RESULTS
Identification of the carotenoid biosynthetic gene cluster inPantoea ananatis PA13
We previously reported the whole genome sequence of P .ananatis PA13 (Choi et al ., 2012b), which revealed a carotenoid gene cluster on a plasmid (PAGR_p; CP003086). Figure 1 shows the genetic map and putative pathway responsible for carotenoid biosynthesis in P . ananatis PA13. The open reading frames (orfs ) in the carotenoid biosynthetic gene cluster were analysed and annotated as crtE , crtX , crtY , crtI ,crtB , and crtZ in sequence. When comparing the crtgene clusters between P . ananatis and the genetically close species P . agglomerans , there is a significant difference in the position of idi , which is located in the chromosome in the former strain (PAGR_g2908) and between crtEand crtX in the latter. The structure of the other genes is identical in the two strains (Fig. 1A).
The putative carotenoid biosynthetic pathway of P .ananatis was inferred from the pathways of Pantoea species (Misawa et al ., 1995; Hundle et al ., 1994) and plants (Guerinot, 2000). Carotenoid biosynthesis begins with isomerisation of isopenthyl diphosphate (IPP) from the mevalonate pathway to produce dimethylallyl diphosphate (DMAPP) in a reaction catalysed by IPP isomerase encoded by idi . Carotenoids are produced from the common precursor farnesyl diphosphate (FPP). Addition of a further IPP molecule yields geranylgeranyl diphosphate (GGPP) in a reaction catalysed by GGPP synthetase (encoded by crtE ). The next step in the carotenoid pathway is the head-to-head condensation of two molecules of GGPP to produce phytoene in a reaction catalysed by phytoene synthase (encoded by crtB ). Sequentially, the involved enzymes include phytoene desaturase (encoded by crtI) , lycopene β-cyclase (crtY ), β-carotene hydroxylase (crtZ ), and zeaxanthin glucosyl transferase (crtX ) (Fig. 1B).
Carotenoid biosynthetic cluster genes crtEXYIB of P. ananatis are polycistronic
We performed reverse transcription-polymerase chain reaction (RT-PCR) to determine if the wild-type P . ananatis carotenoid biosynthetic cluster genes are polycistronic. We used five sets of primers to amplify crtEX, XY, YI, IB , and BZ . RT-PCR followed by Southern hybridisation indicated that the P . ananatis carotenoid biosynthetic cluster genes crtEXYIB are transcribed as a single transcript, and crtZ is transcribed as an independent single transcript in the opposite direction (Fig. 2).