Influence of Varying Water Quality Parameters on the Acute Toxicity of Silver to the Freshwater Cladoceran, Ceriodaphnia dubia.

The acute toxicity of silver to Ceriodaphnia dubia was investigated in laboratory reconstituted waters as well as in natural waters and reconstituted waters with natural organic matter. The water quality characteristics of the laboratory reconstituted waters were systematically varied. The parameters that demonstrated an ability to mitigate the acute toxic effects of silver were chloride, sodium, organic carbon, and chromium reducible sulfide. Factors that did not have a consistent effect on the acute toxicity of silver to C. dubia, at least over the range of conditions tested, included hardness, alkalinity, and pH. The biotic ligand model was calibrated to the observed test results and found to be of use in quantifying the effect of changing water quality characteristics on silver bioavailability and toxicity. The model generally predicted silver toxicity within a factor of two and should be useful in modifying water quality criteria.

The effect of food on the acute toxicity of silver nitrate to four freshwater test species and acute-to-chronic ratios.

Acute silver toxicity studies were conducted with and without food for four common freshwater test species: Daphnia magna, Ceriodaphnia dubia, Pimephales promelas (fathead minnow-FHM), and Oncorhynchus mykiss (rainbow trout-RBT) in order to generate acute-to-chronic ratios (ACR). The studies were conducted similarly (i.e., static-renewal or flow-through) to chronic/early-life stage studies that were previously performed in this laboratory. The acute toxicity (EC/LC50 values) of silver without food ranged from 0.57 µg dissolved Ag/l for C.dubia to 9.15 µg dissolved Ag/l for RBT. The presence of food resulted in an increase in EC/LC50 values from 1.25× for RBT to 22.4× for C. dubia. Invertebrate food type was also shown to effect acute silver toxicity. Food did not affect EC/LC50s or ACRs as greatly in fish studies as in invertebrate studies. ACRs for both invertebrate species were <1.0 when using acute studies without food but were 1.22 and 1.33 when using acute studies with food. ACRs for FHMs ranged from 4.06 to 7.19, while RBT ACRs ranged from 28.6 to 35.8 depending on whether food was present in acute studies. The data generated from this research program should be useful in re-determining a final ACR for silver in freshwater as well as in risk assessments.

Validation study of the acute biotic ligand model for silver.

An important final step in development of an acute biotic ligand model for silver is to validate predictive capabilities of the biotic ligand model developed for fish and invertebrates. To accomplish this, eight natural waters, collected from across North America, were characterized with respect to ionic composition, pH, dissolved organic carbon, and sulfide. Tests were conducted with the cladoceran Ceriodaphnia dubia (48-h static) and the fish Pimephales promelas (96-h static renewal) to determine the concentrations causing lethality to 50% of the organisms (LC50s) for silver in each of these waters. Overall, the biotic ligand model adequately predicted silver toxicity to C. dubia; however, in some cases, predicted LC50 values exceeded measured values. The accuracy of the biotic ligand model predictions was less convincing for silver toxicity to P. promelas with pronounced problems in low-ionic strength waters. Another issue was the use of acclimated organisms in toxicity studies because the biotic ligand model has been developed with the use of a mix of studies with acclimated and nonacclimated test organisms of varying ages and sizes. To evaluate whether effects of acclimation to test waters influence biotic ligand model predictions, a subset of the natural waters were also tested with P. promelas that had been acclimated to the natural water for 7 d before testing. These experiments revealed no differences in toxicity between acclimated and nonacclimated P. promelas. To determine the influence of organism size, which has been previously correlated to Na(+) turnover and acute silver toxicity across multiple species, Na(+) and Cl(-) influx rates were measured in P. promelas of different sizes. Our results show that Na(+) and Cl(-) influx rates were inversely related to fish mass and positively correlated with silver sensitivity.

S-layer variation in Bacillus stearothermophilus PV72 is based on DNA rearrangements between the chromosome and the naturally occurring megaplasmids.

Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions. No stretches of significant sequence identity between sbsA and sbsB were detected. In order to investigate S-layer gene regulation in B. stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6. For sbsB expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2. The sbsA upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.

Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein.

The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.

The transposable element IS4712 prevents S-layer gene (sbsA) expression in Bacillus stearothermophilus and also affects the synthesis of altered surface layer proteins.

Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA-) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.

Two-unit cantilevered resin-bonded fixed partial dentures--a retrospective, preliminary clinical investigation.

PURPOSE: The aim of this study was to retrospectively evaluate the clinical retention and abutment movement of 2-unit cantilevered resin-bonded fixed partial dentures (FPD) that were inserted at Prince Philip Dental Hospital in Hong Kong. MATERIALS AND METHODS: Of 45 patients who were identified from a hospital computer search after receiving a 2-unit cantilevered resin-bonded FPD, 31 were clinically examined (33 FPDs). For each patient the following data were recorded: gender, age, cementation date, endodontic treatment if performed, bone support, tooth mobility, and FPD tipping or drifting. Data about any debonds with subsequent treatment and patient satisfaction on a 10-point scale were also recorded. RESULTS: The mean service life for the 33 prostheses was 30 +/- 18 months, with a range of 72 days to 67 months. One prosthesis debonded, resulting in a clinical retention rate of 97%. No rotation, drifting, or tipping was observed for any of the prostheses during the short period of this study. CONCLUSION: Two-unit cantilevered resin-bonded FPDs are successful in the short term, but further research is required to determine if they offer a viable alternative to fixed-fixed resin-bonded FPD designs.

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis.

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.

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.

Molecular characterization of the Bacillus stearothermophilus PV72 S-layer gene sbsB induced by oxidative stress.

S-layer protein variation from a hexagonally ordered (SbsA; 130 kDa) to a obliquely ordered (SbsB; 98 kDa) protein in Bacillus stearothermophilus PV72 is mediated by an increased oxygen supply. To elucidate the molecular basis of S-layer protein variation in B. stearothermophilus PV72, the sbsB gene, coding for the 98-kDa protein, was cloned by means of inverse PCR technology and sequenced. The sbsB coding region cloned in pUC18 was expressed in Escherichia coli, without its own regulatory upstream sequences but with its putative transcriptional terminator. The reading frame of sbsB (2,760 nucleotides) is predicted to encode a protein of 920 amino acids, including the signal sequence. Amino acid sequence comparison of SbsA and SbsB did not reveal any significant homology. The expression of sbsB in E. coli resulted in an accumulation of SbsB self-assembly products in the cytoplasm.

Bacterial ghosts as multifunctional vaccine particles.

Expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.

Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition.

Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer.

Heterologous expression and self-assembly of the S-layer protein SbsA of Bacillus stearothermophilus in Escherichia coli.

The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S-layer) composed of single species of protein, SbsA, with a molecular weight of 130,000. Recently, the sequence of the corresponding gene (sbsA) has been determined. The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low-copy-number vector under the transcriptional control of the lambda pL promoter. Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the lambda cl857 from the chromosome. As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA- specific antibodies, expression of sbsA in E. coli led to accumulation of sheet-like self-assembling products of the protein in the cytoplasm. No SbsA protein was detected either in the periplasm or in the supernatant fractions. Long-term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA.

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).