Genetic Manipulation of Carotenoid Biosynthesis in the Green Sulfur Bacterium Chlorobium tepidum

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), -carotene desaturase (crtQ/CT1414), ␥-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.

A novel carotenoid 1,2-hydratase (CruF) from two species of the non-photosynthetic bacterium Deinococcus

“…3 and Table 2). Based on the retention time, absorbance spectrum and molecular mass, peak 7 was identified as 19-OH-c-carotene (Frigaard et al, 2004). These results demonstrated that DR0091 and Dgeo2309 can also convert monocyclic ccarotene into 19-OH-c-carotene.…”

“…Combining gene expression and disruption analysis, we confirmed that dr0091 and dgeo2309 encode a novel carotenoid 1,2-hydratase in D. radiodurans and D. geothermalis. The genes dr0091 and dgeo2309 showed little homology to the CrtC-type carotenoid 1,2-hydratase, which is found mainly in photosynthetic bacteria (Armstrong et al, 1989;Frigaard et al, 2004;Giraud et al, 2004;Kovács et al, 2003;Lang et al, 1995;Ouchane et al, 1997;Scolnik et al, 1980) and in the non-photosynthetic bacterium Myxococcus xanthus (Botella et al, 1995). Recently, a carotenoid 1,2-hydratase homologue of DR0091 and Dgeo2309 from the cyanobacterium Synechococcus sp.…”

“…Carotenoid 1,2-hydratase, a carotenoid biosynthetic enzyme, is required to catalyse the synthesis of deinoxanthin by hydration at the C-19,29 double bond. CrtC-type carotenoid 1,2-hydratases have been found in photosynthetic bacteria (Armstrong et al, 1989;Frigaard et al, 2004;Kovács et al, 2003;Ouchane et al, 1997), primarily involved in the biosynthesis of spirilloxanthin, spheroidene, chlorobactene and its derivatives. However, no homologue of the CrtC-type carotenoid 1,2-hydratase has been detected in the D. radiodurans genome (Makarova et al, 2001;White et al, 1999).…”

Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2′-dirhamnoside

“…The functions of carotenoid 1,2-hydratase have been confirmed in two distinct enzymes: CrtC in both purple photosynthetic bacteria (Proteobacteria) (Takaichi, 2009) and Chlorobaculum (previously Chlorobium) tepidum (Chlorobi) (Frigaard et al, 2004); and CruF for myxol in Synechococcus sp. strain PCC 7002 (Cyanobacteria) (Graham & Bryant, 2009) and for deinoxanthin in Deinococcus radiodurans (Deinococcus-Thermus) (Sun et al, 2009).…”

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

“…The cyclization of the linear compound lycopene to produce ␣-, ␤-, ␥-, or -carotene is a branch point in carotenoid biosynthetic pathways in many species of bacteria, plants, and fungi (3,4). The monocyclic ␥-carotene is a precursor to myxoxanthophylls in cyanobacteria (5,6), is both an intermediate and an end product of carotenogenesis in orange and yellow flowers and fruits (7)(8)(9)(10), and is an intermediate in chlorobactene biosynthesis in green sulfur bacteria (GSB) (11)(12)(13). Dicyclic ␤-carotene is a major component of photosystems (PS) I and II in cyanobacteria and plants (14,15) and is modified to isorenieratene in brown-colored GSB and some actinomycetes (16,17).…”