A second transcript in the direction Selleck SC79 complementary to the large transcript in the jamaicamide pathway is probably needed to include jamQ, a gene encoding a condensation like protein that is likely involved with the creation of the pyrrolinone ring of the molecule. According to our RT-PCR experiments, the regions between jamQ and the three genes closest upstream
(ORF5 and ORF6, both transposases, and ORF7, a hypothetical protein), are all transcribed. Quisinostat In addition, the upstream region of jamQ does not appear to serve as a strong promoter in β-galactosidase reporter assays (see below), despite the presence of possible conserved promoter domains (Table 1). From these data, it appears that jamQ could be part of a larger transcript including these transposases. A larger intergenic region (approximately 1070 bp) lies upstream of ORF7, which could contain the TSS and a promoter for this transcript. The reason for including at least one
transposase in the jamQ transcript is unclear, but this may be a way of ensuring transposable elements have remained associated with the cluster so as to facilitate horizontal gene transfer and pathway evolution. The hectochlorin biosynthetic gene cluster from L. majuscula JHB  contains a transposase (hctC) located between two of the initial genes (hctB and hctD) in the pathway, which is also thought to contribute to the plasticity of the cluster. Biosynthetic investigations using Lyngbya majuscula strains have been highly successful in identifying secondary metabolite ACY-738 supplier gene clusters, in part because L. majuscula readily incorporates isotopically labeled precursors in feeding studies [5, 6]. However, further experimentation by way of gene knockout or overexpression in L. majuscula is not yet possible because a viable means of genetic transformation has not been developed. Due to this limitation,
we used genetic constructs in E. coli to determine whether the promoters identified in this study, including the primary pathway promoter upstream of the TSS and GPX6 those predicted in intergenic regions, were functional. Although some differences exist in the structure of RNAP between the two bacteria , promoter structures in cyanobacteria are often compared to consensus sequences in E. coli [22, 41]. Furthermore, a strong E. coli promoter has been shown to function in the cyanobacterium Synechococcus  and the psb2 promoter from Microcystis can be used in E. coli to drive β-galactosidase production . The reporter assay proved effective in verifying the promoter identified upstream of the jamaicamide pathway TSS, as well as several internal promoters located at various regions throughout the gene cluster (Figures 4, 5 and 6).