Domenica Farci

New features of the cell wall of the radio-resistant bacterium Deinococcus radiodurans

We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.

Purification and characterization of DR_2577 (SlpA) a major S-layer protein from Deinococcus radiodurans

The protein DR_2577 is a major Surface layer component of the radio-resistant bacterium Deinococcus radiodurans. In the present study DR_2577 has been purified and its oligomeric profile characterized by means of size exclusion chromatography and gel electrophoresis. DR_2577 was found to be organized into three hierarchical orders characterized by monomers, stable dimers formed by the occurrence of disulfide bonds, and hexamers resulting from a combination of dimers. The structural implications of these findings are discussed providing new elements for a more integrated model of this S-layer.

The S-layer Protein DR_2577 Binds Deinoxanthin and under Desiccation Conditions Protects against UV-Radiation in Deinococcus radiodurans

Deinococcus radiodurans has the puzzling ability to withstand over a broad range of extreme conditions including high doses of ultraviolet radiation and deep desiccation. This bacterium is surrounded by a surface layer (S-layer) built of a regular repetition of several proteins, assembled to form a paracrystalline structure. Here we report that the deletion of a main constituent of this S-layer, the gene DR_2577, causes a decrease in the UVC resistance, especially in desiccated cells. Moreover, we show that the DR_2577 protein binds the carotenoid deinoxanthin, a strong protective antioxidant specific of this bacterium. A further spectroscopical characterization of the deinoxanthin-DR_2577 complex revealed features which could suggest a protective role of DR_2577. We propose that, especially under desiccation, the S-layer shields the bacterium from incident ultraviolet light and could behave as a first lane of defense against UV radiation.

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

RhVI1 is found to be a vacuolar membrane-anchored invertase that is highly expressed in buds. These findings suggest that RhVI1 has a role in blooming and corolla maturation by the regulation of sink-trafficking.

Isolation of Plant Photosystem II Complexes by Fractional Solubilization

Photosystem II (PSII) occurs in different forms and supercomplexes in thylakoid membranes. Using a transplastomic strain of Nicotiana tabacum histidine tagged on the subunit PsbE, we have previously shown that a mild extraction protocol with β-dodecylmaltoside enriches PSII characteristic of lamellae and grana margins. Here, we characterize residual granal PSII that is not extracted by this first solubilization step. Using affinity purification, we demonstrate that this PSII fraction consists of PSII-LHCII mega- and supercomplexes, PSII dimers, and PSII monomers, which were separated by gel filtration and functionally characterized. Our findings represent an alternative demonstration of different PSII populations in thylakoid membranes, and they make it possible to prepare PSII-LHCII supercomplexes in high yield.

Dual HIV-1 reverse transcriptase and integrase inhibitors from Limonium morisianum Arrigoni, an endemic species of Sardinia (Italy)

Abstract During our search for potential templates of HIV-1 reverse transcriptase (RT) and integrase (IN) dual inhibitors, the methanolic extract obtained from aerial parts of Limonium morisianum was investigated. Repeated bioassay-guided chromatographic purifications led to the isolation of the following secondary metabolites: myricetin, myricetin 3-O-rutinoside, myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside, (-)-epigallocatechin 3-O-gallate, tryptamine, ferulic and phloretic acids. The isolated compounds were tested on both HIV-1 RT-associated RNase H and IN activities. Interestingly, (-)-epigallocatechin-3-O-gallate and myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside potently inhibited both enzyme activities with IC50 values ranging from 0.21 to 10.9 μM. Differently, tryptamine and ferulic acid exhibited a significant inhibition only on the IN strand transfer reaction, showing a selectivity for this viral enzyme. Taken together these results strongly support the potential of this plant as a valuable anti HIV-1 drugs source worthy of further investigations.

S-layer proteins as a source of carotenoids: Isolation of the carotenoid cofactor deinoxanthin from its S-layer protein DR_2577

S-layers are regular paracrystalline arrays of proteins or glycoproteins that characterize the outer envelope of several bacteria and archaea. The auto-assembling properties of these proteins make them suitable for application in nanotechnologies. However, the bacterial cell wall and its S-layer are also an important binding sites for carotenoids and they may represent a potential source of these precious molecules for industrial purposes. The S-layer structure and its components were extensively studied in the radio-resistant bacterium Deinococcus radiodurans, which for long time represented one of the model organisms in this respect. The protein DR_2577 has been shown to be one of the naturally over-expressed S-layer components in this bacterium. The present report describes a high scale purification procedure of this protein in solution. The purity of the samples, assayed by native and denaturing electrophoresis, showed how this method leads to a selective and high efficient recovery of the pure DR_2577. Recently, we have found that the deinoxanthin, a carotenoid typical of D. radiodurans, is a cofactor non covalently bound to the protein DR_2577. The pure DR_2577 samples may be precipitated or lyophilized and used as a source of the carotenoid cofactor deinoxanthin by an efficient extraction using organic solvents. The procedure described in this work may represent a general approach for the isolation of S-layer proteins and their carotenoids with potentials for industrial applications.

On the S-layer of Thermus thermophilus and the assembling of its main protein SlpA

We have isolated and analysed the cell envelope of the thermophilic bacterium Thermus thermophilus HB8. Isolated cell walls, characterized by the dominance of the S-layer protein SlpA, are found to be constituted by several protein complexes of high molecular weights. Further isolation steps, starting from the cell wall samples, led to the selective release of the S-layer protein SlpA in solution as confirmed by mass spectrometry. Blue Native gel electrophoresis on these samples showed that SlpA is organized into a specific hierarchical order of oligomeric states that are consistent with the complexes at high molecular weight identified on the total cell wall fraction. The analysis showed that SlpA bases this peculiar organization on monomers and exceptionally stable dimers, leading to the formation of tetramers, heptamers, and decamers. Furthermore, the two elementary units of SlpA, monomers and dimers, are regulated by the presence of calcium not only for the assembling of monomers into dimers, but also for the splitting of dimers into monomers. Finally, the SlpA protein was found to be subjected to specific proteolysis leading to characteristic degradation products. Findings are discussed in terms of S-layer assembling properties as bases for understanding its structure, turn-over and organization.

New insights into the operative network of FaEO, an enone oxidoreductase from Fragaria x ananassa Duch.

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.