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Link to original content: http://pubmed.ncbi.nlm.nih.gov/15292122/
Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum - PubMed Skip to main page content
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. 2004 Aug;186(16):5210-20.
doi: 10.1128/JB.186.16.5210-5220.2004.

Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

Niels-Ulrik Frigaard  1 , Julia A Maresca, Colleen E Yunker, A Daniel Jones, Donald A Bryant
Affiliations

Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

Niels-Ulrik Frigaard et al. J Bacteriol. 2004 Aug.

Abstract

The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.

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Figures

FIG. 1.
FIG. 1.
Proposed pathway of carotenoid biosynthesis in C. tepidum. The HPLC peak numbers are shown in parentheses after the chemical names. All genes shown in this scheme have been inactivated. Geranylgeranyl-PP, geranylgeranyl diphosphate.
FIG. 2.
FIG. 2.
Maps illustrating the DNA constructs used to inactivate genes. The small labeled arrows indicate the binding sites of the primers used to generate PCR products. The gene-inactivating constructs were made by fusion of the PCR products with a fragment containing the aadA antibiotic resistance marker by megaprimer PCR or by ligation. See text and reference for details.
FIG. 3.
FIG. 3.
Unrooted neighbor-joining phylogenetic trees of different enzymes and proteins. (A) CrtB-type phytoene synthases; (B) CrtU-type β-carotene and γ-carotene desaturases; (C) CrtI-type phytoene desaturases, CrtH-type cis-trans isomerases, and related proteins; and (D) CrtP- and CrtQ-type desaturases. Bootstrap values were calculated on the basis of 1,000 replicates; only values less than 70% are shown. The sequences used were obtained from the following organisms: Arabidopsis thaliana, Rubrivivax gelatinosus, Brevibacterium linens, Chlorobium tepidum, Chloroflexus aurantiacus, Erwinia herbicola, “Gloeobacter violaceus” strain PCC 7421, Mycobacterium aurum, Myxococcus xanthus, Nostoc (formerly Anabaena) sp. strain PCC 7120, Rhodobacter sphaeroides, Streptomyces griseus, Synechococcus sp. strain PCC 7942, Synechocystis sp. strain PCC 6803, and Trichodesmium erythraeum. For further information on the protein sequences, see Table S3 in the supplemental material.
FIG. 4.
FIG. 4.
HPLC chromatograms of cell extracts of the wild-type and mutant strains of C. tepidum with detection wavelengths of 270 nm (- - - -), 400 nm (— — —), and 490 nm (———). The amount of extract injected corresponded to about 10 μg of BChl c. Peaks are identified in Table 1. Other peaks are as follows: peak 13, OH-γ-carotene glucoside (35.6 min); peak 14, OH-chlorobactene glucoside (30.3 min); peak 15, chlorobiumquinone 7 (44.8 min); peak 16, 1′-hydroxymenaquinone 7 (44.5 min); peak 17, menaquinone 7 (52.5 min); peak 18, 1,2-dihydro-ζ-carotene (54.2 min); and peak 19, 1,2-dihydrolycopene (51.8 min).
FIG. 5.
FIG. 5.
HPLC chromatograms of cell extracts of the wild-type and mutant strains of C. tepidum with a detection wavelength of 490 nm. See the legend to Fig. 4 and text for details.
FIG. 6.
FIG. 6.
Absorption spectrum of an unidentified component (the F component) found in C. tepidum. The absorption spectrum was obtained from the HPLC detector at the time of elution. There were three peaks at 285, 325, and 453 nm.
FIG. 7.
FIG. 7.
Growth rates of the wild-type and mutant strains of C. tepidum at various light intensities. The light intensities used were 10 (black bars), 76 (shaded bars), and 588 (white bars) μmol of photons m−2 s−1. The data are the means ± standard deviations (error bars) from at least four separate experiments. WT, wild type.
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