Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum - PubMed (original) (raw)

Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum

Julia A Maresca et al. J Bacteriol. 2006 Sep.

Abstract

The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.

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Figures

FIG. 1.

Biosynthetic pathway of carotenoids in C. tepidum. All genes identified in this pathway have been insertionally inactivated, and the resulting mutant strains have been biochemically and physiologically characterized (, ; this work).

FIG. 2.

Genome neighborhood comparisons. CT0967 (cruD) in C. tepidum and orthologs in other organisms are striped; CT1987 (cruC) in C. tepidum and homologs in other organisms are solid black. Other genes predicted to be involved in carotenoid biosynthesis are light gray; all other genes are white. In two species, orthologs of both CT1987 and CT0967 appear in a genomic region with at least one other carotenoid biosynthesis gene. This arrangement of genes suggests that these two ORFs might encode proteins that function in carotenoid biosynthesis.

FIG. 3.

Restriction maps of constructions used to inactivate CT1987 and CT0967 and confirmation of segregated mutants. (A) Restriction maps and primer locations of constructions used to inactivate CT1987 and CT0967. (B) Electrophoretic analysis of PCR products to evaluate putative mutants. The CT1987 and CT0967 loci were amplified by PCR with the primers indicated in Table 1. The DNA templates were derived from wild-type C. tepidum (lanes 2 and 4), from a CT1987::aadA transformant (lane 3), and from a CT0967::aadA transformant (lane 5). The amplicons from the two transformants are 0.9 kb larger than the corresponding amplicon from the wild type. This demonstrates that wild-type and mutant alleles had segregated fully in both transformants. Lanes 1 and 6 contain DNA size markers (Ladder I; GeneChoice, Frederick, MD).

FIG. 4.

HPLC elution profiles of the C. tepidum wild type (WT) and mutants unable to synthesize various modified carotenoids. Peak 1, OH-chlorobactene (550.4 Da); peak 2, OH-chlorobactene glucoside (729.4 Da); peak 3, OH-chlorobactene glucoside laurate (894.6 Da); peak 4, chlorobactene (532.4 Da); peak 5, γ-carotene (536.4 Da); peak 6, 1′,2′-dihydrochlorobactene (534.5 Da). Elution of carotenoid species was monitored at 491 nm.

FIG. 5.

Growth rates for the wild type (WT) and cruC and cruD mutant strains of C. tepidum at different light intensities. The rates are the averages from three independent determinations, and standard errors are indicated.

FIG. 6.

Neighbor-joining tree showing phylogenetic relationships among carotenoid glycosyltransferases. Amino acid sequences were aligned and used to reconstruct the phylogeny; all labels are the automatically assigned locus tags for that ORF. Only C. tepidum CruC and S. aureus SA2350 have been genetically characterized. Another class II glycosyltransferase, CT0362, from C. tepidum was used as the outgroup for this comparison. Numbers for open reading frames in C. aggregans, H. aurantiacus, R. castenholzii, and Roseiflexus sp. strain (R. strain) RS-1 are the numbers currently assigned in the draft genome sequences.

FIG. 7.

Neighbor-joining tree showing phylogenetic relationships among various acyltransferases. Amino acid sequences were aligned and used to reconstruct the phylogeny. Bold lines indicate proteins whose functions have been genetically or biochemically confirmed. Numbers for open reading frames in C. aggregans, H. aurantiacus, R. castenholzii, and Roseiflexus sp. strain RS-1 are the numbers currently assigned in the draft genome sequences. E. coli, Escherichia coli.

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Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum - PubMed (original) (raw)