Biomimetic assembly and activation of [FeFe]-hydrogenases.

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production.

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]­hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H2 production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants.

[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe-4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2-, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2-. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2-, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe-4S] subcluster. The reduced form of HydA1 containing only the [4Fe-4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]-PDT/ADT complex.

Spin dynamics of light-induced charge separation in composites of semiconducting polymers and PC60BM revealed using Q-band pulse EPR.

Light-induced processes in composites of semiconducting polymers and fullerene derivatives have been widely studied due to their usage as active layers of organic solar cells. However the process of charge separation under light illumination - the key process of an organic solar cell is not well understood yet. Here we report a Q-band pulse electron paramagnetic resonance study of composites of the fullerene derivative PC60BM ([6,6]-phenyl-C61-butyric acid methyl ester) with different p-type semiconducting polymers regioregular and regiorandom P3HT (poly(3-hexylthiophene-2,5-diyl), MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]), PCDTBT (poly[N-9'-heptadecanyl-2,7-carbazole-alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)]), PTB7 (poly({4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl}))), resulting in a detailed description of the in-phase laser flash-induced electron spin echo (ESE) signal. We found that in organic donor-acceptor composites the laser flash simultaneously induces species of two types: a polymer˙+/fullerene˙- spin-correlated polaron pair (SCPP) with an initial singlet spin state and (nearly) free polymer˙+ and fullerene˙- species with non-equilibrium spin polarization. Species of the first type (SCPP) are well-known for polymer/fullerene blends and are usually associated with a charge-separated state. Also, spin polarization of long-living free species (polarons in deep traps) is affected by the laser flash, which is the third contribution to the flash-induced ESE signal. A protocol for extracting the in-phase ESE signal of the SCPP based on the dependence of the microwave nutation frequency on the strength of the spin coupling within the polaron pair was developed. Nutation experiments revealed an unusual pattern of the SCPP in RR-P3HT/PC60BM composites, from which the strength of the exchange interaction between the polymer˙+ and fullerene˙- was extracted. In composites with low-efficient polymers the contribution of the SCPP to the in-phase ESE signal is high, while in composites with high-efficient polymers it is low. This finding can be used as a selection criterion of charge separation efficiency in the polymer/fullerene composites.

Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR.

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Bioconversion of crude glycerol by fungi.

The production of synthetic glycerol from petrochemical feedstocks has been decreasing in recent years. This is largely due to increasing supplies of crude glycerol derived as a co-product from the oleochemical industry, especially biodiesel production. The price of glycerol is at historic lows, and the supply of crude glycerol is projected to grow faster than its industrial uses. This oversupply is driving the transition from glycerol as a product to glycerol as a precursor for new industrial applications, including its use as a substrate for bioconversion. This article reviews the use of fungi for the bioconversion of crude glycerol to the value-added products 1,2-propanediol, ethanol, single cell oil, specialty polyunsaturated fatty acids, biosurfactants, and organic acids. Information on the impurities of crude glycerol from different industrial processes is also included.

Tumor lysate-loaded Bacterial Ghosts as a tool for optimized production of therapeutic dendritic cell-based cancer vaccines.

Cancer immunotherapy with dendritic cell (DC)-based vaccines has been used to treat various malignancies for more than two decades, however generally showed a limited clinical success. Among various factors responsible for their modest clinical activity is the lack of universally applied, standardized protocols for the generation of clinical-grade DC vaccines, capable of inducing effective anti-tumor immune responses. We investigated Bacterial Ghosts (BGs) - empty envelopes of Gram-negative bacteria - as a tool for optimized production of DC vaccines. BGs possess various intact cell surface structures, exhibiting strong adjuvant properties required for the induction of DC maturation, whereas their empty internal space can be easily filled with a source tumor antigens, e.g. tumor lysate. Hence BGs emerge as an excellent platform for both the induction of immunogenic DC maturation and loading with tumor antigens in a single-step procedure. We compared the phenotype, cytokine secretion profile, functional activity and ability to induce immunogenic T-cell responses in vitro of human monocyte-derived DCs generated using BG platform and DCs matured with widely used lipopolysaccharide (LPS) plus interferon-γ cocktail and loaded with tumor lysate. Both approaches induced DC maturation, however BG-based protocol was superior to LPS-based protocol in terms of the ability to induce DCs with a lower tolerogenic potential, resulting in a more robust CD8+ T cell activation and their functional activity as well as significantly lower induction of regulatory T cells. These superior parameters are attributed, at least in part, to the ability of BG-matured DCs to resist potential immunosuppressive and pro-tolerogenic activity of various tumor cell lysates, including melanoma, renal carcinoma and glioblastoma.

Orientation-resolving pulsed electron dipolar high-field EPR spectroscopy on disordered solids: I. Structure of spin-correlated radical pairs in bacterial photosynthetic reaction centers.

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Chalcogenide substitution in the [2Fe] cluster of [FeFe]-hydrogenases conserves high enzymatic activity.

[FeFe]-Hydrogenases efficiently catalyze the uptake and evolution of H2 due to the presence of an inorganic [6Fe-6S]-cofactor (H-cluster). This cofactor is comprised of a [4Fe-4S] cluster coupled to a unique [2Fe] cluster where the catalytic turnover of H2/H+ takes place. We herein report on the synthesis of a selenium substituted [2Fe] cluster [Fe2{µ(SeCH2)2NH}(CO)4(CN)2]2- (ADSe) and its successful in vitro integration into the native protein scaffold of [FeFe]-hydrogenases HydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum yielding fully active enzymes (HydA1-ADSe and CpI-ADSe). FT-IR spectroscopy and X-ray structure analysis confirmed the presence of structurally intact ADSe at the active site. Electrochemical assays reveal that the selenium containing enzymes are more biased towards hydrogen production than their native counterparts. In contrast to previous chalcogenide exchange studies, the S to Se exchange herein is not based on a simple reconstitution approach using ionic cluster constituents but on the in vitro maturation with a pre-synthesized selenium-containing [2Fe] mimic. The combination of biological and chemical methods allowed for the creation of a novel [FeFe]-hydrogenase with a [2Fe2Se]-active site which confers individual catalytic features.

Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli.

As a tool for determining the topology of the small, 91-amino acid phi X174 lysis protein E within the envelope complex of Escherichia coli, a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

P700: the primary electron donor of photosystem I.

The primary electron donor of photosystem I, P700, is a chlorophyll species that in its excited state has a potential of approximately -1.2 V. The precise chemical composition and electronic structure of P700 is still unknown. Recent evidence indicates that P700 is a dimer of one chlorophyll (Chl) a and one Chl a'. The Chl a' and Chl a are axially coordinated by His residues provided by protein subunits PsaA and PsaB, respectively. The Chl a', but not the Chl a, is also H-bonded to the protein. The H-bonding is likely responsible for selective insertion of Chl a' into the reaction center. EPR studies of P700(+*) in frozen solution and single crystals indicate a large asymmetry in the electron spin and charge distribution towards one Chl of the dimer. Molecular orbital calculations indicate that H-bonding will specifically stabilize the Chl a'-side of the dimer, suggesting that the unpaired electron would predominantly reside on the Chl a. This is supported by results of specific mutagenesis of the PsaA and PsaB axial His residues, which show that only mutations of the PsaB subunit significantly alter the hyperfine coupling constants associated with a single Chl molecule. The PsaB mutants also alter the microwave induced triplet-minus-singlet spectrum indicating that the triplet state is localized on the same Chl. Excitonic coupling between the two Chl a of P700 is weak due to the distance and overlap of the porphyrin planes. Evidence of excitonic coupling is found in PsaB mutants which show a new bleaching band at 665 nm that likely represents an increased intensity of the upper exciton band of P700. Additional properties of P700 that may give rise to its unusually low potential are discussed.

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)A state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state phi(*-)AQ(*-)A is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BCh1 Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.

ENDOR studies of the intermediate electron acceptor radical anion I-. in Photosystem II reaction centers.

The EPR and ENDOR characteristics of the intermediate electron acceptor radical anion I-. in Photosystem II (PS II) are shown to be identical in membrane particles and in the D1D2 cytochrome b-559 complex (Nanba, O. and Satoh, K. (1987) Proc. Natl. Acad. Sci. USA 84, 109-112). These findings provide further evidence that the D1D2 complex is the reaction center of PS II and show that the pheophytin binding site is intact. A hydrogen bond between I-. and the protein (GLU D1-130) is postulated on the basis of D2O exchange experiments. The ENDOR data of I-. and of the pheophytin a radical anion in different organic solvents are compared and the observed differences are related to structural changes of the molecule on the basis of molecular orbital calculations (RHF-INDO/SP). The importance of the orientation of the vinyl group (attached to ring I) on electron transfer is discussed.

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides.

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Blastochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (Phi(A)(&z.rad;-)) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BChl), B(A), and the BPh, Phi(A), that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of Phi(A)(&z.rad;-) at 160 K indicates that two distinct states of Phi(A)(&z.rad;-) are present in the wild type and the mutant YF(M208). Based on a comparison with Phi(A)(&z.rad;-) in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of Phi(A). Only one Phi(A)(&z.rad;-) state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state Phi(A)(&z.rad;-)Q(A)(&z.rad;-) is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BChl Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of Phi(A) changes its orientation. It is concluded that this distinct structural relaxation of Phi(A) can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.

Cell-cell contacts trigger programmed necrosis and induce cyclooxygenase-2 expression.

Necrosis was induced by cell-cell contacts of human dermal fibroblasts in three-dimensional culture. Dramatic induction of cyclooxygenase-2 (COX-2) expression was found throughout these necrotizing cell clusters, whereas no increase in expression of apoptosis markers was seen. The cells were rapidly committed to necrosis, and the process could not be reversed by allowing them to spread and adhere on a solid substrate. Induction of COX-2 expression was accompanied by greatly enhanced production of the prostaglandins E(2), I(2), and F(2alpha). When applied exogenously on necrotizing clusters, these prostaglandins delayed cell clustering and further enhanced COX-2 expression. Abolishing prostaglandin production by NS-398 or indomethacin reduced cell membrane damage (as measured by lactate dehydrogenase release into the culture medium). We also identified alpha-enolase-mediated plasminogen activation as the major extracellular proteolytic executor of necrotic cell death. In contrast to inhibition of COX-2, inhibition of plasminogen activation failed to inhibit membrane damage associated with necrosis. Intracellular proteolysis, by caspases, was shown to take part in COX-2 induction. Taken together, our results indicate that cell-cell contacts induce an actively programmed necrotic process that functionally involves COX-2, a known hallmark of inflammation and cancer.

Culture free DGGE and cloning based monitoring of changes in bacterial communities of salad due to processing.

To assess the possibilities of a culture-independent monitoring of bacterial communities in the food chain, samples of salad from farming sites as well as corresponding, processed products in stores were analysed. The bacterial DNA was extracted using a modified soil extraction protocol. Amplification of 16S rDNA was carried out using primers specific for eubacteria and enterobacteriaceae. Fingerprints of 200/370 bp respectively were obtained by denaturing gradient gel electrophoresis (DGGE) analysis following PCR and nested PCR amplification. In parallel to DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. DGGE analysis indicated a high diversity of bacterial communities in salad samples. Fingerprints indicated clearly reduced diversity of bacterial communities in processed samples from markets compared to field-grown salads. Surprisingly, primers pointed out in literature as specific for enterobacteriaceae did amplify pseudomonadeceae as well. Therefore, the more specific primers fD2 and rP1 were used subsequently in this study to amplify specific members of the family enterobacteriaceae. A total of 11 different 16S rDNA sequences were obtained and subjected to sequencing and phylogenetic affiliation. Sequences derived from the eubacterial clone library from organically farmed salad were affiliated to the family microbacteriaceae and pseudomonadaceae. In addition, a potential new genus within the family of enterobacteriaceae was detected. Furthermore, a sequence showing 98.9% similarity to Pseudomonas libaniensis (fluorescence subgroup) was found in a processed salad sample but not in the corresponding field samples. This species is generally known as an opportunistic pathogen. Whereas molecular based monitoring of bacterial communities in food still may need more experience and standardisation to detect specific bacteria present, the monitoring strategy presented in this paper, combining DGGE analysis with the construction of clone libraries, is an attractive method for culture-independent monitoring of changes of bacterial communities in the food chain.

Sequence analysis of the sbsA gene encoding the 130-kDa surface-layer protein of Bacillus stearothermophilus strain PV72.

Bacillus stearothermophilus (Bs) contains a surface-layer (S-layer) protein (SbsA), which forms a hexagonal array on the cell wall. In order to understand the structural/functional relationship of SbsA from Bs PV72, the entire nucleotide (nt) sequence of the sbsA gene was determined from three overlapping fragments. The 3'-end was cloned and expressed in Escherichia coli, whereas the 5'-region was amplified from the genome of Bs PV72 by the polymerase chain reaction using two overlapping fragments. The open reading frame (3684 nt) of sbsA is predicted to encode a protein of 1228 amino acids (aa). The SbsA is synthesized with a leader sequence of 30 aa. The predicted SbsA aa profile was similar to most other sequenced S-layer proteins, containing more acidic than basic aa (pI 5.1) and a very low amount of sulfur-containing aa. Based on aa sequence data, SbsA has weak homology of with the S-layer proteins from B. sphaericus, Rickettsia rickettsii, B. brevis HPD31 and B. brevis 47 (OWP).

Cold-sensitive E-lysis systems.

The release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms. The aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures. To obtain cold-sensitive lysis vectors, the lambdacI857 repressor/pR promoter expression system was combined with either the lacI/lacZpo or the phage 434 cI/pR system that control the expression of the lysis gene E of bacteriophage phiX174. Escherichia coli strains harbouring such suicide vectors are able to grow at 37 degrees C, but cell lysis takes place at temperatures below 30 degrees C. By replacing gene E with a beta-galactosidase reporter gene we also showed that the onset of beta-galactosidase activity corresponds with the onset of lysis at 28 degrees C. Results indicate that these newly combined promoter/repressor systems can also be used to confer cold-sensitive expression to any gene of interest.