and causes increased microcystin

production to enhance localized toxicity [26]. As with microcystin, many of the toxins found in L. majuscula are also produced by gene clusters comprised of PKS/NRPS architecture. PKS/NRPS gene clusters in other bacteria have been found to include imbedded regulatory proteins, such as the S treptomyces OICR-9429 price Antibiotic Regulatory Proteins (SARPs) found within the confines of several antibiotic Cobimetinib order pathways in Streptomyces [27]. However, cyanobacterial natural product gene clusters identified to date do not contain any apparent associated regulatory proteins. Insight into the mechanisms used by L. majuscula in the transcription of secondary metabolite gene clusters could be of significant value in enhancing the overproduction of potential drug leads in laboratory culture. Increased compound yield would reduce the need and environmental impact of repeated large scale field collections or the time and expense of chemical synthesis. Additionally, because the secondary metabolite biosynthetic gene clusters identified thus far from L. majuscula have been from different strains of the same species, transcription of each pathway could be under similar

mechanisms of regulation. This paper provides an analysis of transcriptional regulatory elements associated with the jamaicamide gene cluster from Lyngbya majuscula, and to our knowledge is the first such effort for a secondary metabolite gene cluster from a marine cyanobacterium. The jamaicamides are mixed BIBF 1120 cost PKS/NRPS neurotoxins that exhibit sodium channel blocking activity and fish toxicity. The molecules contain unusual structural features including a vinyl chloride and alkynyl bromide [6]. The gene cluster encoding jamaicamide biosynthesis is 57 kbp in length, and is composed of 17 ORFs that encode for proteins ranging in length from 80 to 3936 amino acids. Intergenic regions between 5

and 442 bp are located between all but two of the ORFs, and a region of approximately Dimethyl sulfoxide 1700 bp exists between the first jamaicamide ORF (jamA, a hexanoyl ACP synthetase) and the closest upstream (5′) ORF outside of the cluster (a putative transposase). In this study, we used RT-PCR to locate the transcriptional start site (TSS) of the jamaicamide gene cluster. Because it is not yet possible to perform genetics in filamentous marine cyanobacteria such as Lyngbya, we used a reporter gene assay to identify several possible internal pathway promoters. We also isolated at least one possible regulatory protein using pulldown experiments that is able to bind to the region upstream of the transcription start site in gel shift assays. Bioinformatic analyses conducted with the protein sequence suggest a correlation between secondary metabolite production and complementary chromatic adaptation (CCA) in cyanobacteria. Results RT-PCR using L.