Biodiversity of Actinobacteria from the South Pacific and the Assessment of Streptomyces Chemical Diversity with Metabolic Profiling

Abstract

Recently, bioprospecting in underexplored habitats has gained enhanced focus, since new taxa of marine actinobacteria can be found, and thus possible new metabolites. Actinobacteria are in the foreground due to their versatile production of secondary metabolites that present various biological activities, such as antibacterials, antitumorals and antifungals. Chilean marine ecosystems remain largely unexplored and may represent an important source for the discovery of bioactive compounds. Various culture conditions to enrich the growth of this phylum were used and 232 bacterial strains were isolated. Comparative analysis of the 16S rRNA gene sequences led to identifying genetic affiliations of 32 genera, belonging to 20 families. This study shows a remarkable culturable diversity of actinobacteria, associated to marine environments along Chile. Furthermore, 30 streptomycete strains were studied to establish their antibacterial activities against five model strains, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa, demonstrating abilities to inhibit bacterial growth of Gram-positive bacteria. To gain insight into their metabolic profiles, crude extracts were submitted to liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis to assess the selection of streptomycete strains with potentials of producing novel bioactive metabolites. The combined approach allowed for the identification of three streptomycete strains to pursue further investigations. Our Chilean marine actinobacterial culture collection represents an important resource for the bioprospection of novel marine actinomycetes and its metabolites, evidencing their potential as producers of natural bioproducts.

Keywords: Chilean marine actinobacteria; antimicrobial activity; chemical diversity.

Figure 1

Map of Chile showing the various sampling locations. First insets depict the Chilean regions that were sampled, from top to bottom: Atacama Region (III, samples C1–C4), Valparaíso Region (V, samples V1–V12 and Easter Island, samples I1–I8), Los Lagos Region (X, samples H1–H4) and Aysén del General Carlos Ibáñez del Campo Region (XI, samples G1 and G2). Second insets show the locations sampled in red dots. The Comau fjord in Los Lagos Region and part of the Valparaíso Bay sampling (samples H1–H4 and V7–V12, respectively) have been previously described [24,25]. Scale located in the first inset represents approximately 500 km.

Figure 2

Culturable biodiversity of actinobacteria. (A) Pie chart showing respective abundance of the 32 genera isolated throughout all sampling locations. (B) Diversity in sediment samples, showing exclusive and shared numbers of genera among Valparaíso, Chañaral Aceituno, Comau fjord and Easter Island samplings. (C) Diversity in sponge samples, showing genera abundance between Chañaral de Aceituno and Easter Island samplings.

Figure 3

Phylogenetic tree of representative Streptomyces isolated along the coasts of Chile. Phylogenetic tree, based on the V1 to V9 region of the 16S rRNA gene sequences, was conducted with the PhyML 3.0 using the maximum likelihood algorithm with bootstrap values based on 1000 replications. The statistical selection of the nucleotide model substitution was performed with jModelTest-2.1, supporting the proposed branching order shown at consistent nodes (values below 50% not shown). Gene sequence positions 101–1395 were considered, according to the Escherichia coli K12 (AP012306) 16S rRNA gene sequence numbering. Outgroup is defined as Streptacidiphilus neutrinimicus JL206T. GenBank accession numbers of 16S rRNA sequences are given in parentheses. Scale bar corresponds to 0.05 substitutions per nucleotide positions.

Figure 4

Chemical barcoding and hierarchical cluster analysis based on liquid chromatography-high resolution mass spectrometry metabolic profiles from selected Streptomyces strains. Retention times and m/z values were used as variables. Vertical axis depicts Streptomyces strains and horizontal axis shows variables that represent a single compound, and its presence is represented as a black square.