All of the intergenic regions in the jamaicamide pathway tested were amplified into second strand cDNA, including the intergenic Bafilomycin A1 clinical trial region between jamP and jamQ. Intergenic regions between

the two ORFs downstream of jamQ (putative transposases) were also transcribed. These results indicated that the majority of the jamaicamide gene cluster is composed of the operon CDK assay jamABCDEFGHIJKLMNOP. Because no apparent breaks in transcription occurred between jamQ and at least the two neighboring downstream transposases (ORF5 and ORF6) and a hypothetical protein (ORF7), one contiguous transcript may encode the translation of all of these proteins. Transcription of the intergenic region between jamP and jamQ indicated that a transcript including jamP must extend at least into the complementary strand of jamQ before termination, although transcription in the opposite direction would be necessary to generate jamQ mRNA. Use of promoter prediction and β-galactosidase reporter gene assays to search for promoter activity The large size (approximately 55 kbp) of the main jamaicamide operon (jamA-P) suggested that multiple promoters would likely be needed click here for

efficient jamaicamide transcription. Because transcripts were found for each of the intergenic regions between the ORFs, these promoters may function intermittently and could be subject to promoter occlusion [22]. A software prediction program (BPROM, http://​www.​softberry.​com) was used to predict whether the intergenic regions from the jamaicamide pathway Montelukast Sodium contained conserved promoter binding regions. Several of these regions were predicted to contain at least one potential pair of -35 and -10 binding sites (Table 1). Because transformation methods into L. majuscula have not yet been developed, we used a

reporter gene assay in E. coli to determine whether any of these upstream (up-) regions could function as promoters. Each region predicted to contain a promoter (upjamA, upjamB, upjamC, upjamD, upjamG, upjamI, upjamN, and upjamQ), as well as the promoter upstream of the jamaicamide TSS, was amplified with specific primers from fosmids containing different portions of the jamaicamide biosynthetic pathway ([6]; Additional file 1: Table S1). Each of these regions were individually ligated into the pBLUE TOPO vector (Invitrogen) and transformed into TOP-10 E. coli. The resulting constructs were evaluated for relative promoter activity using the β-galactosidase reporter gene assay (Invitrogen), standardized against total soluble protein content measured by BCA assay (Pierce). For upjamA, two regions were evaluated, including the region predicted to contain the initial promoter, as well as immediately upstream of the jamA gene (a region with high activity in preliminary assays). The arabinose promoter from E.