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1 Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Apdo. 187, 12.400 Segorbe, Castellón, Spain
2 Departamento de Química, Bioquímica y Biología Molecular, Universidad Cardenal Herrera-CEU, 46113 Moncada, Valencia, Spain
3 Instituto de Agrobiotecnología, CSIC-Universidad Pública de Navarra, 31006 Pamplona, Navarra, Spain
4 Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
Correspondence
José R. Penadés
jpenades{at}ivia.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Another step in colonization is the formation of a biofilm. Biofilm formation is a major concern in nosocomial infection because it protects microorganisms from opsonophagocytosis and antibiotics, leading to chronic infection and sepsis (Gotz, 2002). Two major surface components have been implicated in biofilm formation by S. aureus: (i) the product of the icaADBC operon which encodes proteins involved in the synthesis of the polysaccharide intercellular adhesin, the composition of which is poly-N-acetylglucosamine (PIA/PNAG; Cramton et al., 1999; Maira-Litran et al., 2002); and (ii) Bap, a surface protein of 2276 aa that contains 13 repeats of 86 residues (Cucarella et al., 2001). Bap promotes both primary attachment to inert surfaces and intercellular adhesion, whereas PIA/PNAG seems to be involved in intercellular adhesion alone.
Interestingly, the bap gene is contained in a mobile pathogenicity island (Ubeda et al., 2003), and so far, it has only been found in bovine mastitis isolates (Cucarella et al., 2001) and in isolates of several coagulase-negative staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus (Tormo et al., 2005a). However, to date, none of the human S. aureus isolates tested harbours the bap gene (Cucarella et al., 2001), suggesting that ruminant and human staphylococcal strains differ in their host-specific pathogenic strategies.
We have previously investigated the relationship between biofilm formation and the functionality of the MSCRAMM proteins in S. aureus. Our results have demonstrated that Bap interferes with the activity of the MSCRAMM proteins and decreases: (i) adherence to immobilized fibrinogen and fibronectin; (ii) adherence to mammary gland tissue ex vivo; (iii) internalization by epithelial cells; and (iv) initial colonization of the mammary glands of ewes (Cucarella et al., 2002). Similarly, we have observed in a mouse foreign-body-infection model that Bap-deficient mutants colonize catheters more rapidly than does the wild-type at the initial stages of infection, strongly suggesting that the presence of Bap hinders the interaction between bacterial cell receptors and the host proteins on the catheter (Cucarella et al., 2001). However, although initial colonization is affected by Bap, we have demonstrated in several animal models that Bap facilitates long-term persistence of S. aureus (Cucarella et al., 2001, 2004; Ubeda et al., 2003).
Phase variation is one of the many strategies employed by pathogenic bacteria to avoid detection by the host immune system. It involves the ability to switch on the expression of proteins when they are needed, and to switch them off when they are likely to trigger immune responses. In S. epidermidis, reversible inactivation of the ica, sarA and sigB genes by the insertion sequence IS256 can result in the production of biofilm-negative variants (Conlon et al., 2004; Ziebuhr et al., 1999). In S. aureus, a similar phase-variation process controls expression of the ica genes (Valle et al., 2007). However, nothing is known about the role that phase variation might have in the expression of the Bap protein.
In the present study we characterized the phase-variable expression of the Bap protein and demonstrated that this process modulates the functionality of different Staphylococcus adhesins.
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METHODS |
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. Staphylococcal strains were cultured in trypticase soy agar (TSA) and in trypticase soy broth (TSB), supplemented with glucose (0.25 %, w/v) when indicated.
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). Bap-expressing strains grow on this medium as rough colonies, whereas Bap-negative strains exhibit a smooth colony phenotype. Agar plates were incubated overnight at 37 °C, with an additional 24 h at room temperature.
Bacterial adherence to immobilized fibrinogen and fibronectin.
Binding of cells to fibrinogen or fibronectin immobilized on plates was measured as described by Hartford et al. (1997). Briefly, fibrinogen or fibronectin (Calbiochem) were diluted in sodium carbonate buffer (40 mM, pH 9.6) at the indicated concentration, and 100 µl was used to coat 96-well flat-bottomed ELISA plates (Sarstedt) overnight at 4 °C. Control wells contained carbonate buffer only. After washing with PBS, the plates were blocked for 2 h at 37 °C in 2 % BSA in PBS. Cells from an overnight culture (stationary phase) were washed and diluted in PBS (OD600 1.0). A volume of 100 µl of this cell suspension was added, and the plates incubated for 2 h at 37 °C. After gentle washing, adherent cells were fixed by adding 100 µl 25 % aqueous formaldehyde, and incubating at room temperature for 30 min. The plates were then washed gently, stained with crystal violet, washed again, and read on an ELISA reader at 570 nm.
SDS-PAGE and Western immunoblotting or Western ligand affinity blotting.
For ClfA detection, S. aureus cells from a stationary-phase culture were suspended to OD600 40 in 100 mM PBS containing 5 mM EDTA and 1 mM PMSF. Cells were centrifuged and suspended in 1 ml digestion buffer (50 mM Tris/HCl, pH 7.5, 20 mM MgCl2 and 30 % raffinose; Sigma) (Hartford et al., 1997). To each 1 ml sample, 60 µl protease inhibitors (Complete Cocktail; Boehringer Mannheim) and 60 µl 2 mg/ml solution of lysostaphin (Sigma) were then added, and the suspension was incubated in a 37 °C water bath for 30 min. Protoplasts were sedimented by centrifugation at 6000 g, and the supernatant fraction, which contained the wall-associated proteins, was analysed by SDS-PAGE (10 % separation gel, 4.5 % stacking gel).
For Western blot analysis, protein extracts were prepared and analysed by SDS-PAGE as described above, and blotted onto an Immobilon-P membrane (Millipore). Primary anti-ClfA antibody (McDevitt et al., 1994) was used at 1 : 1000 dilution with Tris-buffered saline (TBS; 50 mM Tris/HCl, pH 7.5, 150 mM NaCl) with 1 % skimmed milk. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) diluted 1 : 10 000 in TBS with 1 % skimmed milk was used, and the subsequent chemiluminescence reaction [with disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo' [3.3.1.13,7]decan}-4-yl) phenyl phosphate (CSPD, Roche)] was recorded.
The Bap immunoblotting assay was performed as described previously (Cucarella et al., 2001). Briefly, protein extracts were prepared and analysed by SDS-PAGE as described above, and blotted onto an Immobilon P membrane (Millipore). Anti-Bap serum was diluted 1 : 2500 with TBS (50 mM Tris/HCl, pH 7.5, 150 mM NaCl) and immuno-absorbed with 5 % skimmed milk. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) diluted 1 : 10 000 in TBS/5 % skimmed milk was used, and the subsequent chemiluminescence reaction (CSPD, Roche) was recorded.
For detection of fibronectin binding proteins (FnBPs), the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce) was used to biotinylate human fibronectin (Calbiochem). Bacteria grown to OD600 0.2 in 20 ml TSB in a 250 ml Erlenmeyer flask shaken at 200 r.p.m. at 37 °C were harvested by centrifugation at 12 000 g, resuspended, and treated as above. FnBps were detected by Western ligand affinity blotting by incubation for 1 h with biotinylated human fibronectin (50 µg ml–1) in PBS containing 0.1 % Tween 20 (PBST). They were then given three washes in PBST and incubated for 1 h with Streptavidin–POD conjugate (Roche; 1 : 3000 dilution). The membranes were washed as before, and developed using enhanced chemiluminescence (Roche).
DNA methods.
General DNA manipulations were performed by standard procedures (Ausubel et al., 1990; Sambrook et al., 1989). Oligonucleotides bap-6m and bap-7c (Cucarella et al., 2001) were used in the Southern and Northern blot experiments.
For Southern hybridization, chromosomal DNA was purified, digested with HindIII, and separated by agarose gel electrophoresis. Gels were blotted onto nylon membranes (Hybond-N, 0.45 µm pore-size filters; Amersham Life Science) using standard methods (Ausubel et al., 1990; Sambrook et al., 1989). Labelling of the probe and DNA hybridization were performed according to the protocol supplied with the PCR-DIG DNA-labelling and chemiluminescent detection kit (Roche).
RNA extraction was performed according to the method described by Cheung et al. (1994). Bacterial cells were grown at 37 °C in TSB to the mid-exponential phase. After harvesting, cell disruption and subsequent RNA extraction were performed by using the FastRNA kit, Blue (BIO 101) and the FP120 FastPrep Cell Disruptor apparatus (Savant Instruments), according to the instructions of the manufacturers.
Forty micrograms RNA of each bacterial strain was applied to a 1.5 % agarose/2.2 M formaldehyde gel in MOPS running buffer. RNA was blotted onto Nylon membranes, UV cross-linked, hybridized with a 32P-labelled bap probe in 50 % formamide at 42 °C overnight, washed, and autoradiographed according to standard protocols (Ausubel et al., 1990; Sambrook et al., 1989).
Quantitative assay of biofilm formation on polystyrene.
A late adherence assay was carried out essentially as described previously (Cucarella et al., 2001). Briefly, S. aureus strains were grown overnight in TSB at 37 °C. The culture was diluted 1 : 40 in TSB/0.25 % glucose, and 200 µl was used to inoculate sterile, 96-well polystyrene microtitre plates (Iwaki). After 18 h, the wells were gently washed three times with 200 µl sterile PBS, dried in an inverted position, and stained with 0.25 % safranin for 1 min. Wells were rinsed again, and A495 was determined (Micro-ELISA Autoreader Elx800; Bio-Tek instruments). Each assay was performed in triplicate and repeated five times.
Experimental infection.
The experimental infection was carried out essentially as described previously (Cucarella et al., 2002). Twelve healthy lactating Rasa Aragonesa ewes were inoculated with 1 ml bacterial suspension (5x102 c.f.u.) 20–25 days after parturition. Mothers were separated from their offspring 2 h before inoculation, to ensure the presence of milk (as a natural lubricant) in the teat duct at the time of inoculation. After teats were disinfected with 70 % ethanol, the inoculum was introduced in both glands through a 21-gauge cannula.
Since suckling favoured removal of bacteria, lambs were separated from their mothers for 120 min, in order to study the capacity of bacteria to colonize the mammary gland.
Milk samples for bacteriological analysis were obtained 2, 5 and 8 days after inoculation. Aliquots of milk samples were placed directly on CRA plates. In addition, to exclude the possibility of contamination, bacteria recovered at the end of the experimental period were compared with the parental strains by DNA typing.
Molecular typing of S. aureus strains.
Typing of the different strains was performed using three genetic markers: the coagulase, staphylococcal protein A and clumping factor B genes. This combination resulted in a discriminatory power closely approaching that of the whole-genome microarray (Koreen et al., 2005).
PCR amplification of the coa gene was performed as described by Hookey et al. (1998). Briefly, oligonucleotides coa-1m (5'-ATAGAGATGCTGGTACAGG-3') and coa-2c (5'-GCTTCCGATTGTTCGATGC-3'), encompassing the entire 3' repeats were utilized. Each amplification comprised 100 ng DNA template; 100 pmol each primer; 200 µM (each) dATP, dGTP, dCTP and dTTP; 1x buffer (Netzyme); 1 mM MgCl2 and 1 U thermostable DNA polymerase (Netzyme). Water was added to a final volume of 25 µl. Thermal cycling was performed as described (Hookey et al., 1998). An initial denaturation step at 94 °C for 2 min was followed by 30 cycles of 94 °C for 20 s, 57 °C for 15 s and 72 °C for 30 s, with a final step at 72 °C for 5 min. The size of the PCR products (5 µl aliquot) was analysed by electrophoresis on 1 % (w/v) agarose gels.
Restriction endonuclease analysis of the PCR-amplified coa gene was performed as described (Hookey et al., 1998). Approximately 500 ng PCR product was digested with 5 U restriction endonuclease CfoI (Roche) at 37 °C for 2 h. The digested PCR product was analysed by electrophoresis on 2 % (w/v) agarose gels.
PCR amplification of the spa gene was performed as described by Frenay et al. (1994), with modifications. Briefly, the oligonucleotides spa-1m (5'-GATTTTAGTATTGCAATACATAATTCG-3') and spa-2c (5'-CCACCAAATACAGTTGTACCG-3') were utilized. An initial denaturation step at 94 °C for 4 min was followed by 40 cycles of 94 °C for 20 s, 50 °C for 20 s and 72 °C for 90 s, with a final step at 72 °C for 5 min. The size of the PCR products was analysed by electrophoresis on 1 % (w/v) agarose gels. Restriction endonuclease analysis of the PCR-amplified spa gene was performed to increase the power of discrimination, as described for the coa gene. In both cases, to validate the patterns obtained after CfoI digestion, the different coa and spa PCR-amplified products were sequenced, searching for CfoI sites. In all cases, the observed patterns were identical to those predicted after DNA analysis.
PCR amplification of the clfB gene was performed as described by Koreen et al. (2005).
Statistical analysis.
The data were analysed by Student's t test for unpaired data to determine statistically significant differences. Differences were considered statistically significant at P<0.05 in all cases.
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RESULTS |
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). In three different experiments, a single colony of the wild-type strain was diluted, grown in liquid culture to exponential phase, and plated onto CRA plates. Ten smooth colonies were picked (Fig. 1a) and tested for biofilm formation on polystyrene tissue-culture plates. All the Congo red-negative variants were biofilm negative (data not shown). The frequency of stable biofilm-negative colonies ranged from 4x10–4 to 6x10–4. The wild-type strain and its biofilm-negative variants were analysed by DNA typing, which revealed no detectable difference between the parental strain and its variants (Fig. 1b–d).
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shows the absence of Bap in the biofilm-negative variants V329/1 and V329/2. Identical results were obtained with all the biofilm-negative variants (data not shown). Since biofilm formation in strain V329 is Bap-dependent (Cucarella et al., 2001, 2004), the biofilm-negative phenotype can be explained by the absence of Bap protein.
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), we hypothesized that biofilm-negative variants might be generated by the deletion of the element. To investigate this, chromosomal DNA of biofilm-negative variants was tested by Southern blot hybridization using the bap gene as a probe. This indicated that the bap gene was present in each of the biofilm-negative variants (Fig. 3). Therefore, the lack of Bap expression cannot be explained by the absence of the bap pathogenicity island. Similarly, PCR amplification of segments of the bap gene failed to reveal any differences between the wild-type and the variants (data not shown).
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). To determine if a mutation in the promoter was responsible for the decreased transcription, the bap promoter region of various biofilm-negative variants was amplified by PCR and sequenced. There was no modification in the sequence of this region (data not shown), suggesting that decreased transcription might be due to changes in a controlling element.
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, Table 2). However, we were unable to obtain revertants from strains V329/3 or V329/4 (Table 2), suggesting that different mechanisms are involved in creating biofilm-negative variants. Revertant strains V329/rev1–V329/rev4 (Table 1) expressed Bap and produced biofilm at the same level as the wild-type strain (Figs 2 and 5), although they exhibited different levels of bap transcription relative to that in the wild-type strain (Fig. 4).
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), the Bap-mutant m556 cells showed a significantly higher fibrinogen and fibronectin adherence than did the wild-type strain V329 (Fig. 6). Interestingly, the variant V329/1 and V329/2 cells adhered at a higher level to fibrinogen and fibronectin than did the wild-type V329 and its derivative V329/rev1 and V329/rev2 revertant strains (Fig. 6).
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, the different strains produced similar amounts of ClfA and FnBP. These results indicate that the increased adherence to fibrinogen or fibronectin in the Bap-negative strains is not due to higher levels of expression of the MSCRAMMs.
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). The rough colony revertants were biofilm positive (data not shown). In contrast, we were unable to isolate biofilm-negative variants from sheep infected with wild-type V329. These results contradict the similar frequencies of phase variation observed in both directions in vitro. One possible explanation is that increased adherence of Bap-negative variants to the epithelium of the mammary gland gives a selective advantage to hide the Bap-negative variants. Alternatively, it is also likely that environmental conditions biased phase variation toward the Bap-positive phenotype, which favoured persistence in the mammary gland, as previously described (Cucarella et al., 2004; Ubeda et al., 2003). This increased persistence in the Bap-positive isolates could explain the apparently higher reversion observed in vivo, particularly at later time points (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tormo et al., 2005a). The presence of Bap reduces infectivity in the short term by blocking early adherence of MSCRAMMs to host proteins (Cucarella et al., 2002). In contrast, Bap plays the opposite role in late adherence, allowing longer bacterial persistence in the mammary gland, most likely through the formation of biofilms (Cucarella et al., 2001; Ubeda et al., 2003). Despite the advantages that bacteria in biofilms have over their planktonic counterparts, dispersal from the biofilm allows sessile bacteria to colonize new niches. In this study, we reported that Bap-negative bacteria arose spontaneously at a high frequency in vitro. Phase variation is a strategy used by many pathogenic bacteria to avoid the immune system (Hacker, 1992; Seifert & So, 1988; Weiser et al., 1996). Alternatively, as Bap-negative bacteria have lost the capacity to produce biofilms, phase variation might represent a mechanism for dispersal of S. aureus from biofilms.
The synthesis of a large protein like Bap is presumably strictly regulated and coordinated with the production of other elements of the biofilm matrix. A recent study has shown that the global virulence regulator SarA acts as an activator of bap expression (Trotonda et al., 2005). SarA can modulate gene transcription through direct interaction with the target gene promoter, or by activating the agr regulatory cascade (Cheung & Zhang, 2002). In the case of the bap gene, gel shift and DNase I footprinting assays have shown that SarA directly binds to the bap promoter region (Trotonda et al., 2005). Also, disruption of agr does not have any effect on bap transcription, indicating that the mechanism by which SarA regulates bap is agr independent (Trotonda et al., 2005). Apart from transcriptional regulation, the function of Bap is subject to modulation by Ca2+. The presence of calcium in the growth medium has an inhibitory effect on biofilm formation (Arrizubieta et al., 2004). The concentrations of Ca2+ responsible are similar to those of free Ca2+ in milk. The interaction of Bap with Ca2+ occurs through EF-hand domains, since disruption of two of the four putative EF-hand motifs results in a Bap protein that is able to induce biofilm formation, even in the presence of Ca2+ (Arrizubieta et al., 2004). We now propose an alternative strategy to regulate the function of Bap, based on the ON-to-OFF phase variation of Bap expression. The mechanisms underlying phase variation are unknown. There is no evidence for the involvement of global regulators such as sarA, agr and 
B, since the expression of surface adhesins ClfA, ClfB, FnBPA and FnBPB, the expression of which is dependent on these regulators, does not change in Bap-negative variants (Cheung et al., 2004; Novick, 2003).
A mechanism of phase variation affecting biofilm development has been described in S. epidermidis, and involves reversible insertion/excision of the IS256 in the icaADBC operon (Ziebuhr et al., 1999). The icaADBC operon encodes proteins responsible for the synthesis of the PIA/PNAG exopolysaccharide, which is an important component of the staphylococcal biofilm matrix (Cramton et al., 1999; Maira-Litran et al., 2002). More recently, it has been reported that IS256 can cause biofilm-negative variants in S. epidermidis, through insertion/excision in the sarA and rsbU genes (Conlon et al., 2004), which are positive regulators of the ica operon (Knobloch et al., 2004; Tormo et al., 2005b). The appearance of biofilm-negative variants was significantly more frequent than reversion to biofilm production. In the case of Bap phase variation, similar frequencies in either direction occurred during growth in vitro. However, the frequency of Bap-positive variants seems to be higher than the reverse during infection of the mammary gland, suggesting that phase variation responds to environmental conditions. In contrast, Baselga et al. (1993) have analysed phase variation in slime production of S. aureus, and observed that the severity of ruminant mastitis decreases, but the bacterial capacity to colonize the mammary gland increases, when the infection is caused by a mucoid (slime producer) rather than non-mucoid variant. Samples from the glands infected with the biofilm-negative variant (strain c104–) revealed that none of the 180 500 colonies on CRA showed a biofilm-positive morphology, indicating that reversion from biofilm non-production to production had not taken place. However, glands infected with the biofilm-positive strain c104+ revealed biofilm-negative variants in three animals (in five of the six infected glands of these animals). Reversion was observed at 24 and 48 h after inoculation, suggesting that it occurred immediately after bacterial inoculation. We know that the mucoid strain used in that study (c104+) carries the bap gene (Ubeda et al., 2003), while the non-mucoid variant c104– is bap negative, since it has lost SaPIbov2. This explains the fact that no biofilm-positive (bap-positive) isolates were obtained from the biofilm-negative variants. In view of these results, we used here strain V329, in which the SaPIbov2-integrase gene, responsible for the deletion of the island, is not functional (Ubeda et al., 2003).
The biofilm structure may depend on the nature of the molecules involved. BLAST searches (Altschul et al., 1997) for sequences homologous to Bap have shown the existence of a novel family of proteins, which are important for biofilm formation in both Gram-positive and Gram-negative bacteria (Lasa & Penades, 2006). Members of this family have been described in S. aureus (Cucarella et al., 2001), coagulase-negative staphylococci (Tormo et al., 2005a), Enterococcus faecalis (Esp; Shankar et al., 1999; Toledo-Arana et al., 2001), Burkholderia cepacia (Bap; Huber et al., 2002), Pseudomonas putida (mus20; Espinosa-Urgel et al., 2000) and Salmonella typhimurium (Stm2689; Latasa et al., 2005). All members of the Bap family share similar characteristics, including a high molecular mass, a signal sequence for secretion, and a core domain of repeats, the number of which varies among different isolates (Shankar et al., 1999) and during the course of infection (Cucarella et al., 2004). We have not checked whether expression of these proteins is phase variable, but this could be an interesting avenue of further enquiry.
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ACKNOWLEDGEMENTS |
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Edited by: J. H. Cove
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Received 7 November 2006;
revised 8 February 2007;
accepted 20 February 2007.
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