In Situ Determination of the Effects of Lead and Copper on Cyanobacterial Populations in Microcosms

BACKGROUND: Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth. METHODOLOGY/PRINCIPAL FINDINGS: Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations. CONCLUSIONS/SIGNIFICANCE: The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants

Cellular Electron Microscopy Imaging Reveals the Localization of the Hfq Protein Close to the Bacterial Membrane

Background: Hfq is a bacterial protein involved in several aspects of nucleic acid transactions, but one of its bestcharacterized functions is to affect the post-transcriptional regulation of mRNA by virtue of its interactions with stressrelated small regulatory (sRNA). Methodology and Principal Finding: By using cellular imaging based on the metallothionein clonable tag for electron microscopy, we demonstrate here that in addition to its localization in the cytoplasm and in the nucleoid, a significant amount of Hfq protein is located at the cell periphery. Simultaneous immunogold detection of specific markers strongly suggests that peripheral Hfq is close to the bacterial membrane. Because sRNAs regulate the synthesis of several membrane proteins, our result implies that the sRNA- and Hfq-dependent translational regulation of these proteins takes place in the cytoplasmic region underlying the membrane. Conclusions: This finding supports the proposal that RNA processing and translational machineries dedicated to membrane protein translation may often be located in close proximity to the membrane of the bacterial cell

Impacto del petróleo en la distribución y biomasa de las cianobacterias en ecosistemas naturales y artificiales

Consultable des del TDXTítol obtingut de la portada digitalitzadaEn el presente trabajo se ha estudiado el efecto del petróleo en las cianobacterias, bacterias fototróficas oxigénicas que forman las poblaciones dominantes de los tapetes microbianos. Se trata de ambientes bentónicos estratificados situados en costas litorales y que se encuentran en ocasiones expuestos a los vertidos accidentales de petróleo. El papel de las cianobacterias en la bioreparación del crudo es un tema que suscita mucho interés, aunque no se han dado, hasta el presente momento, datos concluyentes sobre si la degradación del petróleo se produce exclusivamente por un tipo de cianobacteria o por un consorcio de microorganismos. Considerando el objetivo anteriormente expuesto, se ha analizado la diversidad, y determinado los perfiles de biomasa individual y total de las cianobacterias, mediante microscopio láser confocal (CLSM) en los ambientes naturales contaminados y no contaminados por petróleo (delta del Ebro, Salins-de-Giraud, Colònia de Sant Jordi, Waulkmill bay, Swanbister bay y Etang de Bêrre) y en ecosistemas artificiales (mesocosmos). Así mismo, se ha aislado e identificado un consorcio de microorganismos con capacidad para degradar el petróleo. En los tapetes microbianos estudiados, se observan cambios tanto en la diversidad de las cianobacterias, como en su biomasa total. En los ambientes no contaminados se han identificado cianobacterias filamentosas como Microcoleus chthonoplastes, Oscillatoria sp., Lyngbya sp., Limnothrix sp. y cianobacterias unicelulares como Gloeocapsa sp., Chroococcus sp., Synechocystis sp., localizándose la concentración máxima de biomasa total principalmente en la capa óxica de los tapetes. En los ecosistemas poco contaminados, se han identificado principalmente cianobacterias unicelulares correspondientes al grupo Pleurocapsa, siguiendo la biomasa total de estos microorganismos un perfil parecido al de los ambientes anteriormente mencionados. En los muy contaminados, se identificaron exclusivamente cianobacterias del tipo filamentoso, observándose una reducción de la biomasa total a lo largo del tapete. En los ecosistemas artificiales (mesocosmos), las cianobacterias del tipo unicelular se detectaron solo en los contaminados por petróleo (aunque a muy baja concentración de biomasa), mientras que Microcoleus chthonoplastes fue la cianobacteria dominante, tanto en las muestras control, como en las contaminadas por el crudo. Dada la ubicuidad de esta cianobacteria en los diferentes tipos de ambientes estudiados, y por su reconocido papel en la estabilización de los sedimentos, el objetivo anterior se complementó con un análisis de la distribución de los perfiles de biomasa de este microorganismo durante un ciclo día-noche en los tapetes microbianos de Salins-de-Giraud. El estudio demostró la versatilidad metabólica de esta cianobacteria, al presentar máximos de biomasa, en capas sometidas a parámetros ambientales muy distintos: presencia de luz y O2 (31.22 mgC/cm3 de sedimento), presencia de luz y H2S (28.91 mgC/cm3 de sedimento). Finalmente, uno de los principales objetivos del trabajo, fue el aislamiento de Microcoleus sp. en cultivos de laboratorio para analizar el efecto sobre el crecimiento de este microorganismo, de dos tipos de petróleo: el Casablanca (con alto contenido de hidrocarburos alifáticos) y el Maya (rico en azufre y en compuestos aromáticos) y muy tóxico. A partir de dichos cultivos se aisló un consorcio de microorganismos, al que se denominó Microcoleus consorcio. La caracterización de este consorcio, se realizó utilizando técnicas microscópicas de alta resolución. El CLSM, permitió caracterizar e identificar a la cianobacteria filamentosa, mientras que la caracterización de las bacterias heterotróficas que formaban parte del consorcio, se realizó mediante microscopía electrónica de transmisión (TEM) y de barrido (SEM). La identificación de las bacterias antes mencionadas se efectuó además mediante técnicas moleculares (Reacción en Cadena de la Polimerasa-Electroforesis en Gel de Gradiente Desnaturalizante). Los resultados obtenidos mostraban que el consorcio estaba formado por una cianobacteria, Microcoleus chthonoplastes y diferentes bacterias heterotróficas incluidas en la envuelta de exopolisacáridos de la cianobacteria. Las bacterias heterotróficas identificadas, fueron en su mayoría fijadoras de nitrógeno y pertenecían a diferentes grupos filogenéticos como a las ?, ? y ?, subclases de Proteobacteria, y al grupo CFB. Es importante mencionar, que el análisis químico del petróleo, después del crecimiento del consorcio, demostró que éste degradaba el crudo Maya; principalmente los alquiltianos, alquiltiolanos y carbazoles, lo que podría tener un gran interés en estudios futuros de ecotoxicidad.The present work studies the effect of oil on cyanobacteria, oxygenic phototrophic bacteria that form the dominant populations of microbial mats. These are stratified benthonic environments located in coastal sites and that are sometimes exposed to accidental oil spills. The role of the cyanobacteria in the biorepair of oil is an issue that has raised considerable interest, although to date no conclusive data has been forthcoming on whether the degradation of oil is exclusively produced by a given cyanobacterium or by a consortium of micro-organisms. Bearing in mind the objective raised above, we have analysed the diversity-and determined the profiles-of individual and total cyanobacteria biomass, through confocal laser microscopy (CLSM), in natural environments (the Ebro delta, Salins-de-Giraud, Colònia de Sant Jordi, Waulkmill bay, Swanbister bay and Etang de Bêrre) and in artificial environments (mesocosms). At the same time, we have isolated and identified a consortium of micro-organisms capable of degrading oil. In the microbial mats studied, changes are observed both in the diversity of the cyanobacteria and in their total biomass. In the non-polluted environments, we have observed filamentous cyanobacteria such as Microcoleus chthonoplastes, Oscillatoria sp., Lyngbya sp., Limnothrix sp. and unicellular cyanobacteria such as Gloeocapsa sp., Chroococcus sp., Synechocystis sp., situating the maximum concentration of total biomass basically in the oxic layer of the mats. In those environments that are only slightly polluted, we have principally identified unicellular cyanobacteria corresponding to the Pleurocapsa group, the total biomass of these micro-organisms following a profile similar to that of the above-mentioned environments. In heavily polluted environments, we exclusively identify cyanobacteria of the filamentous type, observing a reduction in total biomass throughout the mat. In artificial ecosystems (mesocosmos), unicellular type cyanobacteria are only detected in oil contaminants (although at very low biomass concentrations), whilst Microcoleus chthonoplastes was the dominant cyanobacterium, both in control samples as well as in oil contaminants. Given the ubiquity of this cyanobacterium in the various environment types studied, and because of its recognised role in sediment stabilization, our earlier objective has been complemented with an analysis of the biomass-profile distribution for this micro-organism during the day-night cycle in the Salins-de-Giraud microbial mats. The study demonstrated the metabolic versatility of this cyanobacterium, on showing biomass maximums, in those layers subjected to very distinct environmental parameters, namely, the presence of light and O2 (31.22 mgC/cm3 of sediment), the presence of light and H2S (28.91 mgC/cm3 of sediment). Finally, one of the principal objectives of this work was that of isolating Microcoleus sp. In laboratory cultures so as to analyse the effect on the growth of this micro-organism of two types of oil: Casablanca (with a high content of aliphatic hydrocarbons) and Maya (rich in sulphur and aromatic compounds) and highly toxic. On the basis of these cultures, a consortium of micro-organisms was then isolated, which was given the name of the Microcoleus consortium. The characterization of this consortium was undertaken by high-resolution microscope techniques. CLSM allows the characterization and identification of the filamentous cyanobacterium, whilst characterization of the heterotrophic bacteria that formed part of the consortium was undertaken by transmission-electronic microscopy (TEM) and sweep microscopy (SEM). Identification of the above-mentioned bacteria was additionally carried out through molecular techniques (Polymerase-Electrophoresis Chain Reaction in Denaturalizing Gradient Gel). The results obtained show that the consortium was formed by a cyanobacterium, Microcoleus chthonoplastes, and different heterotrophic bacteria included within the exopolisaccharide sheath of the cyanobacterium. The heterotrophic bacteria identified were, in their majority, nitrogen fixers belonging to different phylogenetic groups such as ?, ? and ?-subclasses of Proteobacteria-and the CFB group. It is of importance to observe that the chemical analysis of oil, after the growth of the consortium, showed that the consortium degraded the Maya oil, principally alkylthiolanes, alkylthianes and carbazoles, which could be of considerable interest to future studies of ecotoxicity

In situ determination of the effects of lead and copper on cyanobacterial populations in microcosms

This research is supported by Spanish grant DGICYT (Ref. CGL2005-03792/BOS and CGL2008-01891/BOS) and by Generalitat de Catalunya grant ITT-CTP (Ref. 2007ITT 00003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Background: Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth. Methodology/Principal Findings: Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations. Conclusions/Significance: The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants

Visualization of proteins in intact cells with a clonable tag for electron microscopy

12 pages, 9 figures.-- PMID: 19114107 [PubMed].-- Supplementary information (Suppl. movies S1-S3) available at: http://dx.doi.org/10.1016/j.jsb.2008.11.009Available online Dec 10, 2008 [Epub ahead of print].Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecAMT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.Authors want to thank the support from the UPMCIFR83 electron microscopy service and Rocío Arranz (CNB-CSIC) for assistance with cryoEM studies. This work has been funded by Grants PIF06-004/200620F0024 from the Consejo Superior de Investigaciones Científicas (CSIC), BFU2006-04584/BMC from the Ministerio de Educación y Ciencia of Spain (C.R.), 3DEM European network (LSHG-CT-2004-502828) and ANR (PCV06 142771) (S.M.). E.D. is funded by the JAE-CSIC program of Spain and J.F. by the Comunidad de Madrid.Peer reviewe

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP-MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells

Changes in the composition of polar and apolar crude oil fractions under the action of Microcoleus consortia

7 pages, 6 figures, 1 table.-- PMID: 15300420 [PubMed].-- Printed version published Dec 2004.Cultures of Microcoleus consortia polluted with two different types of crude oil, one with high content in aliphatic hydrocarbons (Casablanca) and the other rich in sulphur and aromatic compounds (Maya), were grown for 50 days and studied for changes in oil composition. No toxic effects from these oils were observed on Microcoleus consortia growth. In fact, the interface layer between the oils and the water culture medium proved to be the ideal site for consortia development, leading to a wrapping effect of the oil layers by these organisms. Despite this affinity of cyanobacteria for the oil substrate, the changes in oil composition were small. Microcoleus consortia did not induce transformation in the aliphatic-rich oil, and the modifications in the sulphur and aromatic-rich oil were small. The latter essentially involved degradation of aliphatic heterocyclic organo-sulphur compounds such as alkylthiolanes and alkylthianes. Other groups of compounds, such as the alkylated monocyclic and polycyclic aromatic hydrocarbons, carbazoles, benzothiophenes and dibenzothiophenes, also underwent some degree of transformation, involving only the more volatile and less alkylated homologues.Financial contribution from the European Union MATBIOPOL project EVK3-CT-1999-00010.Peer reviewe

Hfq-MT expressed in <i>E. coli</i> wild-type <i>E. coli</i> cells and induced with 0.0025% ARA.

<p>Localization patterns of the small electron-dense particles associated with the fusion protein expressed in the presence of endogenous Hfq show some differences with respect to Hfq⁻ cells. (A) Arrows point to a double layer of particles separated by a gap clearly seen on the cell periphery. (B) In this gap particles arranged with a filamentous pattern are frequently seen at higher magnification (arrow). (C) Immunogold labeling with anti-LamB antibodies and a 15 nm colloidal gold conjugate confirms that the external layer of electron-dense particles corresponds to the outer bacterial membrane. (D–F) Labeling with anti-RNase E antibodies and a 10 nm colloidal gold conjugate shows labelling associated with the internal layer of electron-dense particles. Inset in (D) is a higher magnification view of the area marked with the dashed rectangle. (F) Higher magnification of the area marked with a dashed rectangle in (E). Black Arrows point to RNase E-conjugated colloidal gold particles associated with the inner layer of small particles (Hfq-MT). Bars: 50 nm (A, C, inset in D, and F), 25 nm (B), 100 nm (D and E).</p

Intracellular distribution of Hfq-MT-associated electron-dense particles.

<p>Transformed MC4100 <i>hfq⁻ E. coli</i> cells were induced with different concentrations of arabinose (ARA) and grown in the presence of gold salts. Ultrathin-sections of vitrified and freeze-substituted cells are shown in all panels. (A) A bacterium induced with 0.001% ARA. This concentration reproduces the expression levels of Hfq during the stationary growth phase. Electron-dense particles are abundant both in the interior (asterisk) and the periphery (arrow) of the cell. The inset is an enlargement of the peripheral region marked with the dashed rectangle. (B) A transformed bacterium induced with 0.01% ARA showing a heavy accumulation of particles both in the interior (asterisk) and periphery (arrows) of the cell. The inset is a higher magnification view of the area marked with the dashed rectangle where it can be appreciated that some particles are slightly bigger than in bacteria induced with lower ARA concentrations. (C) Detail of the wall of a bacterium induced with 0.01% ARA and stained for 30 s with uranyl acetate (UA). Staining shows the real location of the outer membrane (OM) and confirms that the peripheral electron-dense particles delineate the inner membrane (IM). Extracellular granules are due to deposition of uranium on resin protrusions. (D) and (E) A transformed bacterium induced with 0.001% ARA showing intense signal in intracellular regions compatible with being the nucleoid (asterisk). (E) A high magnification view of the central area of the cell in (D) shows abundant small electron-dense particles, apparently arranged with a filamentous-like pattern (the arrows point to some of several such fibers). Bars: 100 nm (A, B, C and D), 50 nm (E and insets in A and B).</p

Total cyanobacterial biomass and <i>Microcoleus</i> sp. biomass expressed in mg C· cm⁻² of sediment.

<p>Total cyanobacterial biomass and <i>Microcoleus</i> sp. biomass expressed in mg C· cm⁻² of sediment.</p