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F. Monteiro Publications
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Cantareus aspersus metallothionein metal binding abilities: The unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins

Cantareus aspersus metallothionein metal binding abilities: The unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins | F. Monteiro Publications | Scoop.it
Scooped from: Biochim Biophys Acta. 2014. Authors: Sílvia Pérez-Rafael, Freddy Monteiro, Reinhard Dallinger, Sílvia Atrian, Òscar Palacios and Mercè Capdevila Abstract: In Proteomics, gene/protein families including both specialized and non-specialized paralogs are an invaluable tool to study the evolution of structure/function relationships in proteins. Metallothioneins (MTs) of the pulmonate gastropod molluscs (snails) offer one of the best materials to study the metal-binding specificity of proteins, because they consist of a polymorphic system that includes members with extremely distinct metal preferences but with a high protein sequence similarity. Cantareus aspersus was the first snail where three paralogous MTs were isolated: the highly specific cadmium (CaCdMT) and copper (CaCuMT) isoforms, and an unspecific CaCd/CuMT isoform, so called because it was natively isolated as a mixed Cd and Cu complex. In this work, we have thoroughly analyzed the Zn2 +-, Cd2 +- and Cu+-binding abilities of these three CaMTs by means of the spectroscopic and spectrometric characterization of the respective recombinant, as well as in vitro-substituted, metal-complexes. The comparison with the orthologous HpMTs and the study of the isoform-determinant residues allow correlating the protein sequence variability with the coordination capabilities of these MTs. Surprisingly, the CaCuMT isoform exhibits a stronger Cu-thionein character than the HpCuMT ortholog, and the CaCd/CuMT isoform could be defined as a non-optimized Cu-thionein, which has not attained any defined functional differentiation in the framework of the snail MT gene/protein family.
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A Luminescent Reporter Evidences Active Expression of R. solanacearum T3SS Genes Throughout Plant Infection, by Monteiro et al., in Microbiology

A Luminescent Reporter Evidences Active Expression of R. solanacearum T3SS Genes Throughout Plant Infection, by Monteiro et al., in Microbiology | F. Monteiro Publications | Scoop.it

Monteiro, F., Genin, S., Van Dijk, I. & Valls, M. A Luminescent Reporter Evidences Active Expression of Ralstonia Solanacearum Type III Secretion System Genes Throughout Plant Infection. Microbiology (2012). doi:10.1099/mic.0.058610-0

IF: 2.957 (2010); 3.061 (2011)

 

Summary:

Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen R. solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo -either after re-isolation of pathogens from infected plants or directly in situ- and confirm that the promoter controlling exopolysaccharide synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB -the master regulator of type III secretion system genes- is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection suggesting that their function is required throughout disease.

Our results challenge the widespread view in R. solanacearum pathogenicity that the type III secretion system –and thus injection of effector proteins- is only active to manipulate plant defences at the first stages of infection, and that their expression is turned down when bacteria reach high cell densities and exopolysaccharide synthesis starts.

 

Cited by (2):

- Genin, S. & Denny, T. P. Pathogenomics of the Ralstonia solanacearum Species Complex. Annual Review of Phytopathology 50, 67–89 (2012).

http://www.annualreviews.org/doi/abs/10.1146/annurev-phyto-081211-173000

 

- Vinatzer, B. A. ‘Listening In’ on How a Bacterium Takes Over the Plant Vascular System. mBio 3, (2012).

http://mbio.asm.org/content/3/5/e00269-12

 

 

 

Last Update: 2012/09/14

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Physiol. relevance and contribution to metal balance of specific and non-specific MT isoforms in the garden snail, C. aspersus, by Höckner et al., in BioMetals

Physiol. relevance and contribution to metal balance of specific and non-specific MT isoforms in the garden snail, C. aspersus, by Höckner et al., in BioMetals | F. Monteiro Publications | Scoop.it

Höckner, M., Stefanon, K., de Vaufleury, A., Monteiro, F., Pérez-Rafael, S., Palacios, O., Capdevila, M., Atrian, S., Dallinger, R. Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus. Biometals (2011).doi:10.1007/s10534-011-9466-x

IF: 2.320 (2010); 2.823 (2011)

 

Summary:

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80–90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30–50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

 

Cited by (3):

- Höckner, M., Dallinger, R. & Stürzenbaum, S. R. Nematode and snail metallothioneins. J. Biol. Inorg. Chem. 16, 1057–1065 (2011).

http://www.springerlink.com/content/m2122754750q26h1/

- Palacios, O., Atrian, S. & Capdevila, M. Zn- and Cu-thioneins: a functional classification for metallothioneins? J. Biol. Inorg. Chem. 16, 991–1009 (2011).

http://www.springerlink.com/content/f088t5kv57000r86/

- Pérez-Rafael, S. et al. The metal binding abilities of Megathura crenulata metallothionein (McMT) in the frame of gastropoda MTs. J. Inorg. Biochem. 108, 84–90 (2012).

http://dx.doi.org/10.1016/j.jinorgbio.2011.11.025

 

 

Last Update: 2012/06/30

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Environmental cues controlling the pathogenicity of Ralstonia solanacearum on plants / Señales ambientales que determinan la patogenicidad de Ralstonia solanacearum en plantas 

Environmental cues controlling the pathogenicity of Ralstonia solanacearum on plants / Señales ambientales que determinan la patogenicidad de Ralstonia solanacearum en plantas  | F. Monteiro Publications | Scoop.it

PhD Thesis Author: Freddy Monteiro

Defence date: June 28th 2013

Defence place: Aula Magna. Facultat de Biologia. Universitat de Barcelona. Catalonia. Spain

Field: Genetics

Jury: Dr. Nemo Peeters (LIPM, INRA-CNRS), Dr. Carlos Balsalobre (UB), Dr. Juan José López-Moya (CRAG, CSIC-IRTA-UAB-UB)

Advisor: Dr. Marc Valls

 

Abstract:

Ralstonia solanacearum is a soil-borne beta-proteobacterium that causes wilting disease on a wide range of plants with economic importance like tomato, potato, pepper, eggplant and banana. Each year, bacterial wilt pose important threats to agriculture by producing significant economic losses to small-scale producers in developing countries and, lately, the geographical distribution of the pathogen is spreading to temperate regions of the globe. The long-term aim of the work developed was the determination of the genetic program used by R. solanacearum during plant colonization and at the different stages of disease, in order to provide a biologically relevant understanding of the repression/activation regulatory switches controlling R. solanacearum pathogenicity. We noticed that new molecular tools for functional genetic studies adapted to R. solanacearum were needed, because the widely used mutants obtained by transposon mutagenesis contain gene disruptions rendering, in some cases, bacteria with affected virulence, pathogenicity and unable to multiply inside susceptible plants. In addition, a common issue in R. solanacearum studies was the difficulty to trans-complement gene disruptions. So far, the only alternative available was the use of plasmids, which provided a means of overexpression rather than stoichiometrical complementation, Moreover, the use of antibiotics to maintain plasmids during plant infection is not an option due to the complexity of the system. In this thesis we developed a novel system – pRC, after Ralstonia chromosome –, based in targeted and stable insertions in a precise and permissive location of the bacterial chromosome. We proposed the use of our versatile set of suicide plasmids for the study of transcriptional output (promoter probing) during plant infection, effector overexpression and purification, and monocopy gene complementation in any R. solanacearum strain. The use of the pRC system in any strain will allow the standardization of the genetic studies made in the field. We also investigated gene activities in planta. To that end, we successfully applied a luminescent reporter in the bacterial chromosome to visualize and quantify in real time the activity of pathogenicity-related promoters. We fused the promoter regions controlling two major virulence determinants to the luxCDABE reporter and followed light emission at different stages of plant infection. This strategy allowed us to establish both the timing and the exact location in the plant where these bacterial genes were expressed. Our main finding was that the T3SS is active throughout plant infection and not only at the first colonization stages. It is likely that during plant infection many overlapping signals are perceived by the bacteria, adding complexity to the gene regulatory model proposed in the literature. Together with the two articles published in peer-review journals, two additional drafts, describing the current progress of two other projects are also provided. The first draft reports a novel regulatory feedback loop governing hrpB expression when R. solanacearum is grown in the presence of plant cells. This work is part of a collaboration with Stéphane Genin (Laboratoire des Interactions Plantes Micro-organismes (LIPM, INRA-CNRS, Castanet Tolosan, France).The second draft reports the use of the pRC system to decipher “cool-adaptation” on strain UW551. This work is part of a collaboration with Caitilyn Allen research group (University of Wisconsin – Madison, Wisconsin, USA).

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A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in R. solanacearum, by Monteiro et al., in MPMI

A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in R. solanacearum, by Monteiro et al., in MPMI | F. Monteiro Publications | Scoop.it

Monteiro, F., Solé, M., van Dijk, I. & Valls, M. A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in Ralstonia solanacearum. Molecular Plant-Microbe Interactions 25, 557–568 (2012).

IF: 4.010 (2010); 4.431 (2011)

 

Summary:

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors—the Ralstonia chromosome (pRC) series—that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).

 

 

Cited by (1):

- Solé, M., Popa, C., Mith, O., Sohn, KH., Jones, JD., Deslandes, L., and Valls, M. The awr Gene Family Encodes a Novel Class of Ralstonia solanacearum Type III Effectors Displaying Virulence and Avirulence Activities. Molecular Plant-Microbe Interactions 25 941-953 (2012).

http://dx.doi.org/10.1094/MPMI-12-11-0321

 

 

Last Update: 2012/06/30

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Evidence of Native Metal–S2−–Metallothionein Complexes Confirmed by the Analysis of Cup1 Divalent-Metal-Ion Binding Properties, by Orihuela et al. in Chemistry - A European Journal

Evidence of Native Metal–S2−–Metallothionein Complexes Confirmed by the Analysis of Cup1 Divalent-Metal-Ion Binding Properties, by Orihuela et al. in Chemistry - A European Journal | F. Monteiro Publications | Scoop.it

Orihuela, R., Monteiro, F., Pagani, A., Capdevila, M. & Atrian, S. Evidence of Native Metal–S2−–Metallothionein Complexes Confirmed by the Analysis of Cup1 Divalent-Metal-Ion Binding Properties. Chem. Eur. J. 16, 12363–12372 (2010).

IF: 5.476 (2010); 5.925 (2011)

 

Summary:

It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S2−-containing complexes in addition to the S2−-devoid canonical metal–MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S2− ligands are also constituents of native metal–MT complexes. Because, although practically universal, the highest S2− content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd–Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd–S2−–Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn–Cup1, Cd–Cup1, and Cu–Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research.

 

Cited by (6):

- Orihuela, R. et al. Ferritin and metallothionein: dangerous liaisons. Chem. Commun. (Camb.) 47, 12155–12157 (2011).

http://pubs.rsc.org/en/content/articlelanding/2011/cc/c1cc14819b

- Palacios, O., Atrian, S. & Capdevila, M. Zn- and Cu-thioneins: a functional classification for metallothioneins? J. Biol. Inorg. Chem. 16, 991–1009 (2011).

http://www.springerlink.com/content/f088t5kv57000r86/

- Höckner, M. et al. Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus. Biometals 24, 1079–1092 (2011).

http://www.springerlink.com/content/fv7t62jm758862x7/

- Capdevila, M., Bofill, R., Palacios, Ò. & Atrian, S. State-of-the-art of metallothioneins at the beginning of the 21st century. Coordination Chemistry Reviews 256, 46–62 (2012).

http://dx.doi.org/10.1016/j.ccr.2011.07.006

- Gusmão, R., Prohens, R., Díaz-Cruz, J. M., Ariño, C. & Esteban, M. Combination of chemometrically assisted voltammetry, calorimetry, and circular dichroism as a new method for the study of bioinorganic substances: application to selenocystine metal complexes. J. Biol. Inorg. Chem. 17, 321–329 (2012).

http://www.springerlink.com/content/d604u201648m3833/

- Pérez-Rafael, S. et al. The metal binding abilities of Megathura crenulata metallothionein (McMT) in the frame of gastropoda MTs. J. Inorg. Biochem. 108, 84–90 (2012).

http://dx.doi.org/10.1016/j.jinorgbio.2011.11.025

 

 

Last Update: 2012/06/30

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