Rare actinobacteria isolated from the hypersaline Ojo de Liebre Lagoon as a source of novel bioactive compounds with biotechnological potential.

The Ojo de Liebre Lagoon is a Marine Protected Area that lies within a UNESCO World Heritage Site and is a critical habitat for important migratory species such as the grey whale and bird species. Unique hypersaline environments, such as the Ojo de Liebre Lagoon, are underexplored in terms of their bacterial and chemical diversity, representing a potential source for new bioactive compounds with pharmacological properties. Actinobacteria are one of the most diverse and prolific taxonomic bacterial groups in terms of marine bioactive compounds. This study aimed to identify the culturable actinobacterial community inhabiting the Lagoon, as well as to test their potential as new sources of anticancer compounds with pharmacological potential. A selective isolation approach focused on spore-forming bacteria from 40 sediment samples generated a culture collection of 64 strains. The 16S rRNA gene analyses identified three phyla in this study, the Actinobacteria, Firmicutes and Proteobacteria, where the phylum Actinobacteria dominated (57%) the microbial community profiles. Within the Actinobacteria, nine different genera were isolated including the Actinomadura, Micromonospora, Nocardiopsis, Plantactinospora and Streptomyces sp. We observed seasonal differences on actinobacteria recovery. For instance, Micromonospora strains were recovered during the four sampling seasons, while Arthrobacter and Pseudokineococcus were only isolated in February 2018, and Blastococcus, Rhodococcus and Streptomyces were uniquely isolated in June 2018. Ethyl acetate crude extracts derived from actinobacterial cultures were generated and screened for cytotoxic activity against six cancer cell lines. Strains showed promising low percentages of viability on lung (H1299), cervical (SiHa), colon (Caco-2) and liver (HepG2) cancer lines. Molecular networking results suggest many of the metabolites produced by these strains are unknown and they might harbour novel chemistry. Our results showed the Ojo de Liebre Lagoon is a novel source for isolating diverse marine actinobacteria which produce promising bioactive compounds for potential biotechnological use as anticancer agents.

Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites.

While specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames. IMPORTANCE Microbial natural products are traditionally exploited for their pharmaceutical potential, yet our understanding of the evolutionary processes driving BGC evolution and compound diversification remain poorly developed. While HGT is recognized as an integral driver of BGC distributions, we find that the effects of vertical inheritance on BGC diversification had direct implications for species-level specialized metabolite production. As such, understanding the degree of genetic variation that corresponds to species delineations can enhance natural product discovery efforts. Resolving the evolutionary relationships between closely related strains and specialized metabolism can also facilitate our understanding of the ecological roles of small molecules in structuring the environmental distribution of microbes.

Extending the Salinilactone Family.

Five new members of the salinilactone family, salinilactones D-H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A-C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth-inhibiting effect of this compound family on multiple biological systems including non-Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds.