A comparison of the sequenced genomes of corynebacteria (Figure 1, Additional file 1: Table S1) revealed that C. glutamicum WT is the only species possessing two crtB and crtI like
genes, while the organization of the large gene cluster is comparable in C. glutamicum WT, C. glutamicum R (and ATCC 14067 and S9114) and C. efficiens YS-314. In C. glutamicum R, no crtY e Y f is annotated as likely a G- > T mutation at position 814478 of the C. glutamicum R genome altered the start codon of an open reading frame coding for a protein with 99% amino acid identity to crtY e Y f of C. glutamicum WT to a leucine codon. A second group of corynebacterial species (e.g. C. diphteriae, C. aurimucosum and C. Apoptosis Compound Library supplier pseudotuberculosis) only possess the clustered www.selleckchem.com/products/ca3.html genes crtB and crtI (50 to 55% amino acid identity to the C. glutamicum enzymes; Additional file 1: Table
S1). An intermediate situation is found in C. lipophiloflavum, which possesses a gene cluster with crtB, crtI, crtY e/f and crtEb, as well as in C. genitalium possessing crtB, crtI and crtY e/f but lacking crtEb (Additional file 1: Table S1). Members of a third group (C. kroppenstedtii, C. jeikeium, C. urealyticum as examples) also lack crtY e/f and crtEb orthologs, but possess crtB and crtI, however not clustered. Although the overall amino acid sequence identities of the crtB and crtI gene products are below 50% as compared to the respective CrtB and Selleck CX-5461 CrtI from C. glutamcium WT, their domain structure includes the crtI domain (TIGR02734) as well as an N-terminal NAD(P)-binding Rossmann-like domain (NCBI Domain structure). As an exception, C. variabile only
possesses CrtI with an amino acid identity to CrtI from C. glutamicum WT of 58%. The phylogeny of the crtI gene product (Additional file 2: Figure S1), which is present Ribonucleotide reductase in all analysed corynebacteria, is congruent to the grouping of cornyebacterial species with respect to occurrence and clustering of crt genes as shown in Figure 1 and Additional file 1: Table S1. Analysis of the transcriptional organization of the carotenogenic gene clusters Annotation of the carotenogenic gene cluster of the C. glutamicum WT for the biosynthesis of decaprenoxanthin from the precursor GGPP suggests co-transcription of crtB, crtI, crtY e and crtY f and crtEb, while the upstream GGPP synthase gene crtE appears to be monocistronic. To characterize the transcriptional organization of this cluster RT-PCR experiments have been carried out. PCR analysis of cDNA synthesized from total RNA of the C. glutamicum WT using primer crtEb-rv (see Additional file 3: Table S2) revealed that the entire gene cluster is co-transcribed since fragments overlapping adjacent genes could be amplified in each case. A cDNA preparation without the addition of reverse transcriptase served as a negative control (Figure 3). Figure 3 Transcriptional organization of the carotenogenic gene clusters in C. glutamicum ATCC 13032.