Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.     

Physical mapping of bchG, orf427, and orf177 in the photosynthesis gene cluster of Rhodobacter sphaeroides: functional assignment of the bacteriochlorophyll synthetase gene

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999). An operonal arrangement of the genes is proposed.     

appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.     

Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria.

Four chemically different bacteriochlorophylls (Bchls) a esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol, and phytol have been detected by high-pressure liquid chromatography in cell extracts from Rhodopseudomonas sphaeroides and Chromatium vinosum. Bchl a containing phytol is the principal component, and the other three Bchls a comprise about 4% of the total Bchls a in stationary-phase cells of R. sphaeroides and C. vinosum. The high levels of the minor pigments occur in the beginning of Bchl a phytol formation, indicating that they are not degradation products, but intermediates of Bchl a phytol formation.     

A reaction center-light-harvesting 1 complex (RC-LH1) from a Rhodospirillum rubrum mutant with altered esterifying pigments: characterization by optical spectroscopy and cryo-electron microscopy

Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p4212 space group. A projection map calculated to 9 A shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.     

Das Vorkommen von Phytol und Geranylgeraniol in den Bacteriochlorophyllen roter und grüner Schwefelbakterien

The bacteriochlorophylls a of 38 strains belonging to 15 different species of the purple sulfur bacteria (Chromatiaceae) were studied with respect to the nature of the esterifying alcohol. The classical bacteriochlorophyll a_P containing phytol is the main bacteriochlorophyll in all strains. The new bacteriochlorophyll a_Gg occurs as a minor component in addition to bacteriochlorophyll a_P only in five species.The esterifying alcohol of the bacteriochlorophyll a of the reaction centers of all seven type strains of the Chlorobiaceae was shown to be phytol.The compounds withR _f -values between the bacteriophaeophytins a_P and a_Gg found by thin-layer-chromatography were shown to be artifacts of the preparation technique.All strains of the bacteriochlorophyll b-containing purple bacteria have phytol as the major esterifying alcohol; in addition, small amounts of bacteriochlorophyll b are esterified with another alcohol which is most probably all-trans-geranylgeraniol.

---

Abkürzungen DSM Deutsche Sammlung von Mikroorganismen - Bchl. Bacteriochlorophyll Herrn Prof. Dr. Dr. e. h. Hans Brockmann zum 70. Geburtstag gewidmet.     

Cloning and nucleotide sequence of regA, a putative response regulator gene of Rhodobacter sphaeroides

Abstract A 0.9 kb DNA fragment carrying the Rhodobacter capsulatus reg A gene, which encodes an oxygen-dependent, positively-acting response regulator of photosynthetic gene expression, was used as a probe in Southern hybridisation experiments to determine whether a similar gene occurs in R. sphaeroides . A strongly hybridising DNA fragment isolated from a R. sphaeroides plasmid gene bank was isolated, sequenced and found to contain an open reading frame which exhibits 75% identity with the R. capsulatus reg A gene. The deduced amino acid sequence of 184 residues shows 81% identity and 89% similarity with the R. capsulatus RegA protein, and significant similarities with other response regulators of the two component sensor-regulator type. Introduction of the R. sphaeroides gene into a R. capsulatus reg A mutant, which exhibits abnormally low levels of membrane-bound photosynthetic complexes, resulted in a 22–33-fold increase in these complexes to approximately 62–65% of wild-type levels. This is the first study to identify a putative response regulator in R. sphaeroides and to complement a regulatory mutation in R. capsulatus with a gene from another species. Further studies of associated genes may identify the different mechanisms by which the regulation of photosynthesis complex formation occurs in response to environmental stimuli in R. sphaeroides and R. capsulatus .     

Accumulation of chlorophyllous pigments esterified with the geranylgeranyl group and photosynthetic competence in the CT2256-deleted mutant of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria contain chlorophyllous pigments, chlorophyll (Chl) aPD and bacteriochlorophyll (BChl) aP, esterified with Delta2,6-phytadienol and phytol, respectively, which would be produced by reduction of the geranylgeranyl group at the C-17 propionate residue. In the genome of Chlorobium tepidum, two paralogous genes presumably encoding geranylgeranyl reductase, CT1232 and CT2256, are found. The deletion mutants of the CT1232 and CT2256 genes were constructed using an insertional inactivation method in order to clarify the biosynthetic process of the Delta2,6-phytadienyl and phytyl groups in green sulfur bacteria. The compositions of chlorophyllous pigments in the two mutants were determined by LC-MS analysis. The CT2256-deleted mutant accumulated Chl aGG and BChl aGG esterified with geranylgeraniol, indicating that CT2256 was involved in the production of both Delta2,6-phytadienyl and phytyl groups. The relatively high fluorescence emission from chlorosomes in the mutant also suggested some hindrance of the energy transfer from chlorosomes to the reaction center complex. However, the CT1232-deleted mutant almost showed no apparent phenotype compared to the wild type. Furthermore, the purple bacterium Rhodobacter capsulatus mutant defective in the bchP gene was partially complemented with the CT2256 gene; BChl aP was synthesized in the mutant in addition to accumulating other intermediates.     

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.     

Genetic and physical mapping of the Rhodobacter sphaeroides photosynthetic gene cluster from R-prime pWS2

Plasmid pWS2 is an R68.45 chimera originally isolated as an R-prime which complemented the Rhodobacter sphaeroides bch-420 allele. Our experiments have shown that pWS2 is also able to complement a wide range of R. sphaeroides pigment and photosynthetic mutants employing nitrosoquanidine, transposon or insertion-generated mutations effecting puhA, puc, puf, cycA, bch, and crt genes. A combination of orthogonal-field-alternation gel electrophoresis, transverse alternating field gel electrophoresis, and conventional electrophoresis have been used to estimate the size of pWS2 at congruent to 168.3 +/- 3.5 kb. A restriction map of the congruent to 109 kb of R. sphaeroides insert DNA was generated by partial and complete restriction endonuclease digestion coupled with Southern hybridization analysis using either gene-specific or junction fragment probes. Genes encoding bacteriochlorophyll (Bchl)-binding proteins (pufBALMX, pucBA, and puhA), cytochrome c2 (cycA), and enzymes involved in Bchl (bch) and carotenoid (crt) biosynthesis have been shown to reside within a contiguous 53-kb region of the R. sphaeroides DNA present on pWS2. The puf operon lies at one end of the 53-kb segment, while the genes puhA, cycA, and pucBA, the latter two of which are located within congruent to 12.0 kb of each other, define the other end of this 53-kb region. The genetic and physical mapping data provided in this paper are discussed in terms of the similarities and differences in the organization of the photosynthetic gene cluster between R. sphaeroides and other photosynthetic bacteria as well as highlighting the use of pWS2 in studies of photosynthetic gene structure and function.     

A single flavoprotein,AppA, integrates both redox and light signals in Rhodobacter sphaeroides

Anoxygenic photosynthetic proteobacteria exhibit various light responses, including changing levels of expression of photosynthesis genes. However, the underlying mechanisms are largely unknown. We show that expression of the puf and puc operons encoding structural proteins of the photosynthetic complexes is strongly repressed by blue light under semi-aerobic growth in Rhodobacter sphaeroides but not in the related species Rhodobacter capsulatus. At very low oxygen tension, puf and puc expression is independent of blue light in both species. Photosynthetic electron transport does not mediate the blue light repression, implying the existence of specific photoreceptors. Here, we show that the flavoprotein AppA is likely to act as the photoreceptor for blue light-dependent repression during continuous illumination. The FAD cofactor of AppA is essential for the blue light-dependent sensory transduction of this response. AppA, which is present in R. sphaeroides but not in R. capsulatus, is known to participate in the redox-dependent control of photosynthesis gene expression. Thus, AppA is the first example of a protein with dual sensing capabilities that integrates both redox and light signals.     

Incorporation of exogenous long-chain alcohols into bacteriochlorophyll c homologs by Chloroflexus aurantiacus

Chloroflexus aurantiacus grown in batch culture took up exogenous alcohols and incorporated these into bacteriochlorophyll c as the esterifying alcohol. It was possible to change the distribution of the naturally occurring homologs of bacteriochlorophyll c esterified with phytol, hexadecanol, and octadecanol by adding the appropriate alcohol. The corresponding homolog then made up at least 60% of the cellular bacteriochlorophyll c. It was also possible to obtain novel bacteriochlorophyll homologs not found in detectable amounts in control cells by adding fatty alcohols with short chains (C10, C12) or long chains (C20). These changes in bacteriochlorophyll composition had no detectable effects on the spectral properties of the chlorosomes.Abbreviation BChl Bacteriochlorophyll     

The mechanism of the C-13(3) esterification step in the biosynthesis of bacteriochlorophyll a

5-Aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms was prepared and incorporated into bacteriochlorophyll aphytyl of Rhodopseudomonas sphaeroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum. The biosynthetic samples of the bacteriochlorophylls were separately processed to obtain their isoprenyl alcohol components from the C-17(3) ester linkages and methanol from the C-13(3) methoxycarbonyl group. Methods were developed for the quantification of the isotopic composition of the various alcohols (methanol, phytol, geranylgeraniol). It was shown that the hydroxyl oxygen atoms of all the three alcohols originated from one of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results the in vivo mechanism for the O-methylation reaction at C-13(3) during the biosynthesis of the two species of bacteriochlorophylls is discussed.     

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.     

Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants.

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides.