6 research outputs found
IngenierĂa de proteĂnas fluorescentes y aplicaciones de localizaciĂłn celular en microorganismos termĂłfilos
Tesis doctoral inĂ©dita leĂda en la Universidad AutĂłnoma de Madrid, Facultad de Ciencias, Departamento de BiologĂa Molecular. Fecha de lectura: 05-07-201
Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline Shallow Lake (Laguna de Carrizo, Central Spain)
The phylogenetic and functional structure of the microbial community residing in a Ca2+-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO4âĂâ2 H2O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca2+ and SO 4 2- being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physicalâchemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.This research was supported by the Spanish CSD2007-00005 project and FEDER funds. M-E.G. thanks the CSIC for a JAE fellowship.Peer Reviewe
Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica
M.K. and P.N.G. designed the work; T.N.C. performed physiological studies; M.K., M.F.,
Y.A.-R., A.B., N.L.-C., M.E.G., O.R.K., T.Y.N., S.K., I.L., O.V.G., M.M.Y. R.R. and P.N.G.
were associated with genome annotation; H.J.H. performed lipids and FAME analysis;
M.F., M-l.F., S.J., S.C. and J.P.A performed chaperonin anti-proteome analysis; A.-x. S.,
O.K., O.E., P.A.P., P.S. and Y.K. were associated with structural proteomics; A.T. and R.F.
were associated with functional proteomics; H.L. performed electron microscopy; R.D.
performed real-time PCR; M.M.-G. and M.F. performed DIGE proteome analysis;
M.G. was involved in siderophore production; O.N.R. performed genomic islandsâ
analysis; H.T. performed storage lipid compoundsâ analysis; P.N.G. coordinated
manuscript writing.Accession Codes: The genome sequence of Oleispira antarctica RB-8 has been deposited
in GenBank under accession core FO203512. Protein structures have deposited in PDB
under accession codes 3QVM (a/b hydrolase, OLEAN_C08020), 3QVQ (phosphodiesterase,
OLEAN_C20330), 3M16 (transaldolase, OLEAN_C18160), 3LQY (isochorismatase,
OLEAN_C07660), 3LNP (amidohydrolase, OLEAN_C13880), 3V77/3L53 (fumarylacetoacetate isomerase/hydrolase, OLEAN_C35840), 3VCR/3LAB
(2-keto-3-deoxy-6-phosphogluconate aldolase, OLEAN_C25130), 3IRU (phoshonoacetaldehyde
hydrolase, OLEAN_C33610), 3I4Q (inorganic pyrophosphatase,
OLEAN_C30460), 3LMB (protein with unknown function, OLEAN_C10530).Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment
on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica
and show that compared with Alcanivorax borkumensisâthe paradigm of mesophilic
hydrocarbonoclastic bacteriaâO. antarctica has a larger genome that has witnessed massive
gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants,
siderophores and micronutrient-scavenging pathways. We also show that at low temperatures,
the main protein-folding machine Cpn60 functions as a single heptameric barrel that
uses larger proteins as substrates compared with the classical double-barrel structure
observed at higher temperatures. With 11 protein crystal structures, we further report the
largest set of structures from one psychrotolerant organism. The most common structural
feature is an increased content of surface-exposed negatively charged residues compared to
their mesophilic counterparts. Our findings are relevant in the context of microbial
cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold
environments.We acknowledge the funding from the EU Framework Program 7 to support Projects
MAMBA (226977), ULIXES (266473), MAGIC PAH (245226) and MICROB3 (287589)
This work received the support of the Government of Canada through Genome Canada
and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405 to A.F.Y. and A.S.), and
the U.S. Government National Institutes of Health (grants GM074942 and GM094585
(to A.S. through Midwest Center for Structural Genomics). The study was supported by
the Max Planck Society and the Deutsche Forschungsgemeinschaft through project KU
2679/2-1 and BU 890/21-1. We thank the sequencing team of the AG Reinhardt for
technical assistance and Alfred Beck for computational support. The skilful work of
electron microscopic sample preparation by Mrs. Ingeborg Kristen (Dept. VAM, HZI
Braunschweig) is gratefully acknowledged. Authors thank Professor Ken Timmis for his
critical reading the manuscript and useful comments.http://www.nature.com/naturecommunicationsam201
Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery
Group II introns were developed some time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-positive and Gram-negative bacteria to human cells. Utilizing these introns is advantageous because they are independent of the hostâs DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of additional genes at the userâs will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counterselection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed that directing CRISPR/Cas9 toward the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counterselection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.This work was funded by the SETH Project of the Spanish Ministry of Science RTI 2018-095584âB-C42, MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018), and SYNBIO4FLAV (H2020-NMBP/0500). Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds). E.V. was the recipient of a Fellowship from the Education Ministry, Madrid, Spanish Government (FPU15/04315)
Environmental Performance of Pseudomonas putida with a Uracylated Genome
A variant of the soil bacterium Pseudomonas putida with a genome containing a âŒ20â% replacement of the whole of thymine (T) by uracil (U) was made by deleting genes ung (uracil DNA glycosylase) and dut (deoxyuridine 5âČâtriphosphate nucleotide hydrolase). Proteomic comparisons revealed that, of 281 upâregulated and 96 downâregulated proteins in the Îung Îdut cells, as compared to the wildâtype, many were involved in nucleotide metabolism. Unexpectedly, genome uracylation did not greatly change the gross environmental endurance profile of P.â
putida, increased spontaneous mutagenesis by only twofold and supported expression of heterologous proteins well. As Uâenriched DNA is potentially degraded by the base excision repair of recipients encoding a uracil DNA glycosylase, we then tested the spread potential of genetic material originating in the Îung Îdut cells either within the same species or in a commonly used Escherichia coli strain. Transformation and conjugation experiments revealed that horizontal gene transfer of Uâcontaining plasmids fared worse than those made of standard DNA by two orders of magnitude. Although this figure does not guarantee the certainty of containment, it suggests a general strategy for curbing the dispersal of recombinant genetic constructs.This work was funded by the SETH Project of the Spanish Ministry of Science RTI 2018â095584âBâC42, the MADONNA (H2020âFETâOPENâRIAâ2017â1â766975), BioRoboost (H2020âNMBPâBIOâCSAâ2018), SYNBIO4FLAV (H2020âNMBP/0500) and MIXâUP (H2020âGrant 870294) Contracts of the European Union and the S2017/BMDâ3691 InGEMICSâCM Project of the Comunidad de Madrid (European Structural and Investment Funds)
Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica
© 2013 Macmillan Publishers LimitedUbiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensisâthe paradigm of mesophilic hydrocarbonoclastic bacteriaâO. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.We acknowledge the funding from the EU Framework Program 7 to support Projects MAMBA (226977), ULIXES (266473), MAGIC PAH (245226) and MICROB3 (287589) This work received the support of the Government of Canada through Genome Canada and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405 to A.F.Y. and A.S.), and the U.S. Government National Institutes of Health (grants GM074942 and GM094585 (to A.S. through Midwest Center for Structural Genomics). The study was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft through project KU 2679/2-1 and BU 890/21-1.Peer Reviewe