INTRODUCTION

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For decades, it was widely assumed that purple bacterial anoxygenic photosynthesis is fundamentally an anaerobic metabolic process, resulting in growth under illuminated anaerobic conditions (72, 76, 139). The function of the anoxygenic photosynthetic apparatus is the transformation of light energy into an electrochemical gradient of protons across the photosynthetic membrane (PM), which can be used for ATP production, active transport, motility, and other energy-consuming processes. Oxygen partial pressure is the major factor that regulates the formation of the photosynthetic apparatus and the cell differentiation of most facultative purple phototrophic bacteria capable of respiratory and photosynthetic modes of energy transduction (38), although two species which form the photosynthetic apparatus under both aerobic and anaerobic conditions are exceptions to this generalization (65, 124).

However, many representatives of a new physiological group of bacteria that produce bacteriochlorophyll (Bchl) a and carotenoid pigments have been isolated relatively recently and designated aerobic anoxygenic phototrophic bacteria (48, 146, 148, 208, 212, 214-216, 223, 231). The novel aspect of this increasingly large group of bacteria is the inability to use Bchl for anaerobic growth. It is astounding that, of all the species which synthesize Bchl that have been isolated, there is a marked discontinuity in respect to photosynthetic energy transduction. That is, either an isolate grows robustly in a light-dependent fashion under anaerobic conditions (in which case it is thought to be a "typical" anoxygenic phototrophic bacterium), or it is incapable of anaerobic photosynthesis and light stimulates at best a transient enhancement of aerobic growth after a shift from the dark to illumination (in which case it is grouped with the aerobic anoxygenic phototrophic bacteria).

Although the composition of the photosynthetic apparatus and electron transfer carriers (51, 130, 131, 210, 211, 226), as well as the amino acid sequences of the reaction center (RC) and light-harvesting (LH) I polypeptides (104) of aerobic phototrophic bacteria, is similar to those of anaerobic purple phototrophic bacteria, efficient photoinduced electron transfer is operative only under aerobic conditions in the aerobic phototrophic bacteria (51, 131, 226).

For simplicity and clarity, we frequently refer to anoxygenic purple phototrophic bacteria that are capable of anaerobic photosynthesis as anaerobic phototrophic bacteria and to aerobic bacteria that contain Bchl and photosynthetic complexes as aerobic phototrophic bacteria (see "Conclusions and perspectives" for a discussion of trivial nomenclature).

The presence of Bchl a has also been detected in some physiologically distinct bacterial groups such as aerobic methylotrophic bacteria and rhizobia (43, 149, 184, 205). A review on rhizobial photosynthetic bacteria was recently published (see Addendum in Proof). In this article, only nonmethylotrophic and nonsymbiotic species, the so-called "Erythrobacteria" isolated from aquatic environments, are reviewed. Because of the rich history of studies on facultative anaerobic purple nonsulfur phototrophic bacteria, where appropriate this review compares and contrasts metabolic processes and the physiology and phylogeny of this group with those of the aerobic phototrophic bacteria.

GENERAL CHARACTERISTICS OF THE AEROBIC PHOTOTROPHIC BACTERIA

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Habitats

The discovery of obligately aerobic bacteria containing Bchl a was first reported by T. Shiba et al. (148), in whose work the isolation and enumeration of these microorganisms on seaweed and in seawater, sand, and bottom sediments of Tokyo Bay and adjacent areas were described. Sixteen strains of aerobic pink or orange bacteria that contained Bchl a were isolated from these aerobic marine environments with a rich medium (148) and were found to be abundant on thalli of Enteromorpha linza and Sargassum horneri and in beach sand. The proportions of these bacteria among the species that formed colonies on the medium employed ranged from 0.9 to 1.1% in the seaweed samples and from 1.2 to 6.3% in the beach sand samples (148).

Subsequent reports broadened the geographical area and ecological niches in which obligately aerobic Bchl a-containing bacteria are found. The presence of aerobic heterotrophic Bchl a-synthesizing strains in high proportions (10 to 30%) of the total heterotrophic bacterial strains cultivated was described for marine environments on the west and east coasts of Australia (146) and at the Pacific Ocean inlet English Bay, in Vancouver, Canada (209).

Investigation of samples taken from freshwater cyanobacterial mats in hot springs of the Bol'shoi River valley (Lake Baykal region) and from Neskuchninskii Spring situated on Southern Kurily in Russia led to the discovery of freshwater strains of obligately aerobic bacteria that synthesize Bchl a (212, 214-216). The isolation and growth of freshwater species were obtained in a rich organic medium (212). The mats from which these isolates came were largely composed of cyanobacteria (e.g., Oscillatoria subcapitata), diatoms, and the purple phototrophic bacteria Thiocapsa roseopersicina and Rhodopseudomonas palustris. The mats were located at the boundary of anaerobic and aerobic zones at alkaline pH values ranging from 8.0 to 9.4 and hydrogen sulfide concentrations from 0.6 to 7.4 mg/liter, depending on the spring (213, 217). The samples of these mats contained up to 10⁶ cells of aerobic bacteria containing Bchl a per ml. Some strains were isolated from environments with considerably hot temperatures: strain KR-99 was isolated from an environment with a temperature of about 40°C and strains RB3 and RB7 came from a site with a temperature of 54°C (214, 215). However, in pure laboratory cultures all of these strains demonstrated typical mesophilic properties and grew optimally at 28 to 30°C (214, 215). Such thermotolerancy has been found for purple nonsulfur bacteria such as R. palustris, Rhodomicrobium vannielii, and Rhodopseudomonas viridis, which have also been detected in high-temperature environments and which have temperature optima of 30 to 35°C in pure culture (21, 57, 58). Why and how these latter species and obligately aerobic species survive in thermal environments are unclear.

Mildly thermophilic (growth temperature optima at 42 to 45°C) species of purple phototrophic bacteria, Rhodospirillum centenum, Rhodopseudomonas cryptolactis, and Rhodopseudomonas strain G1, have been isolated (21). Two moderately thermophilic or thermotolerant aerobic anoxygenic representatives, strain OT3 and strain JF-1, were discovered recently (63, 228). Strain OT3 was isolated from bacterial mats in the brackish Usami hot spring (Japan). The temperature at the sampling site was 42.7°C, the pH was 5.8, and the bacterial mat consisted mainly of a dark green layer of thermophilic filamentous cyanobacteria. The new isolate OT3 grew at temperatures up to 50°C, and optimal growth occurred at 40 to 48°C (63). Aerobic anoxygenic phototrophic strains containing Bchl a were discovered in hydrothermal black smoker plume waters of the Juan de Fuca Ridge in the Pacific Ocean (208). Water samples taken from about 2,000 m beneath the ocean surface were found to contain aerobic bacteria producing Bchl a in numbers of 20 to 40 cells/ml of the samples, about 30% of the pigmented strains that formed colonies on the rich medium used. The representative strain JF-1 revealed a broad tolerance for culture conditions such as salinity, temperature, and pH. Thus, growth was obtained in a freshwater medium and a medium supplemented with 10% NaCl, at temperatures ranging from 5 to 42°C and at pH values of 5.5 to 10.0. Therefore, JF-1 is a salt- and pH-tolerant and thermotolerant strain (222).

Several strains of pelagic bacteria were purified from the surface of a freshwater subtropical pond in Australia (48), and acidophilic heterotrophic bacteria that synthesize Bchl a were isolated from an acidic mine drainage system (190). Aerobic phototrophic bacteria were detected in high numbers relative to the numbers of other heterotrophic strains in the North Adriatic Sea, where they comprised 5 to 55% of the total cells cultivated (103).

The strains isolated from freshwater cyanobacterial mats in Russia are obligately freshwater species. For example, the growth of strain RB16-17 was strongly inhibited by salt concentrations higher than 1% (212). Salt-tolerant strains (T1 through T7) were isolated from a cyanobacterial mat located in the supralittoral zone on the West Frisian island of Texel in The Netherlands and comprised 2 to 23% of the aerobic pigmented strains that formed colonies on the medium used (231). This microbial mat was known to be flooded twice a month by the North Sea. Because of alternating heavy rainfall and evaporation, the salinity of this environment varies from 8 to 10 to more than 100. The organisms isolated from this mat are able to grow over a broad salinity range, from 5 (freshwater) to 96 (Table 1). This ability may reflect an adaptation to an environment with fluctuating salinity. Similarly salt-tolerant strains of obligately aerobic Bchl a-containing bacteria (strains 15s.b. and 23s.b.) were isolated from English Bay in Vancouver. The strains were found on the surfaces of seaweeds and sand alternately exposed to air or covered by water during low or high tides, respectively. During summer low tides, the bacterial environment is dried for several hours, consequently presenting an econiche with fluctuating salinity (207). Such salt-tolerant strains can be described as facultatively marine or freshwater organisms.

In summary, most strains of aerobic anoxygenic phototrophic bacteria isolated so far inhabit a wide variety of eutrophic aquatic environments and seem to comprise a significant part of the aerobic heterotrophic bacterial population. An exception is strain JF-1, isolated from apparently oligotrophic deep-sea hydrothermal vent plume waters. In spite of the broad geographical distribution of aerobic phototrophic bacteria in different ecological niches and their presence in high numbers, the ecological importance of this group of organisms (their role in microbial populations) has not been studied.

A wide variety of media rich in organic components such as yeast extract, peptone, Casamino Acids, salts of tricarboxylic acids (TCAs), or sugars have been used to isolate pure cultures of different aerobic phototrophic species (48, 63, 146-148, 189, 208, 212, 230). They can be isolated by direct inoculation of water samples (for free-floating strains) or homogenized mat or sand samples (for the strains found in cyanobacterial communities or on solid surfaces) with dilutions on agar plates of rich organic media (48, 63, 146-148, 189, 208, 212, 230). As a rule, inoculated plates have been incubated in the dark at temperature and pH values similar to those of the environment from which samples were collected. Pigmented colonies are streaked on agar plates to obtain pure isolates. Therefore, pure cultures are easily obtainable. When a pure culture is obtained, a single colony is transferred into liquid medium and cultivated aerobically in the dark. Aerobic phototrophic strains are distinguished from other heterotrophic bacteria by the presence of Bchl a, as indicated by absorption peaks in the region from 800 to 880 nm in cell suspensions or by an absorption peak around 770 nm in acetone-methanol extracts of cells.

It has been found that liquid (taken from late-logarithmic growth phase) and agar surface cultures of most aerobic phototrophic species remain viable after storage at 4°C for at least 2 months (207). Long-term preservation is possible by storage in liquid nitrogen or freezing at 70°C. For this purpose, dense cell suspensions of liquid cultures (mid-logarithmic growth phase) are supplemented with glycerol (30%) as a cryoprotective agent. Lyophilization can also be used as a method of preservation.

At present, aerobic phototrophic bacteria are taxonomically classified in the two marine genera Erythrobacter and Roseobacter (144, 147) and the six freshwater genera Erythromicrobium, Roseococcus (214-216, 223, 230), Porphyrobacter (48), Acidiphilium (190), Erythromonas, and Sandaracinobacter (229) (Table 2).

The discovery of obligately aerobic heterotrophs that synthesize Bchl a (148) stimulated research on their taxonomic and phylogenetic positions, to address the questions of their closest relatives and their evolutionary origin. Preliminary analyses of marine strains (144, 145, 147) and strains isolated from freshwater environments (223) used DNA GC content and DNA-DNA hybridization. These analyses showed that purple nonsulfur phototrophic bacteria seem to be the closest relatives. The GC content of DNA of aerobic phototrophic bacteria calculated from thermal melting points ranges from 57 to 60 mol% in Erythrobacter species to 70.4 mol% in Roseococcus thiosulfatophilus (Table 2). Although similar GC composition does not necessarily indicate relatedness, a high GC content is characteristic of DNA purified from purple nonsulfur bacteria, ranging from 59 to 60 mol% for Rhodoferax fermentans to 70 to 72 mol% for Rubrivivax gelatinosus (73). DNA-DNA hybridization in combination with morphological and physiological observations led to proposals for the establishment of four new genera: the marine Erythrobacter and Roseobacter (144, 147) and the freshwater Erythromicrobium and Roseococcus (214-216, 223).

The isolation of new strains, and detailed biochemical, physiological, and molecular biological analyses (e.g., 5S rRNA and 16S ribosomal DNA [rDNA] sequence comparisons) in comparison to existing strains, resulted in the current classification of aerobic phototrophic bacteria and phylogenetic relations with other bacterial taxa (48, 80, 159, 183, 229, 230). Phylogenetically, all aerobic phototrophic species are associated with members of the  subclass of the class Proteobacteria. R. thiosulfatophilus is a member of subclass -1 and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and members of the genus Acidiphilium (Fig. 1). Erythromicrobium, Erythrobacter, and Porphyrobacter are very closely related genera and are clustered in the -4 subclass, more distant from other aerobic phototrophs. The relatively isolated position of this subgroup in regard to other aerobic phototrophic species and its placement close to the branching point of the  subclass from the other subclasses of the Proteobacteria were shown in a study of Porphyrobacter strains (48) and subsequently (230) in a study of Erythromicrobium species (Fig. 1). The 16S rRNA sequence data placed Roseobacter denitrificans in a branch separate from -4 and -1 representatives and in a relatively close phylogenetic relationship with Rhodobacter sphaeroides and Rhodobacter capsulatus (Fig. 1) (48, 80, 159, 229, 230). A phylogenetic study performed on a psychrophilic gas vacuolate bacterium isolated from polar sea ice, Octadecabacter sp., placed this organism as a close nonphototrophic relative of Roseobacter species (59) (Fig. 2). Recently, the nonphotosynthetic Sphingomonas group was included in the -4 subclass (201), such that the Erythromicrobium-Erythrobacter-Porphyrobacter cluster is most closely related to members of the genus Sphingomonas (230).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   16S rDNA phylogenetic positions of representatives of the  subclass of the Proteobacteria, as determined by the neighbor-joining method. Scale bar = 10% difference in nucleotide sequences. The total distance between two organisms is the sum of the horizontal branch lengths (230).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   16S rDNA dendrogram of relatedness showing the phylogenetic positions of E. ursincola and S. sibiricus released from the genus Erythromicrobium, within the radiation of members of the genus Sphingomonas and related taxa. Numbers refer to bootstrap values, of which only those above 80% are shown. Bar = 5% inferred sequence divergence. (This tree was created by E. Stackebrandt.)

Based on phenotypic similarities, the five isolated strains RB16-17, KR-99, E1, E4(1), and E5 were placed in the same genus, Erythromicrobium, and described as Erythromicrobium sibiricum, Erythromicrobium ursincola, Erythromicrobium ezovicum, Erythromicrobium hydrolyticum, and Erythromicrobium ramosum, respectively. However, DNA-DNA hybridization showed that DNA from the species E. sibiricum and E. ursincola had low homology (11 to 17%) with the other three species of this genus (223). Additionally, an analysis of 5S rRNA sequences indicated a phylogenetic heterogeneity of the Erythromicrobium genus (183). Combination and comparison of new results on the morphology, physiology, biochemistry, molecular biology, and phylogenetic relationships of five Erythromicrobium representatives resulted in the elevation of the tentative species "E. sibiricum" and "E. ursincola" to type species of two new genera: Sandaracinobacter sibiricus and Erythromonas ursincola, respectively (229). These two genera form two separate sublines within the radiation of Sphingomonas species (Fig. 2). The branching point of Erythromonas is between Sphingomonas subclusters 1 and 3 and subcluster 2, while that of Sandaracinobacter is between subclusters 1, 2, 3, and 4. The closest relative of E. ursincola is the nonphotosynthetic Blastomonas natatoria, whereas S. sibiricus stands phylogenetically isolated, with less than 93.5% 16S rDNA sequence identity with any of the reference organisms (Fig. 2) (229).

Phylogenetic 16S rDNA sequence comparisons determined that E. ursincola clusters with B. natatoria (99.8% sequence identity) (229). However, significant physiological differences that exist between E. ursincola and B. natatoria preclude their assignment to the same genus. B. natatoria contains carotenoid pigments but lacks Bchl, whereas E. ursincola is physiologically similar to aerobic anoxygenic phototrophic bacteria because it contains carotenoids and Bchl a. E. ursincola contains Bchl a incorporated into a photochemically active RC and LH complexes and contains electron transfer components of a cyclic photosynthetic pathway (such as a cytochrome [cyt] c bound to the RC, a soluble cyt c₂, the RC quinone primary electron acceptor [Q_A], and the special pair P of the RC). The high 16S rDNA sequence similarity between E. ursincola and B. natatoria indicates a close phylogenetic relationship and a common ancestor. However, due to the existence of significant physiological differences (photosynthesis is a restricted mode of energy generation), they were not designated as members of the same genus (229).

Although 16S rDNA sequences of E. ursincola and B. natatoria had a high level of identity (99.8%), DNA-DNA hybridization of their entire DNA indicated a low level of homology, 40 to 43% (158). This result shows that the taxonomic designation of Erythromonas and Blastomonas as separate genera is correct and that DNA-DNA hybridization analysis should be used in similarly questionable situations.

At present, the taxonomic importance of photosynthetic pigments is controversial. On the one hand, because 16S rRNA sequence analysis indicates close relationships between phototrophic and nonphototrophic bacteria, it has been proposed that a taxonomic rearrangement of phototrophic bacteria which would include phototrophs and nonphototrophs in the same genus should be performed (80). Some authors speculate that, because 16S rRNA differences between phototrophic and nonphototrophic species are very small, this may indicate that independent loss or gain of an essential photosynthesis gene, or cluster coding for the photosynthetic apparatus, occurred in one of two otherwise "identical" species (159). On the other hand, the phylogenetic congruence of most of the genera defined by photosynthetic organisms favors the traditional taxonomic emphasis on this property (74, 159).

In our opinion, the photosynthetic nature of bacteria should be considered a valid and irrefutable taxonomic marker in bacterial classification. Of course, if it is clear that only a small part of the genome, as much as a photosynthesis gene cluster, was lost by a progenitor of a species and that the majority of the genome is still identical, the relationship between two otherwise largely isogenic isolates should be designated taxonomically. The distinction between loss (or gain) of a relatively small part (~1.3%) of a genome and genuinely large differences in genotype (and phenotype) comes down to an exercise in hair splitting. Because of the almost seamless division between some species, it seems that there will always be a certain element of controversy in the assignment of new isolates which are closely related to previous isolates either by taxonomic or by phylogenetic criteria to specific genera. Because the presence of photosynthetic pigments not only results in colors visible to the eye (the presence of absorption peaks is also readily obtained in absorption spectra) but also usually determines the ability of a species to utilize a restricted mode of energy generation, the presences of Bchl, RC and LH pigment-protein complexes, and related cyclic electron transfer components should remain as valid taxonomic criteria.

In the context of this discussion, we think that the assignment of a recently isolated strain to the genus Roseobacter, designated as Roseobacter algicola (99), is not appropriate. If a major phenotypic difference exists between two strains, they should be in different generathis is the rule generally followed. The rules of nomenclature require that the description of a new species of an existing genus should correspond to the main genus characteristics previously published in an original article or in Bergey's Manual of Determinative Bacteriology. Although the genus Roseobacter is described as a genus of the aerobic anoxygenic phototrophic bacteria containing Bchl a and carotenoids, with a photosynthetic apparatus that functions under aerobic conditions (51, 130, 131, 144, 149), the physiologically and morphologically different (as shown in electron micrographs [EMs]) nonphotosynthetic strain, which does not synthesize Bchl or carotenoids, was described as a new member of the genus Roseobacter (99). The main reason given for this unification was the high similarity of 16S rRNA subunit sequences of R. denitrificans to the sequence of the newly isolated strain, although DNA G+C content was not determined. Therefore, we think the designation of "R. algicola" as a Roseobacter species was an inappropriate taxonomic assignment. Although in many cases there is reason to assign two phylogenetically closely related bacteria to the same genus, the two species should not be greatly different with respect to their physiology for determinative and taxonomic purposes. Since there is no standard phylogenetic distance that defines taxonomic ranks, classical taxonomic criteria must be used. Therefore, the new isolate "R. algicola" may be a close phylogenetic neighbor to Roseobacter, but due to the existence of significant physiological differences in comparison to genuine Roseobacter species, this isolate should not be designated as a member of this genus.

In summary, phototrophy is an important and easily recognizable taxonomic marker which should continue to play a significant determinative role in bacterial classification.

Although all aerobic phototrophic bacteria so far examined possess a gram-negative cell wall, their morphologies are very diverse (Table 2; Fig. 3 and 4). Erythrobacter and Sandaracinobacter species are typical rods (very thin in the case of S. sibiricus: 0.3 to 0.5 by 1.5 to 2.5 µm) and produce long chains of up to 10 cells (147, 212, 229, 230) (Fig. 3). The genus Roseococcus contains bacteria with coccoid cells, 0.9 to 1.3 by 1.3 to 1.6 µm in size. The mode of cell division in these three genera is binary fission (147, 212, 214). The cell shape of species in the genera Roseobacter (0.6 to 0.9 by 1.2 to 2.0 µm), Porphyrobacter (0.4 to 0.8 by 1.1 to 2.0 µm), and Erythromonas (0.8 to 1.0 by 1.3 to 2.6 µm) is ovoid rod. Budding in addition to binary division occurs in Porphyrobacter neustonensis and E. ursincola (48, 144, 229). The representatives of Erythromicrobium are very long rods producing characteristic thread-like cells, dividing by symmetric or asymmetric constrictions (Fig. 4). For E. ramosum and E. hydrolyticum, ternary fission and branching were demonstrated (215, 229, 230).

View larger version (148K): [in this window] [in a new window]   FIG. 3.   EMs showing the morphological diversity of aerobic anoxygenic phototrophic bacteria. (A) Distribution of S. sibiricus cells in a microcolony. (B) Single and thread-like cells of S. sibiricus. (C) Pleomorphic cells of strain JF-1 are connected by membranous material (indicated by arrows). (D) A cell of strain JF-1 containing a single flagellum. (E) Coccus cells of R. thiosulfatophilus. (F) Nonmotile cells of the strain 15s.b. embedded in a capsule-like matrix. (A, B, and E) Scanning EMs of carbon-shadowed cells. (C, D, and F) Transmitting EMs of negatively stained cells. Bars, 1 µm.

View larger version (175K): [in this window] [in a new window]   FIG. 4.   Different types of cell division revealed by electron microscopy of thin sections of aerobic anoxygenic phototrophic species. (A) A strain JF-1 Y cell presumably preceding division to form three daughter cells. The nucleoid is seen as light zones of the section, distributed in three directions. (B) A later stage of Y-cell division. One daughter cell is separated by the cell wall from two as-yet-unseparated nascent cells. (C) E. ezovicum dividing by constrictions. (D) Binary division of the strain JF-1. Bars, 0.5 µm.

The new strain (JF-1) of aerobic anoxygenic phototrophic bacteria isolated recently from deep-ocean hydrothermal vent plume waters is unusually pleomorphic. Depending on the age of cultures and composition of liquid medium, the cells can be found as almost coccoid (0.4 to 0.5 by 0.5 to 0.8 µm), as ovoid rods (0.4 to 0.5 by 1.0 to 1.2 µm), as bean shaped, or as thread-like formations of up to five cells. This microorganism is very flexible in its character of cell division, since budding, ternary fission, binary division, and symmetric and asymmetric constrictions were observed. Strain JF-1 forms Y cells, a rare type of bacterial multiplication, which results in the possibility of three daughter cells being produced from one mother cell (Fig. 3 and 4). Cells often remain attached after division, perhaps by a membranous connective material. Therefore, individual cells remain in close contact after division within a free-floating population (Fig. 3) (208).

Most species of aerobic phototrophic bacteria are motile, usually by means of one polar or subpolar flagellum (147, 208, 214-216, 230). R. denitrificans and S. sibiricus (formerly E. sibiricum) have up to three subpolar flagella (144, 212, 229).

The strains 15s.b. and 23s.b., recently discovered at English Bay, Vancouver, Canada (209), and Porphyrobacter tepidarius (63) are the only nonmotile aerobic phototrophic bacteria. The strains 15s.b. and 23s.b. form ovoid cells (0.4 to 0.6 by 1.2 to 1.5 µm) surrounded by capsules and frequently produce a matrix in which cells are embedded (Fig. 3). Therefore, these strains are nonmotile and highly agreggative in liquid culture (209).

In summary, aerobic phototrophic bacteria differ greatly in morphology and in their mode of cell division. However, the diversity of bacterial morphology is not yet exhausted by the group, and the discovery of new strains with vibrio or spirillum morphologies, and conceivably of gram-positive species, is possible.

PIGMENTS AND PHOTOSYNTHETIC PIGMENT-PROTEIN COMPLEXES

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Carotenoids and Bacteriochlorophyll

Carotenoids comprise a diverse class of pigments found in photosynthetic and nonphotosynthetic prokaryotic and eukaryotic organisms. The functions of carotenoids in protection from photooxidative damage and in light absorption and as a structural component of the PM in anoxygenic phototrophic bacteria have been reviewed previously (4, 23).

All species of aerobic phototrophic bacteria synthesize large amounts of carotenoid pigments, which determine the color of the organism and give peaks in the blue and green regions (420 to 550 nm) of absorption spectra (Fig. 5) (48, 144, 147, 212, 214-216, 229-231). The carotenoid composition is species specific, often indicating a large number of different carotenoids of unusual chemical structure (167-170, 211).

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Absorption spectra of membranes isolated from R. thiosulfatophilus (A), E. litoralis (B), E. hydrolyticum (C), and E. ezovicum (D). Cells were cultivated under the same dark-aerobic condition. A. U., absorbance units.

About 20 different carotenoids have been found in the red-orange E. ramosum (207), of which the 10 predominant ones were purified and structurally characterized (Table 3) (211). All of these purified carotenoids were identified as C₄₀ carotenoids, which were classified into four groups: (i) bicyclic carotenoids (-carotene and hydroxyl derivatives such as zeaxanthin, adonixanthin, caloxanthin, and nostoxanthin), (ii) the monocyclic carotenoid bacteriorubixanthinal, (iii) the acyclic spirilloxanthin, and (iv) the polar carotenoid erythroxanthin sulfate (Fig. 6B). A carotenoid composition similar to that of E. ramosum was described for Erythrobacter longus (168-170) and Erythrobacter litoralis (230), with the exception of adonixanthin and 2,3,2',3'-tetrahydroxy-,-carotene-4-one. Zeaxanthin is a major carotenoid in Erythrobacter, whereas in Erythromicrobium, zeaxanthin is a minor component and bacteriorubixanthinal, erythroxanthin sulfate, and 2,3,2',3'-tetrahydroxy-,-carotene-4-one are the major compounds (211).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Chemical structures of highly polar carotenoids (211). (A) C30 carotenoid (4,4'-diapocarotene-4,4'-dioate) (compound I) and the corresponding diglucosyl ester (compound II) of R. thiosulfatophilus. (B) Erythroxanthin sulfate found in cells of E. ramosum, E. longus, and E. litoralis.

Bicyclic carotenoids such as -carotene and its hydroxyl derivatives were found in Erythrobacter and Erythromicrobium species, and the color of Erythromonas and Sandaracinobacter indicates that these bacteria also contain carotene carotenoids, which are rarely present in purple phototrophic bacteria (small amounts of -carotene were detected in R. vannielii [17, 136]). Zeaxanthin and -carotene are widely distributed among oxygenic phototrophs, green plants, cyanobacteria and algae, and some strains of Flavobacterium (90). The highly polar carotenoid sulfates have hitherto been found exclusively in the carotenoids of the aerobic phototrophic bacteria (168, 211, 230) and were recently described as carotenoids of novel structures (168).

The carotenoid composition of two other Erythromicrobium representatives, E. ezovicum and E. hydrolyticum, has not yet been analyzed in detail. Nevertheless, in vivo absorption spectra revealed carotenoid absorption peaks at 466 and 478 nm, as in E. ramosum, indicating similar carotenoid compositions of these species, also apparent in the color of liquid cultures (intensely red-orange) (Table 2) (229).

The carotenoid composition of the pink-red Roseococcus species is not as rich as those of Erythrobacter and Erythromicrobium species but nevertheless is unusual. R. thiosulfatophilus contains mainly two very polar red pigments, C₃₀ carotene-dioate (4,4'-diapocarotene-4,4'-dioate) and the respective diglucosyl ester (di[-D-glucopyranosyl]-4,4'-diapocarotene-4,4'-dioate) (Fig. 6A). Together, they contribute 95% of the total carotenoid content (211). Such highly polar C₃₀ carotenoid glycosides have never before been observed in purple phototrophic bacteria, although the same carotenoid and its diglucosylated form have previously been postulated to exist in Methylobacterium rhodinum (formerly Pseudomonas rhodos) (84).

The most abundant carotenoid species detected in Roseobacter sp. is spheroidenone, which is the major carotenoid of anaerobic purple bacteria such as Rhodobacter species (68, 144, 167).

The only carotenoid of Acidiphilium rubrum is spirilloxanthin, which is found in Rhodospirillum rubrum and several other purple phototrophic bacteria (191).

Bchls have long-lived excited states and appropriate oxidation-reduction potentials that are suited to their function in LH energy transfer and electron transfer reactions. The only Bchl found in aerobic phototrophic bacteria thus far is Bchl a, on the basis of in vivo absorption spectra of intact cells and from organic solvent extracts. Since Bchl a is incorporated into species-specific types of pigment-protein complexes (see "Light-harvesting systems" and "Reaction center"), the corresponding in vivo absorption peaks are in the near-infrared region from about 800 to 870 nm (Fig. 5). Upon extraction with organic solvent, a far-red absorption peak at about 770 nm is obtained, as well as a peak at 370 to 390 nm (48, 63, 144, 145, 147, 148, 190, 212, 214-216, 228-231), consistent with the identity of this pigment as Bchl a.

Typically, cells of aerobic phototrophic bacteria contain small amounts of Bchl relative to the abundance of carotenoids (compared to anaerobic purple phototrophic bacteria). For example, the anaerobic phototrophic bacterium R. sphaeroides may yield about 20 nmol of Bchl/mg (dry weight) of cells, whereas the Bchl content of obligately aerobic species was found to be as follows: E. longus, 2.0 nmol/mg (dry weight) of cells; S. sibiricus and E. hydrolyticum, 1.0 to 4.0 nmol/mg of protein; A. rubrum, 0.7 nmol/mg (dry weight) of cells; R. thiosulfatophilus, 0.1 to 1.0 nmol/mg of protein. Therefore, the ratio of Bchl to carotenoid peaks in whole cells of aerobic phototrophic species is typically about 1:8 to 1:10 (66, 149, 177, 190, 206, 214, 216, 230, 232). (However, see "The influence of light on growth and pigment formation" and "Effect of oxygen on growth and pigment synthesis" for a discussion of oxygen and light effects on Bchl content of cells.)

Bchl a purified from E. longus, R. denitrificans, and Acidiphilium species was found to be Bchl a_p, which contains phytol as the esterifying alcohol, the most common Bchl a form found in purple phototrophic bacteria (66, 70, 90, 97, 190). No Bchl a esterified to geranylgeraniol, as it is in the anaerobic phototrophic bacterium R. rubrum (19), has been detected in aerobic phototrophic species. However, the ester moiety of Bchl from most species of Erythromicrobium, Erythrobacter, Roseococcus, Sandaracinobacter, and Erythromonas has not yet been determined.

Until recently, all natural chlorophylls were thought to be porphyrin derivatives containing a magnesium atom at the center of a chlorin macrocyclic ring. Among the semisynthetic chlorophyll derivatives containing metals other than Mg, only Zn-containing chlorophylls have photochemical properties comparable to those of Mg-chlorophylls (195). Zn-containing Bchl a was introduced artificially into isolated antenna or RC proteins to replace Mg-Bchl a (122, 142), but until recently, natural photosynthesis without Mg-chlorophylls was unknown. However, a natural Zn-containing Bchl a was discovered in the aerobic acidophilic bacterium A. rubrum (191). This Zn-containing Bchl a is esterified with phytol (Zn-Bchl a_p). Chemical analysis of A. rubrum cell extracts yielded a 13:2:1 molar ratio of Zn-Bchl to Mg-Bchl to bacteriopheophytin, and most of these pigments were determined to be photochemically active (191).

In summary, the aerobic phototrophic bacteria contain an unusually diverse variety of carotenoids, but at present Bchl a (containing either Mg or Zn) is the only chlorophyll that has been found. The discovery of Zn-Bchl in A. rubrum raises the possibility that other Bchls or novel chlorins might exist in species that have not yet been carefully analyzed or discovered.

Most of the anaerobic purple phototrophic bacteria have, in addition to the cytoplasmic membrane (CM), an intracytoplasmic membrane (ICM) system, of species-specific morphology (38, 129). It is thought that the ICM is derived from and is contiguous with the CM. The ICM may form vesicles, tubules, or thylakoid-like sheets (38). The pigment-protein complexes of the photosynthetic apparatus of anaerobic phototrophic bacteria are incorporated into the ICM or, in a few cases, seem to be located in the CM (37, 192, 193). In most anaerobic phototrophic bacteria, light and oxygen tension regulate the formation of the ICM, such that ICM formation is induced when the oxygen tension is lowered and the most extensive ICM development occurs during anaerobic growth.

Aerobic phototrophic bacteria contain significantly lower amounts of Bchl than do typical anaerobic phototrophic bacteria (see "Carotenoids and bacteriochlorophyll"). As noted above, Bchl content commonly differs between anaerobic phototrophic and aerobic phototrophic bacteria by factors of 10 to 20. With such a low number of photosynthetic units (RC plus LH system), the absence of an extensive ICM system in obligately aerobic species is not surprising. An ICM system was not detected in thin-section EMs of Erythrobacter, Erythromicrobium, Roseococcus, Porphyrobacter, Erythromonas, or strain JF-1 (48, 63, 208, 214-216, 218, 229, 230). Occasionally, chromatophore-like vesicle structures have been observed in R. denitrificans (69, 149). Thin-section EMs of S. sibiricus (renamed E. sibiricum) revealed rare vesicular or loop-like CM invaginations (225).

Two kinds of intracellular membrane fragments produced by French press disruption of cells were designated in R. thiosulfatophilus and E. ramosum (Table 4) (210, 211), on the basis of two distinct membrane fractions separated in sucrose gradients. Both fractions isolated from R. thiosulfatophilus and E. ramosum were free of peptidoglycan and active in NADH dehydrogenase, confirming the CM nature of these fractions. One fraction banded at a sucrose concentration of 1.0 to 1.2 M (fraction I), and a second fraction banded at 1.2 to 1.5 M (fraction II). The RC and LH complexes were located mainly in the 1.0 to 1.2 M fraction of R. thiosulfatophilus and exclusively in the 1.2 to 1.5 M fraction of E. ramosum, whereas carotenoids were found in both fractions (211). The ratio of Bchl to carotenoids detected in whole cells (1:9 in both species) decreased to 1:4 in purified PMs of R. thiosulfatophilus and to 1:7 in the E. ramosum PMs. Most of the carotenoids were not bound to the PM but were located in cell wall and other peripheral membrane fractions (Table 4) (211). Membrane preparations from cell suspensions of Erythrobacter, Erythromicrobium, Erythromonas, and Sandaracinobacter species also gave rise to two membrane fractions in sucrose gradients (233). It is not clear how the two fractions relate to CM or ICM differentiation, but the data indicate a discontinuous organization of membranes in these species.

In conclusion, it is evident that the photosynthetic apparatus of aerobic phototrophic bacteria is present in lower amounts than that of typical anaerobic phototrophic bacteria and probably is located mainly in the CM or ICM structures that are not visible in thin-section EMs of cells.

The major light-absorbing pigments in anaerobic phototrophic bacteria are Bchl and carotenoids. These pigments are noncovalently attached to two types of integral membrane proteins, forming on the one hand the photochemical RC and on the other hand the LH (antenna) complexes (24). Purple bacteria have a relatively simple LH system consisting of a core antenna protein complex (LHI) closely associated with the RC and, in many species, one or more peripheral antenna complexes (LHII), all of which are located in the ICM (236). The LH complexes absorb light quanta, and the energy migrates through the pigments of the antenna system to the RC (24). The LH complexes are sometimes designated by their long-wavelength light absorption maxima, for example, B870 (LHI) and B800-850 (LHII) in R. capsulatus.

The isolation and characterization of new aerobic phototrophic species have led to the discovery of LH complexes with unusual absorption maxima. For example, in vivo absorption spectra of R. thiosulfatophilus cells yielded a major peak of Bchl a at 859 nm (214), and spectra of E. ramosum yielded two major peaks at 836 and 871 nm (215). Subsequent purification of LH complexes from these bacteria by detergent treatment of membranes and sucrose density gradient centrifugation revealed the existence of an R. thiosulfatophilus LHI complex with an absorption peak at 856 nm and an E. ramosum LHII complex with peaks at 798 and 832 nm (Fig. 7) (210). E. ramosum additionally contains an LHI complex with absorption characteristics (maximum at 871 nm) similar to those measured in many anaerobic phototrophic bacteria and other aerobic phototrophic bacteria.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Absorption spectra of isolated pigment-protein complexes recorded at room temperature (210). (A) LHI-RC complex of R. thiosulfatophilus. (B) E. ramosum LHI-RC. (C) LHII complex of E. ramosum.

The unusual absorption properties of the LHI (B856) complex isolated from R. thiosulfatophilus together with the polypeptide pattern (four polypeptides; two of about 8.0 kDa and two of about 7.0 kDa) indicated an unusual protein environment of Bchl. Preliminary Raman spectroscopy analysis of the R. thiosulfatophilus blue-shifted RC-B856 core complex suggested that the presence of free 2-acetyl carbonyl groups of Bchl may be responsible for the blueshifting in absorbancy (50). However, a blueshift of LHI Bchl a absorbancy in whole cells could be due to the presence of Zn-Bchl instead of Mg-Bchl, giving a blueshift of 5 to 15 nm, as reported for A. rubrum (191) (see "Carotenoids and bacteriochlorophyll"). Although this blueshift could result from a complex, simultaneous effect of several factors, the possibility of the presence of Zn-Bchl in R. thiosulfatophilus should be investigated.

The LHII complex (B798-832) of E. ramosum is composed of three polypeptides of about 16.0, 9.0, and 8.0 kDa (210). Three proteins, designated , , and , with molecular weights of 14,000, 7,000, and 5,000, were previously found to copurify with the LHII (B800-850) complex from the purple nonsulfur bacterium R. capsulatus (36, 155, 165). According to preliminary Raman spectroscopy results, the absorption features of the E. ramosum B798-832 LHII complex indicate the presence of H bonds to the 2-acetyl substituents of both Bchl molecules, which would explain the most redshifted electronic transition (36, 155, 165).

Because of the unusual properties of LH complexes of aerobic phototrophic bacteria, it would be of interest to determine the amino acid sequences of these antenna polypeptides. It also would be interesting to determine if E. ramosum forms a variety of LHII complexes dependent on temperature, oxygen, and light conditions, as do the anaerobic phototrophic bacteria Rhodopseudomonas acidophila and R. palustris (12, 42, 54, 166).

In a recent investigation, E. hydrolyticum and E. ezovicum were found to possess LHI and LHII complexes with absorption characteristics similar to those of E. ramosum (229) (Table 5).

R. denitrificans has a unique type of antenna in addition to the RC-LHI core (RC-B870). This complex exhibits one peak at 806 nm, is composed of two polypeptides (5.0 and 7.0 kDa), and is considered to be a peripheral antenna (LHII), since detergent-sucrose gradient-purified preparations that lacked RC showed a Raman spectrum similar to those of LHII complexes of purple bacteria and transferred energy to the RC-LHI complex with high efficiency (145, 150, 151, 153).

The LHI-RC core complex has been purified from E. longus (151), E. litoralis (233), E. ursincola, and S. sibiricus (229) (Table 5). These LHI-RC core complexes are similar to analogous core complexes of anaerobic phototrophic bacteria on the basis of absorption spectroscopy. A. rubrum produces only an LHI complex with absorption characteristics similar to those of the corresponding complexes of anaerobic phototrophic bacteria. The main difference is a blue-shifted absorption maximum of the main peak in the near-infrared region due to the presence of Zn-Bchl a in this complex (152, 191). The absorption spectra of intact cells or membranes of Porphyrobacter strains indicated the absence of LHII complexes, although pigment-protein complexes have not yet been purified from these bacteria (48, 190).

In summary, the LH systems of aerobic phototrophic bacteria are very diverse in regard to their light absorption properties and frequently differ from analogous complexes found in anaerobic phototrophic bacteria. However, the major principles of LH system organization seem to be similar between these two groups.

The photosynthetic RC is defined as the minimal functional unit that catalyzes light-induced electron transfer processes, leading to a stable charge separation, and, as C. R. D. Lancaster and H. Michel (100) nicely wrote, "their function lies at the heart of the photosynthetic process of converting solar energy into biochemically amenable energy" (for reviews, see references 41, 44, 100, and 133). The purple bacterial anoxygenic RC is an integral membrane pigment-protein complex that contains three protein subunits (L, M, and H) and the following cofactors: four Bchls, two bacteriopheophytins, two quinones, and one nonheme high-spin Fe²⁺ (100). The RC of anaerobic phototrophic bacteria is at present the structurally and functionally best-characterized membrane protein complex. High-resolution three-dimensional structures have been determined by X-ray crystallography for the RC of R. viridis and R. sphaeroides (160), and structure-function questions have been addressed by site-directed mutagenesis (200).

The presence of a functional RC in aerobic phototrophic bacteria was first shown for R. denitrificans and E. longus on the basis of light-induced absorption changes (69), and a purified RC preparation from R. denitrificans was described previously (175). The purification of an RC from Erythrobacter, Erythromicrobium, Sandaracinobacter, and Roseococcus species has been difficult. Explorations of modified techniques allowed successful RC purification from E. litoralis T4, E. ursincola KR99, and S. sibiricus RB16-17 (renamed E. sibiricum) (Fig. 8). The content and overall organization of chromophores in these preparations, as determined by linear dichroism analysis, appear to be very similar to those of anaerobic phototrophic bacteria (224).

View larger version (18K): [in this window] [in a new window]   FIG. 8.   Absorption spectra of the RC purified from E. litoralis (A) and S. sibiricus (renamed E. sibiricum) (B). a. u., absorbance units.

An RC preparation purified from E. litoralis did not contain a tightly bound cyt, whereas the RCs of S. sibiricus and E. ursincola possess tetraheme cyts c. Each of the tetraheme cyts contains two high-potential hemes (+330 and +305 mV for E. ursincola and +380 and +300 mV for S. sibiricus) and two low-potential hemes (+40 and 40 mV for E. ursincola and +30 and 40 mV for S. sibiricus) (224).

An RC preparation containing a bound cyt c was isolated from R. denitrificans, and absorption spectra of reduced and oxidized forms of the RC were similar to those of the RC of R. sphaeroides except for the contributions of cyt c and carotenoids (175). The cofactors of R. denitrificans RC were the same as those of the RC of anaerobic purple bacteria and contained the following numbers of molecules (per RC): four of Bchl, two of bacteriopheophytin, four of cyt c₅₅₄, and two of ubiquinone-10 and carotenoid(s); the cofactors also contained four different polypeptides of 26, 30, 32, and 42 kDa. The 42-kDa protein corresponds to tetraheme cyt c (175). The heme which possesses the highest redox midpoint potential (+290 mV, designated H1) has an  band absorption of 555 nm. The second high-potential heme (+240 mV [H2]) exhibits an  peak at 554 nm. The two low-potential hemes, L1 and L2, had similar high redox midpoint potentials (about +90 mV), with bands at 553 and 550 nm, respectively (51). The values of the midpoint potentials of hemes H1 and H2 are lower than those determined for anaerobic purple phototrophic bacteria (+370 and +320 mV for R. viridis [188]); +350 and +320 mV for Chromatium vinosum [39]). These values are also lower than the values calculated for the H1 and H2 hemes of the RC-bound cyt in the obligately aerobic species E. ursincola and S. sibiricus (see above). Furthermore, the values of the low-potential hemes are relatively high (+90 mV). Similarly high midpoint potentials were measured in R. gelatinosus (39) and Chloroflexus aurantiacus (47, 186).

The RC of anaerobic phototrophic bacteria is thought to be surrounded by a ring-shaped LHI complex at a fixed stoichiometry relative to the RC (24). A constant number of about 30 LHI Bchl molecules per RC has been measured for seven aerobic phototrophic species (233). These results are in good accordance with data reported for anaerobic phototrophic bacteria (1, 32, 46) and suggest a similar overall organization of the photosynthetic apparatus in aerobic and anaerobic phototrophic bacteria. Nevertheless, the LH complexes of some aerobic phototrophic bacteria have unusual spectral properties presumably due to different protein environments of the Bchl (see "Light-harvesting systems").

ELECTRON TRANSFER SYSTEM AND PHOTOSYNTHESIS

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Quinones

Quinones are found in many bacteria, plants, and animals (9). The characteristic feature of quinones is their function as redox carriers in electron transport within membranes in metabolic processes such as aerobic and anaerobic respiration and photosynthesis (79, 187, 235). The composition of quinones was shown to vary among different representatives of phototrophs. Some species of phototrophic bacteria contain only ubiquinone Q₁₀, whereas other species contain Q₈ or Q₉ as well as the menaquinone MK₈ or MK₉ (25, 75).

Most studies of quinone function in phototrophic bacteria have been conducted on anaerobic phototrophic bacteria, and very little is known about the structure, function, and synthesis of quinones in aerobic phototrophic bacteria. Freshwater species of aerobic phototrophs from the genera Erythromicrobium, Sandaracinobacter, Erythromonas, and Acidiphilium (56, 189) and marine species of Erythrobacter and Roseobacter (144, 147, 149) possess Q₁₀ as the major quinone species. No menaquinones or rhodoquinones have been found in these species. The ubiquinone Q₉ was detected as a minor quinone in addition to Q₁₀ in S. sibiricum, E. ezovicum, and E. ramosum. However, in E. hydrolyticum the content of Q₉ was twice as high as that of Q₁₀ (Table 5). The quinone Q₁₀ of E. hydrolyticum and E. ramosum seems to exist as a methylated form (56). The total average amount of ubiquinones in obligately aerobic species (0.02 and 0.7 µmol/g of dry cells in E. hydrolyticum and S. sibiricus, respectively) is much lower than that determined for anaerobic purple bacteria (2 to 4 µmol/g of dry cells in Rhodobacter species) (56). No effect of light on the quinone composition and content of obligately aerobic freshwater species was detected (56).

cyts are found in most organisms that carry out electron transport through membrane-bound chains of carriers, regardless of the ultimate oxidant. Thus, cyts are not only present in chloroplasts and mitochondrial and aerobic bacterial respiratory chains but are also found in facultative anaerobes, obligate anaerobes, facultative photoheterotrophs, and the cyanobacteria (60, 93, 117, 127, 187, 235).

A fundamental process in the transformation of light into chemical energy in anaerobic phototrophic bacteria is a cyclic series of electron transfer reactions linked to the transport of protons across a membrane. This process involves two highly conserved integral membrane multisubunit complexes, the photosynthetic RC (see "Reaction center") and the cyt bc₁ complex. Two diffusible components, ubiquinone in the hydrophobic domain of the membrane and cyt c₂ in the periplasmic space, usually connect these two transmembrane complexes on the acceptor and donor sides of the RC, respectively (30, 139). Electron transfer from the bc₁ complex to the RC may be mediated by more than one type of mobile cyt c in R. sphaeroides, R. capsulatus, R. rubrum, and R. palustris (117). In most species of anaerobic phototrophic bacteria studied, however, a cyt c does not directly reduce the photooxideized special pair (P⁺) of RC but instead donates an electron to a multiheme cyt bound to the RC, which is the immediate electron donor to P⁺ (127). The photosynthetic electron transfer system shares some carriers, such as mobile cyts c, quinone molecules, and the cyt bc₁ complex, with the respiratory electron transfer system (60).

Analyses of light-induced difference spectra in the presence of oxygen in whole cells of Erythromicrobium, Sandaracinobacter, Roseobacter, Roseococcus, Erythromonas, and Erythrobacter species, as well as redox titrations and gel electrophoresis of soluble, membrane, and LHI-RC purified fractions, resulted in the distinction of two different groups on the basis of electron transfer properties and cyt compositions (Table 5). The first group contains R. denitrificans, R. thiosulfatophilus, E. ursincola, and S. sibiricus (175, 226). These species possess a cyt c tightly bound to the RC that serves as the immediate electron donor to the photooxidized RC. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular masses of these cyt species are 42, 44, 37, and 40 kDa in R. denitrificans, R. thiosulfatophilus, S. sibiricus, and E. ursincola, respectively, which is similar to that of the analogous tetraheme cyt c of anaerobic phototrophic bacteria (175, 224, 226, 227). The second group includes E. longus, E. litoralis, E. ramosum, E. hydrolyticum, and E. ezovicum, which contain an RC that is directly reduced by a soluble cyt c, apparently a cyt c₂ (172, 226, 229). The RC of A. rubrum seems to contain tetraheme bound cyt c, which is readily detached in vitro (77).

A comparison of the soluble and membrane-bound cyt c populations of aerobic phototrophic bacteria reveals that all species contain a complex mixture of these two general classes of cyt c (Table 5). In two species (S. sibiricus and E. hydrolyticum), a single soluble cyt c with a midpoint potential of 295 mV was present in the soluble protein fraction and, therefore, probably involved in both respiration and photosynthesis (226). Only one soluble cyt c possesses a redox potential high enough (E_m = 210 mV) to sustain respiration and photosynthesis in R. thiosulfatophilus. Several soluble cyts c are found in E. litoralis, E. ursincola, E. ramosum, and E. ezovicum with midpoint potentials ranging from 340 to 275 mV, and so they could participate in both the respiratory and photosynthetic pathways. Unusually small soluble cyts c were isolated from R. thiosulfatophilus (cyt c₅₄₉ [6.5 kDa] and c₅₅₂ [4.0 kDa]) and E. ursincola (cyt c₅₅₀ of 6.5 kDa) (Table 5) (226). Similarly small cyts c have so far been purified from only Hydrogenobacter thermophilus (cyt c₅₅₀ of 6.0 kDa) and Methylomonas strain A4 (cyt c₅₅₄ of 4.0 kDa) (202).

No evidence of cyt cd₁ in soluble fractions of freshwater species was found, consistent with the inability of these bacteria to reduce nitrite (212, 214-216, 229, 230). In contrast, the soluble fraction of R. denitrificans (which is capable of denitrification) contains a cyt cd₁ reductase (34). Detailed studies showed that there are two types of cyt cd₁, which have slightly different absorption spectra. Purified cyts cd₁ had cyt c oxidase and nitrite reductase activity, although the nitrite reductase activity was much lower than the oxidase activity (172).

In some species of aerobic phototrophic bacteria, several membrane-bound cyts were revealed by redox titration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the RC-bound tetraheme cyt c (49, 171, 226). The cyt bc₁ complex was shown to be present in R. denitrificans, E. litoralis, E. hydrolyticum, E. ramosum, and E. ezovicum (172, 226). In several species, the E_m values for the high- and low-potential cyt b ranged from 70 to 30 mV and from 20 to 150 mV, respectively, in purified membranes (226). Such low values for the E_m of a bc₁ complex cyt b heme were reported for the thermophilic bacterium PS3 and the photosynthetic Heliobacillus species (92). However, in these two organisms the quinone pool is composed of menaquinone, a low-potential quinone, which is not the case in aerobic bacteria, in which this pool is mainly composed of ubiquinone Q₁₀ (see "Quinones"). Therefore, it would be useful to confirm these low E_m values for the cyt b of these aerobic phototrophic bacteria by using purified cyt bc₁ complexes.

A high-potential membrane-bound cyt c (350 mV) was observed in E. hydrolyticum, E. ramosum, and E. ezovicum (226). Since no cyt photooxidation was detected in isolated membranes of these three species, reduction of the photooxidized primary donor of RC seems to involve a soluble cyt c and not this high-potential membrane-bound electron carrier, as postulated for Protaminobacter ruber (173).

cyt oxidases were purified from E. longus (49) and R. denitrificans (34). The E. longus cyt c oxidase is a cyt aa₃ type, which is unusual since previously R. sphaeroides was thought to be the only purple photosynthetic bacterial species that contains a cyt aa₃ oxidase, composed of three polypeptides (53). The E. longus cyt aa₃ is composed of two proteins of the same molecular weight.

In summary, the cyt content and composition of soluble and membrane fractions of aerobic phototrophic bacteria are highly diverse and species specific.

In anoxygenic photosynthetic energy transduction, absorption of photons in the antenna system results in energy transfer to the RC, where the primary donor P (the special pair of Bchl molecules) is excited. Excited P* is a strong reductant. An electron is rapidly transferred (perhaps through an accessory Bchl) from the excited primary donor to a bacteriopheophytin (H). The electron is transferred from H to the quinone Q_A within 220 ps, which results in transformation of the relatively unstable excited state into a relatively stable electrical potential across the membrane (, outside positive, inside negative). The negatively charged primary donor may be directly reduced by a reduced cyt c₂ or by an RC-bound tetraheme cyt (see "Cytochrome composition") (41, 100, 133).

The photochemical activity of the aerobic bacterial photosynthetic apparatus has been analyzed independently in several laboratories by using different species and techniques (51, 63, 131, 132, 174, 191, 226). The results indicate that the photosynthetic apparatus of aerobic phototrophic bacteria, although it has some peculiarities, is functional in terms of a cyclic electron transfer system.

In species of the genera Erythrobacter, Roseobacter, Roseococcus, Erythromicrobium, Erythromonas, and Sandaracinobacter, photoinduced cyclic electron transfer occurs only under relatively oxidized (aerobic) conditions, as elucidated by light-induced absorbance changes in whole cells (51, 131, 226). Under relatively reduced (anaerobic) conditions, no light-induced RC absorbance changes were observed. The lack of photochemistry under anaerobic conditions is consistent with the inability of these bacteria to grow by light-dependent photophosphorylation in the absence of oxygen (130, 144, 145, 147, 212, 214-216, 229, 230).

The very fast cyt c photooxidation observed after flash excitation in E. ursincola, S. sibiricus, R. thiosulfatophilus, and R. denitrificans (51, 131, 226) confirmed that the immediate electron donor to the RC in these species is a cyt c tightly bound to the RC. The difference spectrum measured at 50 µs was centered at 556 nm and correlates with the wavelength position of high-potential hemes of RC-bound cyts c of various photosynthetic bacteria (Fig. 9A) (35, 39, 51, 52). The wavelength of maximum bleaching at 5 ms was centered at 551 nm and corresponds to the photooxidation of soluble cyt c species, as reported for whole cells of the anaerobic phototrophic bacterium R. viridis (52). A large bleaching of cyt c attributed to photooxidation of soluble cyt c was induced in aerobic phototrophic bacterial species under continuous illumination (227).

View larger version (17K): [in this window] [in a new window]   FIG. 9.   Difference absorption spectra obtained for intact cells of aerobic anoxygenic phototrophic bacteria suspended in growth medium under aerobic conditions. (A) E. ursincola, determined at 50 µs and 5 ms after a saturating flash. (B) E. hydrolyticum, determined at 50 µs and 5 ms. (C) E. litoralis, determined at 50 µs and 7 and 20 ms.

As noted above, E. ramosum, E. hydrolyticum, E. ezovicum (226), and E. longus (132) do not possess a cyt c bound to the RC, and their photochemistry is similar to that observed in R. sphaeroides (16). The light-induced difference spectrum detected at 50 µs corresponds to a cyt c₂ photooxidation, with an absorption band at 550 nm superimposed on absorption changes linked to the photooxidation of the RC special pair (Fig. 9B). There was no detectable spectral shift between 50 µs and 5 ms. As observed in cells of R. sphaeroides (78, 135), the cyt c photooxidation detected at 551 to 542 nm consists of two phases: a fast phase (with a half-time of less than 50 µs, not resolved in experiments on obligately aerobic species) and a slower phase with a half-time of 250 µs. The half-time of the subsequent cyt c reduction was about 40 ms (226). Continuous illumination caused the oxidation of a large amount of a soluble cyt c, about five times more than the amount detected after one saturating flash (227).