Bacteriochlorophyll Biosynthesis Research Articles

The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts

Most of the essential biosynthetic and structural genes involved in bacterial photosynthesis are clustered in a 46 kb region of the Rhodobacter capsulatus genome. Previous analyses have demonstrated that the puf operon, which encodes light harvesting and reaction center structural genes as well as a regulatory gene for bacteriochlorophyll biosynthesis, is expressed from a complex set of overlapping transcripts. Differential initiation and processing of these transcripts is thought to be involved in regulating expression of puf-encoded genes. In this study we demonstrate that the puh operon, which is located 39 kb away from the puf operon, also contains overlapping transcripts. One large 11 kb puhA transcript is shown to be a product of read-through from an upstream operon (bchB) which encodes numerous bacteriochlorophyll biosynthesis genes. A second 1.1 kb mRNA is shown to be derived from the 11 kb bchB transcript by processing and a third, highly expressed, 0.95 kb transcript is shown to be initiated from a promoter located within the distal gene of the bchB operon. The occurrence of overlapping transcripts for the puf and puh operons was further shown to influence development of the photochemical apparatus during conditions of environmental shifts in oxygen tension. Evidence for the occurrence of a "superoperonal" organization of overlapping operons in several different species of purple photosynthetic bacteria is discussed.

TETRAPYRROLE‐DEPENDENT PHOTODYNAMIC HERBICIDES

TETRAPYRROLE‐DEPENDENT PHOTODYNAMIC HERBICIDES

Genetic and biochemical characterization of carotenoid biosynthesis mutants of Rhodobacter capsulatus.

We have used genetic and biochemical techniques to study carotenoid biosynthesis (crt) mutants of Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. All nine identified crt genes are located within the 46-kilobase pair photosynthesis gene cluster, and eight of the crt genes form a subcluster. We have studied the operon structure of the crt gene cluster using transposon Tn5.7 mutants. The Tn5.7 insertion sites in 10 mutants have been mapped to high resolution (25-267 base pairs) by Southern hybridization. Two insertions each map within the coding regions of the crtA, crtC, crtE, and crtF genes, and one insertion lies within the crtI gene. The insertion in crtI is not polar on the downstream crtB gene, suggesting that crtI and crtB may form two separate operons. Another insertion located in the 5' noncoding region between the divergent crtA and crtI genes has no effect on wild-type pigmentation and apparently lies between the promoters for these operons. A Tn5.7 mutation in the 3' region of crtA yields a bacteriochlorophyll-minus phenotype, while a 5' insertion affects only carotenoid biosynthesis. Regulatory signals for transcription of a downstream operon required for bacteriochlorophyll biosynthesis may thus overlap the coding region of crtA. We also present the first evidence for the functions of the crtB, crtE, and crtJ gene products using a new in vitro assay for the incorporation of [14C]isopentenyl pyrophosphate into carotenoid precursors and phytoene in cell-free extracts. Extracts from a crtE mutant accumulate [14C]prephytoene pyrophosphate, while those from crtB and crtJ mutants accumulate [14C]geranylgeranyl pyrophosphate. We therefore propose that CrtE is the phytoene synthetase and that CrtB, and possibly CrtJ, are components of the prephytoene pyrophosphate synthetase.

Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides

Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction-centre and light-harvesting LH1 complexes. The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202. We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction-centre H subunit. This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium.

Promoter mapping and nucleotide sequence of the bchC bacteriochlorophyll biosynthesis gene from Rhodobacter capsulatus

Because there are not yet direct assays for most of the proteins required for differentiation of Rhodobacter capsulatus cytoplasmic membrane into photosynthetically competent intracytoplasmic membrane, a molecular inquiry into the mechanism and regulation of this process is difficult. We have, therefore, chosen to isolate R. capsulatus photosynthesis genes by creating in-frame fusions to lac' Z vectors, and selecting for those that direct appropriately regulated levels of β-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. The chromosomal copy of this ORF was mutated by insertion of a kanamycin-resistant cartridge into the cloned fragment and substitution for the chromosomal copy by homologous recombination. The phenotype of the resultant mutant cells showed that the ORF encodes 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase, an enzyme that catalyzes the penultimate step in bacteriochlorophyll a biosynthesis. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence shows that the bchC gene encodes a 33-kDa protein that is less hydrophobic than integral membrane proteins of R. capsulatus, although there are hydrophobic segments that could in principle interact with a lipid membrane. Results of S l nuclease protection and primer extension experiments show that a 5' mRNA end is positioned within the cloned segment, and that this 5' end maps to sequences with significant sequence similarity to the previously characterized puf operon promoter region.

Syntheses of Bile Pigments. Part 16. Synthesis of a vinyl‐substituted 2,3‐Dihydrobilinedione: Possible role of this new class of bile pigments in phycobilin biosynthesis

AbstractThe total synthesis of racemic cis‐2,3,181, 182, ‐tetrahydroprotobiliverdin IXα dimethyl ester (19b), which is identical with the dimethyl ester of rac‐4, is described (Scheme 2). Under virtually neutral conditions, in solution, this bile pigment isomerized within a few min to racemic Z‐phycocyanobilin dimethyl ester (rac‐5b). Likewise, acid‐catalyzed allyl rearrangement of 3‐vinyl‐substituted cis‐ and trans‐2,3‐dihydrodipyrrin‐1(10H)‐ones 11c and 13c, respectively, yielded the corresponding ethylidene derivatives. In this case, however, the E‐isomer was formed stereo selectively from both substrates. The above results prove that, if protobiliverdin IXα (2) is transformed enzymatically to its 2,3,181, 182‐tetrahydro derivative, the latter would isomerize spontaneously to phycocyanobi‐lin. The biosynthesis of bacteriochlorophyll a and b from a common precursor bearing a vinyl group at C(8)may be straightforwardly explained in the same way.

Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus.

Carotenoid pigments are essential for the protection of both photosynthetic and non-photosynthetic tissues from photooxidative damage. Although carotenoid biosynthesis has been studied in many organisms from bacteria to higher plants, little is known about carotenoid biosynthetic enzymes, or the nature and regulation of the genes encoding them. We report here the first DNA sequence of carotenoid genes from any organism. We have determined the complete nucleotide sequence (11,039 bp) of a gene cluster encoding seven of the eight previously known carotenoid genes (crtA, B, C, D, E, F, I) and a new gene, designated crtK, from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. The 5' flanking regions of crtA, I, D and E contain a highly conserved palindromic sequence homologous to the consensus binding site for a variety of prokaryotic DNA-binding regulatory proteins. This putative regulatory palindrome is also found 5' to the puc operon, encoding the light-harvesting II antenna polypeptides. Escherichia coli-like sigma 70 promoter sequences are located 5' to crtI and crtD, suggesting for the first time that such promoters may exist in purple photosynthetic bacteria. The crt genes form a minimum of four distinct operons, crtA, crtIBK, crtDC and crtEF, based on inversions of transcriptional orientation within the gene cluster. Possible rho-independent transcription terminators are located 3' to crtI, B, K, C and F. The 3' end of crtA may overlap transcription initiation signals for a downstream gene required for bacteriochlorophyll biosynthesis. We have also observed two regions of exceptional amino acid homology between CrtI and CrtD, both of which are dehydrogenases.

Inhibition of Bacteriochlorophyll Biosynthesis by Gabaculin (3-Amino, 2,3-dihydrobenzoic Acid) and Presence of an Enzyme of the C5-Pathway of δ-Aminolevulinate Synthesis in Chloroflexus aurantiacus

Abstract Biosynthesis of bacteriochlorophyll c and a in the thermophilic phototrophic prokaryote. Chloroflexus aurantiacus Ok-70-fl, was strongly inhibited by the antibiotic gabaculin (3-amino 2,3- dihydrobenzoic acid), an inhibitor of the glutamate-C5-pathway of ö-aminolevulinate (ALA) synthesis. The key enzyme of the Shemin-pathway of ALA formation, ALA synthase (EC 2.3.1.37), was not detected in cell extracts of Chi. aurantiacus. However, the extracts catalyzed ALA formation from glutamate 1-semialdehyde, a reaction being highly sensitive to gabaculin.

A genetic-physical map of the Rhodobacter capsulatus carotenoid biosynthesis gene cluster

We used the interposon mutagenesis technique to map a cluster of Rhodobacter capsulatus genes that are responsible for the biosynthesis of carotenoids, membrane pigments that protect living organisms from photooxidations. Fifteen interposons define 7 genes (crtA, crtI, crtB, crtC, crtD, crtE and crtF) in an approximately 8 kb of the chromosome. This cluster is flanked by genes affecting bacteriochlorophyll biosynthesis. Based on the analysis of the phenotypes of the insertional mutants and the results of complementation analyses, we present a genetic and physical map of this region. Our data indicate that, with the possible exception of crtI and crtB, all genes analyzed are organized as single transcriptional units. We used nuclease S1 protection analysis to study the influence of oxygen on the regulation of expression of 4 genes (crtA, crtI, crtC and crtE). Under anaerobic conditions, mRNA levels for crtA, crtC and crtE are elevated and a new crtE transcript is detected.

Transcription of oxygen-regulated photosynthetic genes requires DNA gyrase in Rhodobacter capsulatus.

The regulation of the photosynthetic genes by DNA supercoiling in Rhodobacter capsulatus has been studied by using gyrase inhibitors in vivo and by measurement of mRNA levels of more than a dozen genes. The results demonstrate that the levels of mRNA for light-harvesting (I, II) and reaction center (L, M, H) proteins, bacteriochlorophyll biosynthetic enzymes, ribulose-bisphosphate carboxylase (EC 4.1.1.39), and the mRNAs from the open reading frames Q and R decreased immediately and dramatically upon addition of novobiocin and coumermycin. In contrast, the mRNAs for carotenoid biosynthetic enzymes, the cytochrome bc1 complex, and constitutively expressed mRNA under aerobic conditions for light-harvesting I and for reaction center (L, M) proteins are less sensitive to the inhibitors. In accordance with these results, the biosynthesis of bacteriochlorophyll is markedly repressed by gyrase inhibitors novobiocin, coumermycin, nalidixic acid, and oxolinic acid either under anaerobic conditions or during a shift from aerobic to anaerobic conditions. The synthesis of light-harvesting (I, II) bacteriochlorophyll complexes is also inhibited by novobiocin and coumermycin. The kinetics of specific mRNA changes and the differential sensitivity of anaerobic and aerobic genes to the gyrase inhibitors strongly suggest that DNA supercoiling is involved in the differential expression of photosynthetic genes in response to the level of oxygen in R. capsulatus.

Cloning and Oxygen-regulated Expression of the Bacteriochlorophyll Biosynthesis Genes bch E, B, A and C of Rhodobacter sphaeroides

Four mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was transferred into these mutants using the helper plasmid pRK2073. Three clones that produced photosynthetic transconjugants from one or more of the bch mutants were isolated and characterized. These clones were used as probes to estimate levels of specific transcripts in cells undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7% of the eventual level of pigment had been synthesized.

The influence of bacteriochlorophyll biosynthesis on formation of pigment-binding proteins and assembly of pigment protein complexes in Rhodopseudomonas capsulata

The synthesis of bacteriochlorophyll (Bchl) was inhibited in wild type strain of Rhodopseudomonas capsulata by levulinic acid (LA), δ-aminolevulinic acid of α,α′-dipyridyl. The transcription of genes for pigment-binding proteins, the synthesis and incorporation of these proteins into the membrane and the assembly of photosynthetic complexes were studied. Inhibition of Bchl synthesis did not impair formation of mRNA for pigment-binding polypeptides, but inhibited the synthesis of these proteins as well as stable incorporation into the membrane. The results let suggest, that Bchl is necessary for the stabilization of pigment-binding proteins in the membrane, and that intermediates of Bchl-synthesis affect the synthesis of these proteins. The same experiments were carried out with mutant strains blocked in Bchl synthesis. All Bchl-less mutant strains investigated here, showed transcription of photosynthetic genes at a rate which was independent of oxygen tension. The synthesis of polypeptides of reaction center, B870 and B800-850 complexes were differently affected by blocks in Bchl-synthesis. LA inhibited the synthesis of pigment-binding proteins in mutant strains similarly as in wild-type strain.

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.

13C nuclear magnetic resonance studies on bacteriochlorophyll a biosynthesis in Rhodopseudomonas spheroides S

The 13C NMR spectra were analyzed in bacteriochlorophyll a and magnesium protoporphyrin methyl ester formed in Rhodopseudomonas spheroides S. in the presence of l-[1- 13C]glutamate and [2- 13C]glycine. After reassignment of three α-pyrrolic carbons (C-9, -14 and -16) of bacteriochlorophyll a, the spectra showed that C-2 of glycine was preferentially incorporated into the eight-carbon atoms in these tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA). C-2 of glycine was also incorporated specifically into methyl ester carbon of magnesium protoporphyrin IX methyl ester and methoxyl carbon of methoxycarbonyl group attached to isocyclic ring of bacteriochlorophyll a. No enrichment of these nine-carbon atoms was observed in the spectrum of bacteriochlorophyll formed in the presence of l-[1- 13C]glutamate, showing exclusive operation of ALA synthase on bacteriochlorophyll biosynthesis.

Occurrence and some properties of two types of .DELTA.-aminolevulinic acid synthase in a facultative methylotroph, Protaminobacter ruber.

In addition to the α-ALA synthase already reported (Fraction I: molecular weight, 100,000; optimal pH, ca. 8.0), an isozyme (Fraction II: molecular weight, 64,000; optimal pH, ca. 6.4) was found in Protaminobacter ruber grown in the dark after an initial light period (20~30hr). The fraction I- and II-enzymes were separable by gel-filtration through Sepharose 6B. While the former was formed constitutively, the latter was formed inducibly under the conditions for bacteriochlo-rophyll formation. Therefore, the fraction II-isozyme seems to be responsible for the biosynthesis of bacteriochlorophyll.

Variation of levels of mRNA coding for antenna and reaction center polypeptides in Rhodopseudomonas capsulata in response to changes in oxygen concentration.

The effect of oxygen tension on the transcription of genes coding for the photosynthetic apparatus of Rhodopseudomonas capsulata was determined by the Southern hybridization technique. Restriction endonuclease digests of the R-prime plasmid pRPS404 and a subcloned fragment thereof served as DNA probes for genetically defined regions. The results showed that transcripts corresponding to the genes for certain pigment-binding polypeptides increase in amount by about 40-fold after a drop in oxygen tension. Transcripts hybridizing to genes involved in bacteriochlorophyll biosynthesis increase to a much lesser extent, and several genes involved in carotenoid biosynthesis are not affected by pO2.

Transcriptional regulation of several genes for bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata in response to oxygen

Although it has been shown that bacteriochlorophyll synthesis in Rhodopseudomonas capsulata is repressed by oxygen and high light intensity, few details of regulation by these environmental factors are known, primarily owing to a lack of assays for the biosynthetic enzymes. We have examined regulation at the transcriptional level by isolating and studying fusions between the Mu d1(Apr lac) phage and various bch genes. In these strains, the lacZ gene of the phage is under the control of bch gene promoters. We have found that atmospheric oxygen tension (20% O2) reduces the expression of these fusions at least twofold compared with low oxygen tension (2% O2). Therefore, transcription of the bchA, bchB, bchC, bchG, and bchH genes is regulated in response to oxygen.

2-(1-Hydroxyethyl)-2-desvinyl chlorophyllide a: Characterisation by nuclear overhauser enhancement proton magnetic resonance of a novel pigment obtained from mutants of rhodopseudomonas sphaeroides.

The major pigment of mutants of Rp. sphaeroides was isolated and characterized as 2-(1-Hydroxyethyl)-2-desvinyl chlorophyllide a : this compound is a likely intermediate in the biosynthesis of bacteriochlorophyll a .

Optically excited triplet states in the bacteria Rhodopseudomonas sphaeroides ‘wild-type’ detected by magnetic resonance in zero-field

Optically-detected magnetic resonance (ODMR) experiments in zero-field on the photosynthetic bacteria Rhodopseudomonas sphaeroides revealed triplet states belonging to molecules which have a prompt emission in the optical region from 590 to 700 nm and a delayed emission between 700 and 830 nm. The zero-field parameters of these triplet states are 29 <| D|< 34 · 10 −3 cm −3 and 4 <| E|< 8 · 10 −3 cm −1, the decay rates of the | D|+| E| transitions being in the order of 60–340 ms. The correlation between optical emission and radio-frequency was used to separate the total optical emission from 590–700 nm into individual emissions, belonging to molecules whose triplet states were studied by ODMR in this region. Comparing the fluorescence microwave double resonance (FMDR) spectra with the results of excitation spectroscopy, as well as comparing the zero-field parameters and the decay rates with that of Mg-porphyrins in matrices given in the literature, allowed the identification of the emitting molecules as Mg-porphyrins which are produced by the biosynthesis of bacteriochlorophyll in the cells.

Regulation of vitamin B12 and bacteriochlorophyll biosynthesis in a facultative methylotroph, Protaminobacter ruber.

The regulation of vitamin B12 and bacteriochlorophyll formation was studied in a facultative methylotroph, Protaminobacter ruber, classified as a non-photosynthetic bacterium. Vitamin B12 was formed at an almost constant level under various cultivation conditions, while bacteriochlorophyll synthesis was drastically influenced by the growth conditions. Addition of B12 and hemin to the medium and the change of culture conditions from light to darkness in the early growth phases stimulated the pigment synthesis, but no pigment was formed under the continuous illumination and the addition of chloramphenicol inhibited the pigment formation. delta-ALA synthase and delta-ALA dehydratase activities were demonstrated in P. ruber. delta-ALA synthase formation was induced when cultures were transferred from light to dark conditions, which promoted the bacteriochlorophyll formation. Bacteriochlorophyll-protein complex had an absorption maximum at 870 nm and a very small peak at 800 nm.