Comparative Genomics and Biosynthetic Potential Analysis of Two Lichen-Isolated Amycolatopsis Strains

Abstract

Actinomycetes have been extensively exploited as one of the most prolific secondary metabolite-producer sources and continue to be in the focus of interest in the constant search of novel bioactive compounds. The availability of less expensive next generation genome sequencing techniques has not only confirmed the extraordinary richness and broad distribution of silent natural product biosynthetic gene clusters among these bacterial genomes, but also has allowed the incorporation of genomics in bacterial taxonomy and systematics. As part of our efforts to isolate novel strains from unique environments, we explored lichen-associated microbial communities as unique assemblages to be studied as potential sources of novel bioactive natural products with application in biotechnology and drug discovery. In this work, we have studied the whole genome sequences of two new Amycolatopsis strains (CA-126428 and CA-128772) isolated from tropical lichens, and performed a comparative genomic analysis with 41 publicly available Amycolatopsis genomes. This work has not only permitted to infer and discuss their taxonomic position on the basis of the different phylogenetic approaches used, but has also allowed to assess the richness and uniqueness of the biosynthetic pathways associated to primary and secondary metabolism, and to provide a first insight on the potential role of these bacteria in the lichen-associated microbial community.

Keywords: Amycolatopsis; biosynthetic gene clusters; phylogeny; secondary metabolites; whole genome sequence.

Figure 1

Phylogenetic NJ tree based on 16S rRNA gene sequences from 82 Amycolatopsis strains. The scale bar indicates 5 nucleotide substitutions per 1,000 nucleotides, and the node numbers are percentage bootstrap values based on 1,000 resampled datasets. Bootstrap values below 50% are not shown. The AOS, ATS and AMS subclades are indicated, as well as the groups A-J. The strains grouped with CA-126428 and CA-128772 isolates (group C) are blue-shaded.

Figure 2

Phylogenetic NJ trees based on 16S rRNA gene sequences (Left) and four concatenated sequences (atpD, dnaK, recA and rpoB) (MLSA, Right) from 43 genome-sequenced Amycolatopsis strains. The scale bars indicate the percentage of difference in the nucleotide sequences, and the node numbers are percentage bootstrap values based on 1,000 resampled datasets. Bootstrap values below 50% are not shown. The AOS, ATS and AMS subclades are indicated, as well as the groups A–J. The strains grouped with CA-126428 and CA-128772 isolates (group C) are blue-shaded.

Figure 3

16S rRNA (Bottom left) vs. MLSA (Top right) genomic distances heatmap using the Kimura 2-parameter model. Self-genome comparisons occur on a diagonal line stretching from the top left to the bottom right corners. The strains are ordered in the same way as the 16S phylogenetic tree in Figure 2, which has been placed upon both axes for orientation. The strains belonging to group C have been shaded in blue for clarification. The heatmap legend is shown on the right.

Figure 4

Comparative analyses of CA-126428 and CA-128772 draft genomes with 9 complete Amycolatopsis genomes. Horizontal straight lines represent the genomes and vertical colored bars represent different conserved genes. The vertical bars are connected by corresponding colored thin lines. Black arrows represent the position of the secondary metabolite biosynthetic pathways predicted by antiSMASH.

Figure 5

Most frequent secondary metabolite biosynthetic gene clusters predicted by antiSMASH in the genomes of Amycolatopsis strains. The strains are ordered in the same way as the 16S rRNA phylogenetic tree in Figure 2, which has been placed on the left axis for orientation. The strains of group C have been shaded in gray for clarification. The length of each horizontal bar corresponds with the number of BGCs, and the color code is indicated on the right.

Figure 6

Biosynthetic gene clusters predicted in the genomes of Amycolatopsis with more than 70% homology with known BGCs. Yellow: predicted pathways whose encoded metabolites have not been detected in culture; green: predicted pathways whose encoded metabolites have been detected in culture; red: compounds detected in culture but not predicted by antiSMASH. The strains are ordered in the same way as the 16S rRNA phylogenetic tree in Figure 2, which has been placed on the left axis for orientation. The strains of group C have been shaded in gray for clarification.