Actinobacteria from Arctic and Atlantic deep-sea sediments-Biodiversity and bioactive potential

Abstract

The deep-sea covers over 70% of the Earth's surface and harbors predominantly uncharacterized bacterial communities. Actinobacteria are the major prokaryotic source of bioactive natural products that find their way into drug discovery programs, and the deep-sea is a promising source of biotechnologically relevant actinobacteria. Previous studies on actinobacteria in deep-sea sediments were either regionally restricted or did not combine a community characterization with the analysis of their bioactive potential. Here we characterized the actinobacterial communities of upper layers of deep-sea sediments from the Arctic and the Atlantic (Azores and Madeira) ocean basins, employing 16S rRNA metabarcoding, and studied the biosynthetic potential of cultivable actinobacteria retrieved from those samples. Metabarcoding analysis showed that the actinobacterial composition varied between the sampled regions, with higher abundance in the Arctic samples but higher diversity in the Atlantic ones. Twenty actinobacterial genera were detected using metabarcoding, as a culture-independent method, while culture-dependent methods only allowed the identification of nine genera. Isolation of actinobacteria resulted on the retrieval of 44 isolates, mainly associated with Brachybacterium, Microbacterium, and Brevibacterium genera. Some of these isolates were only identified on a specific sampled region. Chemical extracts of the actinobacterial isolates were subsequently screened for their antimicrobial, anticancer and anti-inflammatory activities. Extracts from two Streptomyces strains demonstrated activity against Candida albicans. Additionally, eight extracts (obtained from Brachybacterium, Brevibacterium, Microbacterium, Rhodococcus, and Streptomyces isolates) showed significant activity against at least one of the tested cancer cell lines (HepG2 and T-47D). Furthermore, 15 actinobacterial extracts showed anti-inflammatory potential in the RAW 264.4 cell model assay, with no concomitant cytotoxic response. Dereplication and molecular networking analysis of the bioactive actinobacterial extracts showed the presence of some metabolites associated with known natural products, but one of the analyzed clusters did not show any match with the natural products described as responsible for these bioactivities. Overall, we were able to recover taxonomically diverse actinobacteria with different bioactivities from the studied deep-sea samples. The conjugation of culture-dependent and -independent methods allows a better understanding of the actinobacterial diversity of deep-sea environments, which is important for the optimization of approaches to obtain novel chemically-rich isolates.

Keywords: actinobacteria; anti-cancer; anti-inflammatory; antimicrobial; deep-sea sediments; metabarcoding.

Figure 1

Taxonomic diversity and relative abundance at the phylum level of bacteria from the deep-sea sediment samples. Unassigned sequences and phyla comprising <2% of the total number of ASVs were classified as “Other.”

Figure 2

Heatmap with log transformation of the ASV abundance of Actinobacteria at order and family taxonomic levels in all deep-sea samples.

Figure 3

Absolute abundances (as number of ASVs) of the actinobacterial genera found across the deep-sea samples.

Figure 4

Cytotoxic activity of the deep-sea actinobacterial crude extracts tested at 15 μg/ml. The effects on the viability in the two cancer cell lines: breast ductal carcinoma (T-47D) and liver hepatocellular carcinoma (HepG2), as well as in the non-cancer cell line hCMEC/D3 (human brain capillary endothelial cells) are shown after 48 h of exposure. Only the extracts with statistically significant differences are indicated on the graphs. PC and SC mean positive and solvent controls, respectively. Values are represented as interval displaying the data distribution through their quartiles with 5–95% confidence from two independent assays conducted in triplicate and line within the box-whisker represents the median. Significant differences compared to the solvent control are annotated with asterisks in the graphs (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 5

Anti-inflammatory activity of the deep-sea actinobacterial crude extracts (15 μg/ml) after inflammation induced by LPS exposure (200 μg/ml). The inflammatory effect on the RAW 264.4 cell line as measured by NO production (box-whisker graph) and the effect on the viability of the same cellular line (plot graph) are shown after 48 h of exposure. Only the extracts with statistically significant differences are indicated in the graphs. All plot values represented below the pink line indicate that the extracts significantly reduce cell line viability in addition to having anti-inflammatory activity. Without LPS and LPS + SC denote positive and solvent with lipopolysaccharide controls, respectively. Values are demonstrated as interval displaying the data distribution through their quartiles with 5–95% confidence from two independent assays conducted in triplicate and line within the box-whisker represents the median. Significant differences compared to the solvent control are annotated with asterisks in the graphs (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 6

GNPS-based LC-HRESIMS/MS molecular networking analysis of the actinobacterial crude extract from Microbacterium sp. DS3_6.1, retrieved from the Arctic region, which probably contains news natural products. Unique molecular cluster represented by connected ellipses and marked with the corresponding m/z value. The major compound is estimated from the UV chromatogram and EICs, and shown in a different color from the remaining cluster nodes. The NL (normalization level) value corresponds to the base peak intensity of the m/z value when compared with the mass composition of the actinobacterial crude extract.