The σ54 System Directly Regulates Bacterial Natural Product Genes

Abstract

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences.

Unraveling a Bacterial Starvation Response Through the Direct Targets of a Starvation-Induced Transcriptional Activator

“…It is noteworthy that the 9/12 of the putative σ 54 -RNA polymerase binding sites or core σ 54 promoter regions are located within genes and not in intergenic sequences. This finding is consistent with previous studies that placed many core σ 54 promoter regions in the coding sequences of M. xanthus genes (5,15,18,21) and raises the possibility that intragenic σ 54 promoters might be common in M. xanthus and in bacteria in general. As in previous analyses of M. xanthus σ 54 promoters, some of the intragenic -12-and -24-bp regions are located in the protein coding sequence of an upstream gene (upstream promoters) and some are located in the protein coding sequence of one gene in an operon (internal promoters).…”

“…In the initial experiments, we confirmed twelve potential σ 54 promoter targets of Nla28 and identified the likely promoter binding sites of Nla28 using expression data, in vitro promoter binding assays, and in vitro and in vivo mutational analyses. As in previous studies of Nla EBPs (5,18,21), our analysis placed the vast majority of the Nla28 target promoters inside genes and not in intergenic regions. Several of the previously uncharacterized targets of Nla28 were subsequently analyzed via insertion mutagenesis and all were linked to production of stress-resistant spores during starvation-induced development.…”

“…A fragment of the nla28 gene corresponding to the Nla28 DNA Binding Domain (Nla28-DBD) (5,21) was PCR amplified using gene-specific primers (Table S2) and then cloned into plasmid pMAL-c5x to create an N-terminal maltose binding protein (MBP) fusion to Nla28-DBD. The Nla28-DBD expression plasmid was introduced into E. coli strain BL21 (DE3) using electroporation.…”

“…However, the three Nla28 targets characterized in a recent study (5,20,21) are worth mentioning here, since they are likely involved in production of secondary metabolites and hence could have a defense function or could be involved in signaling. It has been suggested that Nla28 is part of a general stress response induced by nutrient depletion, since Nla28 modulates transcription of many early, starvationresponsive developmental genes and genes that are highly expressed during the transition into stationary phase (5,20,21). That some confirmed targets have defense functions makes sense if Nla28 is viewed as a regulator of starvationinduced stress.…”

“…Although Nla28 is important for expression of hundreds of M . xanthus developmental genes and for the developmental process (5, 14), only two classes of developmental genes have been linked to direct Nla28-mediated regulation: regulatory genes and natural product-associated genes (5, 21). One goal of this study was to better understand how Nla28 identifies its target promoters and to better understand how these promoters are arranged.…”