Transcriptional Regulation of Congocidine (Netropsin) Biosynthesis and Resistance.

The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.

Natural combinatorial biosynthesis involving two clusters for the synthesis of three pyrrolamides in Streptomyces netropsis.

The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.

An iterative nonribosomal peptide synthetase assembles the pyrrole-amide antibiotic congocidine in Streptomyces ambofaciens.

Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.