Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

<p>Abstract</p> <p>Background</p> <p>Polycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination.</p> <p>Results</p> <p>Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (<it>r </it>= 0.834, <it>p </it>< 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical <it>nahAc</it>-like genes, <it>phnAc</it>-like genes as identified in <it>Alcaligenes faecalis </it>AFK2, and <it>phnA1</it>-like genes from marine PAH-degraders from the genus <it>Cycloclasticus</it>.</p> <p>Conclusion</p> <p>These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.</p

Prospecting biotechnologically-relevant monooxygenases from cold sediment metagenomes: An in silico approach

Source at https://doi.org/10.3390/md15040114.The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

Bioprospection of marine microorganisms: biotechnological applications and methods

Environmental microorganisms constitute an almost inexhaustible reserve of genetic and functional diversity, accumulated during millions of years of adaptive evolution to various selective pressures. In particular, the extent of microbial biodiversity in marine habitats seems to grow larger as new techniques emerge to measure it. This has resulted in novel and more complex approaches for the screening of molecules and activities of biotechnological interest in these environments. In this review, we explore the different partially overlapping biotechnological fields that make use of microorganisms and we describe the different marine habitats that are particularly attractive for bioprospection. In addition, we review the methodological approaches currently used for microbial bioprospection, from the traditional cultivation techniques to state of the art metagenomic approaches, with emphasis in the marine environment.Bioprospección de microorganismos marinos: aplicaciones biotecnológicas y métodos. Los microorganismos ambientales constituyen una reserva prácticamente inagotable de diversidad genética, acumulada durante millones de años de evolución adaptativa a varias presiones selectivas. En particular, la magnitud de la biodiversidad microbiana en hábitats marinos parece crecer al emerger nuevas técnicas para medirla. Como resultado, se han comenzado a utilizar enfoques novedosos y más complejos para la búsqueda de moléculas y actividades de interés biotecnológico en estos ambientes. En este artículo de revisión, nosotros exploramos los diferentes campos de la biotecnología que utilizan microorganismos, los cuales se superponen parcialmente, y describimos los diferentes hábitats marinos que resultan particularmente atractivos para la bioprospección. Además, revisamos los enfoques metodológicos actualmente utilizados para la bioprospección microbiana, desde las técnicas de cultivo tradicionales hasta modernos enfoques metagenómicos, con énfasis en el medio ambiente marino

Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ± 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ± 0.28% of the biosludge population in the municipal WWTP and 0.37% ± 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs

Predominance and high diversity of genes associated to denitrification in metagenomes of subantarctic coastal sediments exposed to urban pollution.

The aim of this work was to characterize the microbial nitrogen cycling potential in sediments from Ushuaia Bay, a subantarctic environment that has suffered a recent explosive demographic growth. Subtidal sediment samples were retrieved in triplicate from two urban points in the Bay, and analyzed through metagenomic shotgun sequencing. Sequences assigned to genes related to nitrification, nitrate reduction and denitrification were predominant in this environment with respect to metagenomes from other environments, including other marine sediments. The nosZ gene, responsible for nitrous oxide transformation into di-nitrogen, presented a high diversity. The majority of NosZ sequences were classified as Clade II (atypical) variants affiliated to different bacterial lineages such as Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, Verrucomicrobia, as well as to Archaea. The analysis of a fosmid metagenomic library from the same site showed that the genomic context of atypical variants was variable, and was accompanied by distinct regulatory elements, suggesting the evolution of differential ecophysiological roles. This work increases our understanding of the microbial ecology of nitrogen transformations in cold coastal environments and provides evidence of an enhanced denitrification potential in impacted sediment microbial communities. In addition, it highlights the role of yet overlooked populations in the mitigation of environmentally harmful forms of nitrogen

Emergence of Competitive Dominant Ammonia-Oxidizing Bacterial Populations in a Full-Scale Industrial Wastewater Treatment Plant

Ammonia-oxidizing bacterial populations in an industrial wastewater treatment plant were investigated with amoA and 16S rRNA gene real-time PCR assays. Nitrosomonas nitrosa initially dominated, but over time RI-27-type ammonia oxidizers, also within the Nitrosomonas communis lineage, increased from below detection to codominance. This shift occurred even though nitrification remained constant