Investigation on the diversity and antimicrobial potential of marine predatory bacteria enabled elucidation of the thio-peptide kocurin and aminophenylpyrrole-derived alkaloids biosynthesis

Abstract

Bacteria are a remarkable source of bioactive compounds, of which many served as lead compounds for the development of medicinal drugs currently used for diverse applications in clinic. Despite the immense bacterial diversity, only a few taxa commonly isolated in bioprospecting projects are known as proliferative producers of these compounds (e.g., 70% of antibiotics are derived from Streptomyces species). In recent years, the re-isolation of known compounds became a recurrent problem, mak-ing the search for novel talented taxa a priority. <br /> Predatory bacteria possess large genomes, which provide them with a large metabolic repertoire. The assumption that these bacteria use antibiotics to weaken or even kill their prey implements the great potential of these organisms as source for novel bioactive compounds. In the present work, the isolation of predatory bacterial strains, characterization of their produced specialized metabolites and elucidation of the biosynthesis of these metabolites was pursued. The Peruvian coastline was selected as sampling area, since it represents a biodiverse ecosystem, rich in endemic species. It is of interest, if the grade of biodiversity, usually judged by macroorganisms, can also be transferred to microorganisms. Therefore, a culture-independent metagenomics study was carried out to obtain first insights into the bacterial diversity. The microbi-ome of three different sites at the Peruvian Pacific coast was analyzed, revealing a lower bacterial biodiversity at Isla Foca than at Paracas and Manglares, with 89 bacte-rial genera identified, as compared to 195 and 173 genera, respectively. Only 47 of the bacterial genera identified were common to all three sites. <br /> Then, in order to obtain promising strains for the production of novel antimi-crobials, predatory bacteria were isolated from these sampling sites, using two differ-ent bait organisms. Even though the proportion of predatory bacteria was only around 0.5% in the here investigated environmental microbiomes, by this approach in total 138 bacterial strains were isolated as axenic culture. 25% of these strains showed anti-bacterial activity, thereby nine revealed activity against clinically relevant methicillin resistant Staphylococcus aureus (MRSA) and three against enterohemorrhagic Esche-richia coli (EHEC) strains. Phylogeny and physiological characteristics of the active strains were investigated. Metabolomic analyses enabled first insights into the chemi-cal basis of the antibacterial activity and indicated production of the known com-pounds ariakemicin, kocurin, naphthyridinomycin, pumilacidins, resistomycin, surfac-tin and amino-phenylderived alkaloids. However, most antibacterial compounds re-mained elusive until now. <br /> Among the reported compounds, it was aimed to understand the biosynthesis of the promising antibiotic kocurin. Kocurin is a ribosomally synthetized and posttransla-tional modified (RIPP) thiopeptide produced by Kocuria rosea s17. The genetic basis of this natural product with activity against MRSA was revealed by comparative ge-nome analysis of this new isolate and other reported thiopeptide producer strains. The functionality of the predicted gene locus was experimentally proven by heterologous expression in Streptomyces coelicolor M1146. Expression of the gene cluster under the control of a constitutive promoter enabled the transgenic strain to produce kocurin in selected media. The kocurin biosynthetic gene cluster (BGC) comprises 9 open reading frames and spans around 12 kbp of the genome. <br /> Another class of highly interesting natural products, showing various bioactivi-ties, for which the biosynthesis was investigated in detail, were the highly diverse aminophenyl-derived alkaloids (APPAs). APPAs, like the antifungal pyrrolnitrin, which was already discovered in the 60ies, represent high value lead compounds that were developed into globally important fungicides. More recently, diverse APPAs were discovered from bacteria belonging to the Cytophagales order (phylum Bac-teroidetes). In the present work, a bioinformatics and phylogenetic approach enabled the elucidation of the genetic basis, as well as the non-enzymatic processes involved in the biosynthesis of these natural products. A bioinformatic analysis of the producer genomes (Rapidithrix thailandica s80 and Porifericola rhodea s47) allowed the identi-fication of the putative APPA BGC. The biosynthesis was experimentally validated by heterologous expression of the BGCs in E. coli and revealed that first, one enzyme-dependent biosynthetic step yields the tryptophan-derived precursor 3-(2’-aminophenyl)-pyrrole. Second, a spontaneous Pictet-Spengler-like coupling reaction enables the producer strains to create a library of tricyclic alkaloids. Which alkaloids are formed is highly dependent on the chemical environment, since several aldehydes can be applied as substrates. The diversity of these natural products class is further enlarged by the catalytic action of a methyltransferase (MT), which adds a methyl group to the aminophenyl intermediate. This N-MT is encoded in some of the bacterial BGCs, and was investigated in detail, using in vivo and in vitro experiments. <br /> In summary, predatory bacteria represent an important bioresource for the dis-covery of novel metabolites. In the present study, the combination of bioinformatics and metabolomics analyses enabled the elucidation of the BGCs for kocurin and AP-PAs. The results obtained might facilitate the further development of such compound classes in the future and might contribute to enlarge the toolbox for synthetic biology approaches.