Draft genome sequence of Psychrobacter nivimaris LAMA 639 and its biotechnological potential.

Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.

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.

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.

Evidence of selective pressure in whale fall microbiome proteins and its potential application to industry.

The present study addresses the microbiome of the first whale fall (YOKO 16) that has been described in the deep sea in the southern Atlantic Ocean (São Paulo Plateau; 4204 m depth), in terms of its metabolic uniqueness. Sets of ten thousand protein sequences from YOKO 16 and 29 public domain metagenomes (SRA and GenBank databases) that represent various marine, terrestrial and gut-associated microbial communities were analyzed. The determination of protein functionality, based on the KAAS server, indicated that the YOKO 16 microbiome has industrially-relevant proteins, such as proteases and lipases, that have low similarity (~50%) with previously-described enzymes. The amino acid usage in the YOKO 16 protein sequences (based on blastp and Clustal analysis) revealed a pattern of preference similar to that of extremophiles, with an increased usage of polar, charged and acidic amino acids and a decreased usage of nonpolar residues. We concluded that the targeted microbiome is of potential biotechnological use, which justifies the allocation of resources for the discovery of enzymes in deep-sea whale fall communities.

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria.

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)-a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Prospecting for Marine Bacteria for Polyhydroxyalkanoate Production on Low-Cost Substrates.

Polyhydroxyalkanoates (PHAs) are a class of biopolymers with numerous applications, but the high cost of production has prevented their use. To reduce this cost, there is a prospect for strains with a high PHA production and the ability to grow in low-cost by-products. In this context, the objective of this work was to evaluate marine bacteria capable of producing PHA. Using Nile red, 30 organisms among 155 were identified as PHA producers in the medium containing starch, and 27, 33, 22 and 10 strains were found to be positive in media supplemented with carboxymethyl cellulose, glycerol, glucose and Tween 80, respectively. Among the organisms studied, two isolates, LAMA 677 and LAMA 685, showed strong potential to produce PHA with the use of glycerol as the carbon source, and were selected for further studies. In the experiment used to characterize the growth kinetics, LAMA 677 presented a higher maximum specific growth rate (µmax = 0.087 h-1) than LAMA 685 (µmax = 0.049 h-1). LAMA 677 also reached a D-3-hydroxybutyrate (P(3HB)) content of 78.63% (dry biomass), which was 3.5 times higher than that of LAMA 685. In the assay of the production of P(3HB) from low-cost substrates (seawater and biodiesel waste glycerol), LAMA 677 reached a polymer content of 31.7%, while LAMA 685 reached 53.6%. Therefore, it is possible to conclude that the selected marine strains have the potential to produce PHA, and seawater and waste glycerol may be alternative substrates for the production of this polymer.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

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.

Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

BACKGROUND: Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals. RESULTS: Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater. CONCLUSIONS: Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

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.

Endophytic Methylobacterium extorquens expresses a heterologous ß-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa.

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Heavy metal resistance of microorganisms isolated from coal mining environments of Santa Catarina

A atividade de mineração do carvão é responsável pela geração de diferentes sub-produtos. Entre esses, está a pirita que acidifica a água e acelera o processo de solubilização de metais. Como conseqüência, o ambiente torna-se ácido e rico em metais pesados, os quais selecionam os microrganismos capazes de sobreviver nestas condições. Esses microrganismos podem, por sua vez, serem empregados como agentes para a biorremediação de áreas contaminadas com metais pesados. No presente trabalho é descrito o isolamento e a caracterização de bactérias, fungos e leveduras resistentes aos metais zinco, níquel e cádmio.

Heavy metal resistance of microorganisms isolated from coal mining environments of Santa Catarina

The coal mining activity is characterized by the generation of large amount of by-products. One of them is pyrite, which tends to acidify the water, solubilizing heavy metals. As a consequence the environment becomes acid and rich in heavy metals, selecting microorganisms able to survive in this condition, which are of great interest as bioremediation agents. This work describes the isolation and characterization of microorganisms from a coal mining area in Santa Catarina. These microorganisms comprised bacteria, fungi and yeasts resistant to zinc, nickel and cadmium.

A atividade de mineração do carvão é responsável pela geração de diferentes sub-produtos. Entre esses, está a pirita que acidifica a água e acelera o processo de solubilização de metais. Como conseqüência, o ambiente torna-se ácido e rico em metais pesados, os quais selecionam os microrganismos capazes de sobreviver nestas condições. Esses microrganismos podem, por sua vez, serem empregados como agentes para a biorremediação de áreas contaminadas com metais pesados. No presente trabalho é descrito o isolamento e a caracterização de bactérias, fungos e leveduras resistentes aos metais zinco, níquel e cádmio.