Genome mining of marine bacteria for bioactive metabolites

Abstract

Marine bacteria have come into the focus for natural product discovery as a consequence of the emergence of antibiotic resistance. This was additionally boosted by the limitations encountered in drug developments from traditional producers of drug leads from terrestrial environments. The comparative genomic/metabolomic analysis is considered as a new and powerful tool to disclose the potential of microbes for the biosynthesis of novel specialized metabolites. In the group of marine myxobacteria, only a limited number of isolated species and sequenced genomes is available. However, the few compounds isolated thereof to date show interesting bioactivities and even novel chemical scaffolds, indicating a huge potential for natural product discovery. In this study, all marine myxobacteria with accessible genome data (n=5), including Haliangium ochraceum DSM 14365, Plesiocystis pacifica DSM 14875, Enhygromyxa salina DSM 15201, and the two newly sequenced species Enhygromyxa salina SWB005 and SWB007, were analyzed. All of these genomes are large (~10 Mb), with a relatively small core genome and many unique coding sequences in each strain. Genome analysis revealed a high variety of biosynthetic gene clusters (BGCs) between the strains (Figure 1A, in summary), and several resistance models and essential core genes indicated the potential to biosynthesize antimicrobial molecules. Polyketides (PKs) and terpenes represented the majority of predicted specialized metabolite BGCs and contributed to the highest share between the strains. BGCs coding for non-ribosomal peptides (NRPs), PK/NRP hybrids and ribosomally synthesized and post-translationally modified peptides were mostly strain specific. These results were in line with the metabolomic analysis, which revealed a high diversity of the chemical features between the strains (Figure 1B, in summary). Only 6-11% of the metabolome was shared between all the investigated strains, which correlates to the small core genome of these bacteria (13-16% of each genome). In addition, the compound enhygrolide A, known from E. salina SWB005, was detected for the first time and structurally elucidated from E. salina SWB006. The here acquired data corroborate that these microorganisms represent a most promising source for the detection of novel specialized metabolites.<br /> Figure1. A: Venn diagram displaying biosynthetic gene clusters (BGCs) counts according to distribution in closely related strains of marine myxobacteria. (acquired from the similarity network of the predicted BGCs in the analyzed genomes). B: Venn diagram displaying chemical features counts according to detection from single or multiple strains of marine myxobacteria. (acquired from the molecular network of analyzed strains).<br /> To date, only a few myxobacteria are known as marine, owing to the salt dependency of their growth. Thus, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as a sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains E. salina SWB007 and P. pacifica DSM 14875 had developed different strategies to handle salt stress. P. pacifica DSM 14875, which was grown between 1 and 4% NaCl, relies solely on the accumulation of amino acids, while E. salina SWB007, which was grown between 0.5 and 3% NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine, and hydroxyectoine.<br /> The second group of marine bacteria investigated here belongs to the family Rhodobacteraceae and is frequently encountered in the oceans. The marine bacterium Labrenzia sp. 011 was isolated from the coastal sediment of Kronsgaard, Germany. This strain produces two cyclopropane-containing medium-chain fatty acids, namely cis-4-(2-hexylcyclopropyl)-butanoic acid and cis-2-(2-hexylcyclopropyl)-acetic acid (Figure 2A, in summary), which showed activity against a range of microorganisms. It is of special interest, that these compounds strongly inhibit Pseudoroseovarius crassostreae DSM 16950 (genus Roseovarius), the causative agent of Roseovarius oyster disease (ROD). The latter is a bacterial-induced infection and causes major problems in oyster aquaculture. Bacteria of the genus Labrenzia have been proposed as protective agents against ROD.<br /> Figure 2. A: Cyclopropane fatty acids from Labrenzia sp. 011. B: Proposed biosynthetic gene cluster of the isolated cyclopropane fatty acids. The genome analysis of bacteria of the genus Labrenzia was expected to provide information to understand the mollusk-protective role of Labrenzia spp.. Therefore, the genome of Labrenzia sp. 011 was sequenced and assembled into 65 contigs. It has a size of 5.1 Mbp and a G+C content of 61.6%. Comparative genome analysis defined Labrenzia sp. 011 as a distinct new species within the genus Labrenzia, whereby 44% of the genome was contributed to the Labrenzia core genome. The genomic analysis revealed several conserved cyclopropane fatty acid synthases (CFAS) genes in bacteria of the genus Labrenzia, putatively responsible for methylation and cyclopropanation of long-chain fatty acids. In addition, a gene cluster encoding for two distinct CFAS genes is proposed as the biosynthetic origin of the isolated cyclopropane fatty acids from Labreniza sp. 011 (Figure 2B, in summary). In conclusion, each of the investigated marine bacteria, myxobacteria and Labrenzia, harbors a high unique genetic and metabolic diversity, rendering these groups of microorganisms promising sources for novel specialized metabolites.