Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces.

Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide.

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.