. denitrificans well-established that hydroperoxidases are induced in (Satoh & Kurihara 1987) and since its K,~ for DMSO many bacteria in response to physiological stress (Barr is much lower than for TMAO it is now described as & Kogoma 1991). DMSO reductase. DMSO reductase is able to reduce a wide variety of S-oxides and N-oxides as well as chlo- 3.2 Pathways of denitrification rate. The purified enzyme is a monomer Mr = 82,000 and it contains a pterin molybdenum cofactor as its only There are very few reports of purple non-sulfur bacteria prosthetic group (Satoh & Kurihara 1987; McEwan et which are able to catalyse the complete denitrification al. 1991). This makes it very useful for investigation of nitrate to dinitrogen gas. The best-known example of the molecular properties of molybdoenzymes (Ben- is R. sphaeroides f. sp. denitrificans (Satoh et al. 1976) son et al. 1992). Recently, we have obtained crystals but many strains of R. capsulatus and R. sphaeroides of DMSO reductase from R. capsulatus and a native have been shown to respire using one or more of the data set has been collected to 2.5 A resolution (S. Bai- electron acceptors in the denitrification pathway (Fer- ley & A.G. McEwan, unpublished data). We have also guson et al. 1987). cloned the DMSO respiratory genes of R. sphaeroides 159

NzO~Nz

[ ;:7, I I~0~ NO?, .OMSO

' b -', lib. o' "" 3-Hz , : ,I (llnS ,dI ,.~,~ •--.,.Ol-lz'A~'" ..... OHz ..... bL l(oa~o anfiA. jOHz

cytoptctsm

Fig. 3. Organisationof anaerobic respiratory pathways of electron transfer to nitrous oxide, dimethylsulphoxideand nitrate in R. capsulatus. The b-type cytochromes involved in electron transfer between ubiquinol and the terminal reductases of nitrate and DMSO respiration are indicated by dashed lines since their molecularproperties are not known.

Nitrate respiration in R. capsulatus and R. reductase has been identified in R. sphaeroides f. sp. sphaeroides f. sp. denitrificans involves a periplas- denitrificans (Byrne & Nicholas 1987) and R. capsu- mic nitrate reductase (McEwan et al. 1984; Sawa- latus strain BK5 (Ballard et al. 1990). Nitrite does not da & Satoh 1980). The enzyme from R. capsulatus appear to be used by many strains of photosynthetic can be isolated as a single polypeptide M~ = 90,000 bacteria but a copper-containing nitrite reductase has and contains a pterin molybdenum cofactor but no been purified from the periplasm of R. sphaeroides f. haem centres (McEwan et al. 1987). This enzyme is sp. denitrificans (Sawada et al. 1978). In contrast it relatively insensitive to inhibition by azide and does appears probable that the use of nitrous oxide (McE- not use chlorate as a substrate. A second enzyme wan et al. 1985) and nitric oxide (Bell et al. 1992) preparation from R. capsulatus has been described as electron acceptors is a widely distributed property which contains a cytochrome c552 (M~ = 13,000) as of purple non-sulfur bacteria. A periplasmic nitrous part of a water-soluble nitrate reductase redox com- oxide reductase has been purified from R. capsulatus plex (Richardson et al. 1990). Electron transfer from (McEwan et al. 1985) and R. sphaeroides f. sp. den- ubiquinol to the periplasmic nitrate reductase com- itrificans (Michelski et al. 1986) and it resembles the plex involves a membrane-associated oxidoreductase multiple Cu-containing enzyme of non-phototrophic which is inhibited by HOQNO and low concentrations bacteria. Similarly, it is expected that the NO reduc- of cyanide and contains a cytochrome b559 (Richardson tase of photosynthetic bacteria will be a membrane- et al. 1990) (Fig. 3). Very similar redox components bound cytochrome bc complex as described by Zumft have been identified in the nitrate respiratory pathway (1993). Cytochrome c2 is essential for nitrous oxide of R. sphaeroides f. sp. denitrificans (Yokota et al. respiration in R. capsulatus (Richardson et al. 1991) 1984). Until recently the periplasmic nitrate respira- (Fig. 3) and nitric oxide reductase (Bell et al. 1992). tory pathway was considered to be restricted to pho- The cytochrome bcl complex is a major component totrophic bacteria but it is now known that periplasmic of the ubiquinol-cytochrome ca oxidoreductase activi- nitrate reductase is quite widely distributed among non- ty which is linked to electron transfer to nitrous oxide phototrophic bacteria and allows nitrate respiration to (Fig. 3), nitrite and nitric oxide but the situation is occur under aerobic conditions (Bell et al. 1990). The rather more complicated. respiratory nitrate reductases of E. coli and Paracoc- cus denitrificans are quite distinct from the periplasmic enzyme described above. However, this nitrate 160

3.3 Evidence for a ubiquinol-cytochrome c as Paracoccus denitrificans (Parsonage et al. 1986) it oxidoreductase activity in Rhodobacter which is seems likely that the alternative ubiquinol-cytochrome independent of the cytochrome bcl complex c oxidoreductase is specifically associated with photosynthetic electron transport. Spectroscopic studies of The cytochrome bcl complex is an integral component intact cells of R. capsulatus have shown that the alter- of the cyclic electron transport system of Rhodobacter native ubiquinol-cytochrome c oxidoreductase con- and sincefbc mutants are unable to grow phototroph- tains a b-type cytochrome (c~ma~ = 557,560 nm) and ically (Prince & Daldal 1987) then it can be regard- electron transfer via this cytochrome is sensitive to ed as essential. However, a light-induced membrane HOQNO (Richardson et al. 1989). These properties potential was generated in an fbc mutant of R. cap- are almost identical to those of the cytochrome b- sulatus and was maintained during continuous illumi- containing oxidoreductase which operates in nitrate nation (Richardson et al. 1989). This indicates that respiration (Richardson et al. 1991). Taken togeth- cyclic electron transfer can occur in R. capsulatus er, these data suggest that a single type of alterna- in the absence of a functional cytochrome bcl com- tive ubiquinol-cytochrome c oxidoreductase functions plex. However, the Ap generated is rather small (low- in these pathways of anaerobic respiration (Fig. 3). er than that observed for N20 respiration) and is of Furthermore, the link between nitrate respiration and insufficient magnitude to support phototrophic growth photosynthesis is clearer following the observation of (Richardson et al. 1989). It has been estimated that, Rott et al. (1992) who showed that antibodies raised at concentrations of myxothiazol which are sufficient against isocytochrome c2 from R. sphaeroides cross- to completely block turnover of the cytochrome bcl reacted with cytochrome c552 from R. sphaeroides f. complex, residual cyclic electron transfer activity can sp. denitrificans. This leads to the view that the c- be observed which constitutes about 3% of the unin- type cytochrome which functions in electron transfer hibited rate (Myatt et al. 1987). This cytochrome bcl- to nitrate reductase in R. sphaeroides f. sp. denitrificans independent pathway makes a small contribution to is isocytochrome c2. The above data suggest that in R. cyclic electron transport but its presence is revealed capsulatus and R. sphaeroides a ubiquinol-cytochrome when pathways of lower flux such as nitric oxide res- c oxidoreductase may be present which can operate as piration and nitric oxide respiration were investigated an alternative to the cytochrome bcl complex in photo- in R. capsulatus. The rate of turnover of this alternative synthesis and in anaerobic respiratory pathways other ubiquinol-cytochrome c oxidoreductase was sufficient than DMSO respiration. to support about 30% of the rate of N20 reduction Why should an enzyme with such a low activity in myxothiazol=inhibited cells compared to uninhibit- compared to cytochrome bcl complex be involved in ed cells (Richardson et al. 1989). Since nitrous oxide photosynthetic electron transport? The answer may lie respiration is absolutely dependent upon cytochrome in the need to prevent inhibition of the turnover of c2 in R. capsulatus (Richardson et al. 1991) it seems the cytochrome bCl complex which might occur under probable that this cytochrome accepts electrons from phototrophic conditions at high values of Ap and when the alternative ubiquinol-cytochrome c oxidoreductase the Q-pool was highly reduced. This could lead to an (Fig. 3). The situation regarding nitric oxide respi- attenuation of the Q-cycle. The presence of an alterna- ration is less clear because this respiratory pathway tive pathway of ubiquinol oxidation, probably involv- can operate in mutants whick lack cytochrome bcl and ing a linear pathway of electron flow, would enable cytochrome c2. One possibility is that cytochrome c, the cell to avoid such a situation. The requirement for which is suggested to operate in cyclic electron trans- an alternative ubiquinol-cytochrome c oxidoreductase port in parallel with cytochrome c2 (Jones et al. 1990) activity would be most acute during growth on highly (Fig. 1) can also function in nitric oxide respiration reduced carbon sources such as alcohols and aliphatic as a mediator of electron transfer between the alterna- acids. tive ubiquinol-cytochrome c oxidoreductase and nitric oxide reductase. The observations described above raise the question 4. Concluding remarks of the nature of this alternative ubiquinol-cytochrome c oxidoreductase and its physiological function. Since The molecular characterisation of the protein complex- there is no evidence for an alternative to the cytochrome es involved in photosynthetic electron transfer and CO2 bci complex in non-photosynthetic denitrifiers such fixation is now at an advanced stage. Recent progress in 161 the understanding of the expression of photosynthesis Bauer CE & Marrs BL (1988) Rhodobacter capsulatus pufoper- and CO2 fixation genes should lead to a more com- on encodes a regulatory protein (PufQ) for bacteriochlorophyll synthesis. Proc. Natl. Acad. Sci. 85:7074-7078 plete understanding of how adaptation to phototroph- Beatty JT & Gest H (1981) Biosynthetic and bioenergetic functions ic conditions is coordinated. Although there is less of citric acid cycle reactions in Rhodopseudomonas capsulata. J. detailed information concerning the molecular proper- Bacteriol. 148:584-593 ties of anaerobic respiratory pathways their importance Belasco JG, Beatty JT, Adams CW, Gabain A yon & Cohen SN (1985) Differential expression of photosynthetic genes in in the regulation of phototrophic growth is now estab- Rhodopseudomonas capsuIata results from segmental differences lished. This work has revealed that during phototrophic in stability within a polycistronic transcript. Cell 40:171-181 growth Rhodobacter uses a variety of pathways includ- Bell LC, Richardson DJ & Ferguson SJ (1990) Periplasmic and ing CO2 fixation and anaerobic respiration to achieve membrane-bound respiratory nitrate reductases in Thiosphera pantotropha: the periplasmic enzyme catalyses the first step in redox homeostasis. Finally, it is clear that although aerobic denitrification. FEBS kett. 265:85-87 the metabolic capabilities of purple non-sulfur bacte- -- (1992) Identification of nitric oxide reductase activity in ria appear to be quite well documented new capabilities Rhodobacter capsulatus: the electron transport pathway can continue to be found. In this context it is particularly either use or bypass both cytochrome c2 and the cytochrome bc~ complex. J. Gen. Microbiol. 138:437-443 important that species other than R. sphaeroides and R. 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