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

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

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

Marine biomolecules: a promising approach in therapy and biotechnology

The marine environment is characterized by a wide diversity of microorganisms among which marine bacteria. To insure their survival in hostile conditions where they face high competition with pathogenic microorganisms, they produce various kinds of bioactive molecules within biofilms with unique structural and functional features. As example: marine peptides which provide a broad spectrum of antimicrobial, antitumoral, antiviral and anti-inflammatory activities, in addition to marine exopolysaccharides showing antifouling and antifungal activities, immunomodulatory properties, emulsion stabilization capacity with other various potentials. Some biofilms have shown a beneficial role for aquaculture, among which enhancement of growth performance and improvement of water quality, while others are threatening not only aquaculture and maritime fields, but also medicine and food industry. Thus, marine bioactive compounds are promising preventing agents for the establishment and growth of fouling microorganisms, which may be useful in different fields in order to decrease economic losses and avoid foodborne illnesses. DOI: http://dx.doi.org/10.5281/zenodo.438415

Intraorganellar acidification by V-ATPases: a target in cellproliferation and cancer therapy

V-ATPases are multicomponent proton pumps involved in the acidification of singlemembrane intracellular compartments such as endosomes and lysosomes. They couple thehydrolysis of ATP to the translocation of one to two protons across the membrane. Acidification ofthe lumen of single membrane organelles is a necessary factor for the correct traffic of membranesand cargo to and from the different internal compartments of a cell. Also, V-ATPases are involved inregulation of pH at the cytosol and, possibly, extracellular milieu. The inhibition of V-ATPases hasbeen shown to induce apoptosis and cell cycle arrest in tumour cells and, therefore, chemicals thatbehave as inhibitors of this kind of proton pumps have been proposed as putative treatment agentsagainst cancer. The present review will summarize the major types of V-ATPase inhibitors and theirmechanisms of action and put them in relation to the patents registered so far for the treatment of cancer.Junta de Andalucía PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovación BMC2007-6188

The ciliate protist tetrahymena pyriformis as a cellular adhesion model for the pathogenic bacterium staphylococcus aureus

Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of cna and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains¿ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion¿s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis

Cell stress by phosphate of two protozoa tetrahymena thermophila and tetrahymena pyriformis

Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (glucose, insulin···), in microorganisms whose phosphorus is involved in cell growth. Phosphorus has various forms including pyrophosphate, a by-product of multiple pathways of biosynthesis. Enzymes that hydrolyze pyrophosphate are called inorganic pyrophosphatases (PPases). Two major types of inorganic pyrophosphatases are distinguished: the soluble pyrophosphatases (sPPases) and the membrane pyrophosphatases (mPPases or H+/Na+-PPases). They play a key role in the control of intracellular inorganic pyrophosphate level and produce an important ions gradient (H+ or Na+) to the cells. In this work, we primarily focused on the physiological study in a phosphate-poor medium of two models Tetrahymena thermophile and Tetrahymena pyriformis, following the mobility, the growth and the morphology of cells. Secondly, we evaluated the enzymatic activity of soluble and membrane pyrophosphatases in both species grown in the same complex medium. A decrease of cell growth is correlated with unusual morphologies and different mobility in the stress medium. The measurement of soluble and membrane inorganic pyrophosphatases activities also shows a decrease which illustrates the lack of phosphate found in the stress medium. Deficiency of phosphate is a limiting factor for protozoan growth. These results indicate that Tetrahymena can be used as a model of cellular stress and consists of a target to study inorganic pyrophosphatases for a better understanding of phosphate cycle in higher organisms