Identification of an extracellular infection-induced glyceraldehyde-3-phosphate dehydrogenase of the phytopathogenic proteobacterium Pseudomonas syringae pv tomato DC3000

According to molecular biology, genomic and proteo- mic data, the phytopathogenic gamma-proteobacte- rium Pseudomonas syringae pv. tomato DC3000 pro-duces a number of proteins that may promote infec- tion and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) enzymes with different predic- ted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the ex-pression levels of each paralogous gap gene by real- time PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the in-fection process. Nevertheless, proteomic analysis of cul- ture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extra-cellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know en-zymatic function in the glycolytic metabolic pathway.Peer Reviewe

Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS

The genome of the thermophilic green-sulfur bacterium Chlorobium tepidumTLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppxgenes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K+-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg2+. Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world. © 2014 The Authors.España, Gobierno BFU2004-00843, BFU2007- 61887 and BFU2010-15622Junta de AndalucÍa BIO118

Purificación y caracterización de deshidrogenasa gliceraldehído-3- fosfato de Pseudomonas syringae pv. tomato DC3000

1 página.Pseudomonas syringae pv. tomato causa frecuentemente una enfermedad en el tomate llamada técnicamente mancha bacteriana. Pero su mecanismo de infección sigue siendo muy poco conocido.Peer reviewe

Inorganic Polyphosphate in the Microbial World. Emerging Roles for a Multifaceted Biopolymer

inorganic polyphosphates (polyP) are linear polymers of tens to hundreds orthophosphate residues linked by phosphoanhydride bonds. These fairly abundant biopolymers occur in all extant forms of life, from prokaryotes to mammals, and could have played a relevant role in prebiotic evolution. Since the first identification of polyP deposits as metachromatic or volutin granules in yeasts in the nineteenth century, an increasing number of varied physiological functions have been reported. Due to their "high energy" bonds analogous to those in ATP and their properties as polyanions, polyP serve as microbial phosphagens for a variety of biochemical reactions, as a buffer against alkalis, as a storage of Ca(2+) and as a metal-chelating agent. In addition, recent studies have revealed polyP importance in signaling and regulatory processes, cell viability and proliferation, pathogen virulence, as a structural component and chemical chaperone, and as modulator of microbial stress response. This review summarizes the current status of knowledge and future perspectives of polyP functions and their related enzymes in the microbial world.España, MINECO BFU2004-00843, BFU2007-61887, BFU2010-15622España, Junta de Andalucía BIO-26

Identification of an extracellular infection-induced glyceraldehyde-3-phosphate dehydrogenase of the phytopathogenic proteobacterium Pseudomonas syringae pv tomato DC3000

According to molecular biology, genomic and proteo- mic data, the phytopathogenic gamma-proteobacte- rium Pseudomonas syringae pv. tomato DC3000 pro-duces a number of proteins that may promote infec- tion and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) enzymes with different predic- ted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the ex-pression levels of each paralogous gap gene by real- time PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the in-fection process. Nevertheless, proteomic analysis of cul- ture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extra-cellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know en-zymatic function in the glycolytic metabolic pathway.España AECID (MAEC) A1/043076/1

Solubilization of inorganic phosphate and production of organic acids by bacteria isolated from a Moroccan mineral phosphate deposit

Three efficient inorganic-phosphate solubilizing bacteria (PSB) were isolated from a phosphate rock deposit of a Moroccan mine. The phosphate solubilization index of these isolates, determined in National Botanical Research Institute's phosphate (NBRIP) medium supplemented with tribasic calcium phosphate, ranging from 2.8 to 4.4. The medium pH dropped from 7.0 to 3.5 units after growth under continuous agitation for seven days. PSB6, the most efficient PSB, closely related to Enterobacter hormaechei subsp. steigerwaltii strain NM23-1, permitted the recovery of the maximum soluble orthophosphate concentration in the medium (505 mg/L) after a growth period of 60 to 72 h. PSB4 and PSB5 strains were identified as Enterobacter sp. Strain TSSAS2-48 and Bacterium DR172. The capacity to solubilize inorganic phosphate by these PSB can be attributed to the secretion of organic acids, to determine their presence in the cultures supernatant, reverse-phase high performance liquid chromatography was performed. The presence of 9 identified and three unidentified organic acids was consequently demonstrated. Gluconic acid was strongly produced by all strains, extending to 55.4 mM by PSB6, 46.4 mM by PSB5, and 44.9 mM by PSB4. Besides succinic, acetic, glutamic, oxaloacetic, pyruvic, malic and fumaric acids, a newly detected and identified organic acid was the alpha-ketoglutaric acid. To the best of our knowledge this is the first report mentioning alpha-ketoglutaric acid production by PSB strain

Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), toghether with other transporters, are responsible to a great extent for these symptoms. These multisubunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been determined to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target for control of tumour cells. In the present review we portray the major characteristics of this kind of proton pumps, we provide some recent insights on their in vivo regulation, an overview of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and a brief summary of the studies related to cancer made recently with commercially available inhibitors for this kind of proton pump. Some new approaches to affect V-ATPase function are also suggested in the light of recent knowledge on the regulation of this proton pump.Junta de Andalucía PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovación BFU2007-61887 BFU2010-1562

El Cerquillo (Cerro de Andévalo, Huelva) y la producción metalúrgica prerromana en el suroeste Ibérico

En este trabajo se dan a conocer los resultados analíticos de las escorias metalúrgicas del asentamiento prerromano de El Cerquillo (Cerro de Andévalo, Huelva), que demuestran la producción de plata, cobre y hierro por el laboreo de minerales complejos de los depósitos de piritas de Suroeste Ibérico, y la continuidad de las técnicas metalúrgicas de la Edad del Bronce.__________________________________In this work it give the results of the analytical study of the preromans slags from El Cerquillo (Cerro de Andévalo, Huelva, SW Spain), that demonstrates the silver, copper and iron production by the exploitation of complex ore at the pyrite deposites of the Iberian Southwest, and the metallurgiques techniques continuity of the Bronze Age

N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast

Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. In the present study, we show that the expression level and/or subcellular localization in S. cerevisiae of a heterologous type of multispanning membrane protein, the proton-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimaeric proteins were constructed by adding the putative N-terminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimaeric constructs not only substantially enhanced H+-PPase expression levels in transformed mutant cells, but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses demonstrated further the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana, a protein efficiently expressed in yeast in its natural form, alters the subcellular distribution of the chimaeras, suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP (green fluorescent protein)–H+-PPase fusion proteins.Ministerio de Ciencia e Innovación BMC2007-61887Junta de Andalucía PAIDI BIO-26

Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria

The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families. [Int Microbiol 2005; 8(4):251-258