Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries

Ajuts: Departamento Administrativo de Ciencias, Tecnología e Innovación (Colciencias), República de Colombia; Convocatoria 489 - 2009, Código 657048925406, Contrato de financiación RC. 427 - 2009 Colciencias - CorpoGen; Programa de Asistencias Graduadas de Universidad de los Andes, Bogotá, Colombia; i Programa Jóvenes Investigadores de ColcienciasBACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been develope

Characterization of the Genetic Potential, Catabolic Structure and Degradative Activities against BTEX in Microbial Communities from Aquifers under Adaptation to Organic Contaminants

Developments in molecular techniques have led to rapid and reliable tools to monitor microbial community structures and dynamics under in situ conditions. However, even though various functional genes are localized on mobile genetic elements such that metabolic potential/activity is not necessarily reflected by the community structure, there has been a lack of emphasis on monitoring functional diversity. A more detailed picture of the catabolic gene structure and sequence diversity in environmental samples will significantly increase our knowledge of the functional potential of microbial communities. We adapted suitable techniques to follow functional gene diversity and applied those to target catechol 2,3-dioxygenases as key genes in aromatic hydrocarbon degradation. Catabolic gene diversity in differentially BTEX contaminated environments was assessed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs. The PCR-SSCP technique could be shown to be a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the biodegradation functioning of microbial communities. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish pre-selection or enrichment bias and to assess the function of predominant gene polymorphs. To rapidly determine phylogenetic diversity of functional genes from strain collections or environmental DNA amplifications, a restriction enzyme, theoretically producing characteristic profiles, the similarities of which reassembled the main divergent branches of C23O gene phylogeny, was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and isolates. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation. AFDRA was also used to determine the predominant polymorphism of the C23O gene present in environmental DNA extracts and in combination with a most-probable-number-PCR approach, its abundance. This approach may be useful to differentiate functional genes also for many other gene families. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralising phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralise benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. Complete C23O encoding open reading frames were cloned, sequenced and overexpressed by using conserved regions in operon neighbouring genes. Such strategy also allows the direct access to complete genes from environmental DNA. A single amino acid substitution at position 218 had severe influence on enzyme kinetics, and the Tyr218 variant differed from the His218 variant by lower turnover number but higher affinity. The information in this work underlines the importance to analyze catabolic gene diversity at different scales, from global views of diversity and fitness of genes in the ecosystem, to detailed understanding of sequence variations effects on catalytic activities.Caracterización del potencial genético, estructura catabólica y actividades degradadoras de BTEX en comunidades microbianas autóctonas de acuíferos oligotróficos expuestas a contaminantes orgánicos

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries

BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed

Growth of Pseudomonas chloritidismutans AW-1T on n-alkanes with chlorate as electron acceptor

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day−1, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes

Mangrove microniches determine the structural and functional diversity of enriched petroleum hydrocarbon-degrading consortia

In this study, the combination of culture enrichments and molecular tools was used to identify bacterial guilds, plasmids and functional genes potentially important in the process of petroleum hydrocarbon (PH) decontamination in mangrove microniches (rhizospheres and bulk sediment). In addition, we aimed to recover PH-degrading consortia (PHDC) for future use in remediation strategies. The PHDC were enriched with petroleum from rhizosphere and bulk sediment samples taken from a mangrove chronically polluted with oil hydrocarbons. Southern blot hybridization (SBH) assays of PCR amplicons from environmental DNA before enrichments resulted in weak positive signals for the functional gene types targeted, suggesting that PH-degrading genotypes and plasmids were in low abundance in the rhizosphere and bulk sediments. However, after enrichment, these genes were detected and strong microniche-dependent differences in the abundance and composition of hydrocarbonoclastic bacterial populations, plasmids (IncP-1 alpha, IncP-1 beta, IncP-7 and IncP-9) and functional genes (naphthalene, extradiol and intradiol dioxygenases) were revealed by in-depth molecular analyses [PCR-denaturing gradient gel electrophoresis and hybridization (SBH and microarray)]. Our results suggest that, despite the low abundance of PH-degrading genes and plasmids in the environmental samples, the original bacterial composition of the mangrove microniches determined the structural and functional diversity of the PHDC enriched.Deutsche Forschungsgemeinschaft [SM59/4-1, 4-2]; FAPERJ-Brazil; European Commission [003998, 211684]; Alexander-von-Humboldt-Stiftung; CONICET (Argentina)info:eu-repo/semantics/publishedVersio

Enrichment and characterization of a bilge microbial consortium with oil in water-emulsions breaking ability for oily wastewater treatment

Oily bilge wastewater is one of the main sources of hydrocarbons pollution in marine environments due to accidental or clandestine discharges. The main technical challenge for its effective treatment is the presence of stable oil-in-water (O/W) emulsions. In this work we are reporting an enriched microbial consortium from bilge wastewater with remarkable ability to demulsify oil in water emulsions. The consortium showed emulsion-breaking ratios up to 72.6% in the exponential growth phase, while the values range from 11.9 to 8.5% in stationary phase. A positive association was observed between demulsifying ability and microbial adhesion to hydrocarbons, as well as between cell concentration and demulsifying ability. Also, an interesting ability to demulsify under different temperatures, conditions of agitation, and bilge emulsions from different vessels was observed. The Bacterial and Archaeal composition was analyzed by 16S rRNA gene amplicon lllumina sequencing analyses, revealing an assemblage composed of bacterial types highly related to well characterized bacterial isolates and also to non-yet cultured bacterial types previously detected in marine and sediment samples. Hydrocarbonoclastic microbial types such as Marinobacter, Flavobacteriaceae, Alcanivorax and Gammaproteobacteria PYR10d3 were found in high relative abundance (27.0%– 11.1%) and types of marine oligotrophs and surfactant degraders such as Thallasospira, Parvibaculum, Novospirillum, Shewanella algae, and Opitutae were in a group of middle predominance (1.7–3.5%). The microbial consortium reported has promising potential for the biological demulsification of bilge wastewater and other oily wastewaters

Design and evaluation of synthetic bacterial consortia for optimized phenanthrene degradation through the integration of genomics and shotgun proteomics

Two synthetic bacterial consortia (SC) composed of bacterial strains Sphingobium sp. (AM), Klebsiella aerogenes (B), Pseudomonas sp. (Bc-h and T), Burkholderia sp. (Bk) and Inquilinus limosus (Inq) isolated from a natural phenanthrene (PHN)-degrading consortium (CON) were developed and evaluated as an alternative approach to PHN biodegradation in bioremediation processes. A metabolic network showing the potential role of strains was reconstructed by in silico study of the six genomes and classification of dioxygenase enzymes using RHObase and AromaDeg databases. Network analysis suggested that AM and Bk were responsible for PHN initial attack, while Inq, B, T and Bc-h would degrade PHN metabolites. The predicted roles were further confirmed by physiological, RT-qPCR and metaproteomic assays. SC-1 with AM as the sole PHN degrader was the most efficient. The ecological roles inferred in this study can be applied to optimize the design of bacterial consortia and tackle the biodegradation of complex environmental pollutants.EEA RafaelaFil: Macchi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Festa, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Nieto, Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Irazoqui, Jose Matias. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Vega-Vela, Nelson E. Pontificia Universidad Javeriana; Colombia. Universidad de Bogotá Jorge Tadeo Lozano; ColombiaFil: Junca, Howard. Microbiomas Foundation. Division Ecogenomics & Holobionts. RG Microbial Ecology: Metabolism, Genomics & Evolution; ColombiaFil: Valacco, María Pía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. IQUIBICEN. Consejo Nacional de Investigaciones Científicas y Técnicas. IQUIBICEN; ArgentinaFil: Amadio, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Morelli, Irma S. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires; ArgentinaFil: Coppotelli, Bibiana M. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript