Metataxonomy of acid mine drainage microbiomes from the Santa Catarina Carboniferous Basin (Southern Brazil).

Mining activities generate large quantities of wastes that significantly alter the biogeochemistry and ecological structure of entire river basins. Microbial communities that develop in these areas present a variety of survival and adaptation mechanisms. Knowing this diversity at the molecular level is strategic both for understanding adaptive processes and for identifying genomes with potential use in bioremediation and bioprospecting. In this work, prokaryotic and eukaryotic communities were evaluated by meta-taxonomics (16S and 18S amplicons) in sediments and water bodies impacted by acid mine drainage in an important coal mining area in southern Brazil. Five sampling stations were defined on a gradient of impacts (pH 2.7-4.25). Taxon diversity was directly proportional to pH, being greater in sediments than in water. The dominant prokaryotic phyla in the samples were Proteobacteria, Actinobacteria, Acidobacteria, OD1, Nitrospirae, and Euryarchaeota, and among the eukaryotes, algae (Ochrophyta, Chlorophyta, Cryptophyceae), fungi (Basidiomycota, Ascomycota, and Cryptomycota), and protists (Ciliophora, Heterolobosea, Cercozoa). The prokaryotic genera Leptospirillum, Acidithiobacillus, Acidiphilium, Thiomonas, Thermogymnomonas, and Acidobacterium, and the eukaryotic genera Pterocystis and Poteriospumella were associated with more acidic conditions and higher metal concentrations, while the prokaryotic genera Sediminibacterium, Gallionella Geothrix, and Geobacter were more abundant in transitional environments.

Carotenoids produced by the deep-sea bacterium Erythrobacter citreus LAMA 915: detection and proposal of their biosynthetic pathway.

Technologies based on synthetic biology to produce bacterial natural carotenoids depend on information regarding their biosynthesis. Although the biosynthetic pathway of common carotenoids is known, there are carotenoids whose pathways are not completely described. This work aimed to mine the genome of the deep-sea bacterium Erythrobacter citreus LAMA 915, an uncommon bacterium that forms yellow colonies under cultivation. This work further explores the potential application of the carotenoids found and low-cost substrates for bacterial growth. A combined approach of genome mining and untargeted metabolomics analysis was applied. The carotenoid erythroxanthin sulfate was detected in E. citreus LAMA 915 cell extract. A proposal for carotenoid biosynthesis by this bacterium is provided, involving the genes crtBIYZWG. These are responsible for the biosynthesis of carotenoids from the zeaxanthin pathway and their 2,2'-hydroxylated derivatives. E. citreus LAMA 915 extracts showed antioxidant and sun protection effects. Based on the high content of proteases and lipases, it was possible to rationally select substrates for bacterial growth, with residual oil from fish processing the best low-cost substrate selected. This work advances in the understanding of carotenoid biosynthesis and provides a genetic basis that can be further explored as a biotechnological route for carotenoid production.

Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions.

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

Draft Genome Sequence of the Deep-Sea Bacterium Moritella sp. JT01 and Identification of Biotechnologically Relevant Genes.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Draft Genome Sequence of Bacillus stratosphericus LAMA 585, Isolated from the Atlantic Deep Sea.

Bacillus stratosphericus LAMA 585 was isolated from the Mid-Atlantic-Ridge seafloor (5,500-m depth). This bacterium presents the capacity for cellulase, xylanase, and lipase production when growing aerobically in marine-broth media. Genes involved in the tolerance of oligotrophic and extreme conditions and prospection of biotechnological products were annotated in the draft genome (3.7 Mb).

Phylogenetic identification of marine bacteria isolated from deep-sea sediments of the eastern South Atlantic Ocean.

The deep-sea environments of the South Atlantic Ocean are less studied in comparison to the North Atlantic and Pacific Oceans. With the aim of identifying the deep-sea bacteria in this less known ocean, 70 strains were isolated from eight sediment samples (depth range between 1905 to 5560 m) collected in the eastern part of the South Atlantic, from the equatorial region to the Cape Abyssal Plain, using three different culture media. The strains were classified into three phylogenetic groups, Gammaproteobacteria, Firmicutes and Actinobacteria, by the analysis of 16s rRNA gene sequences. Gammaproteobacteria and Firmicutes were the most frequently identified groups, with Halomonas the most frequent genus among the strains. Microorganisms belonging to Firmicutes were the only ones observed in all samples. Sixteen of the 41 identified operational taxonomic units probably represent new species. The presence of potentially new species reinforces the need for new studies in the deep-sea environments of the South Atlantic.

Bioprospection of cellulolytic and lipolytic South Atlantic deep-sea bacteria

Background: Cellulases and lipases have broad industrial application, which calls for an urgent exploration of microorganisms from extreme environments as valuable source of commercial enzyme. In this context, the present work describes the bioprospection and identification of deep-sea bacteria that produce cellulases and lipases, as well their optimal temperature of activity. Results: The first step of this study was the screening of cellulolytic and lipolytic deep-sea bacteria from sediment and water column, which was conducted with substrates linked with 4-Methylumbelliferyl. Among the 161 strains evaluated, 40 were cellulolytic, 23 were lipolytic and 5 exhibited both activities. Cellulolytic and lipolytic bacteria are more common in sediment than at the water column. Based on the ability to produce cellulases and lipases three isolates were selected and identified (16S rRNA sequencing) as Bacillus stratosphericus, B. aerophilus and B. pumilus. Lipases of strain B. aerophilus LAMA 582 exhibited activity at a wide temperature range (4º to 37ºC) and include psychrophilic behaviour. Strain Bacillus stratosphericus LAMA 585 can growth in a rich (Luria Bertani) and minimal (Marine Minimal) medium, and does not need an inducer to produce its mesophilic cellulases and lipases. Conclusions: Deep-sea sediments have great potential for bioprospection of cellulase and lipase-producing bacteria. The strains LAMA 582 and LAMA 585 with their special features, exhibit a great potential to application at many biotechnology process.