A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond

A [2Fe-2S] ferredoxin (Fd1) from the hyperthermophilic bacterium Aquifex aeolicus has been obtained by heterologous expression of the encoding gene in Escherichia coli. Sequence comparisons show that this protein belongs to the extended family of plant- and mammalian-type [2Fe-2S] ferredoxins but also indicate that it is not closely similar to either the plant-type or mammalian-type subfamilies. Instead, it appears to bear some similarity to novel members of this family, in particular the Isc-type ferredoxins involved in the assembly of iron-sulfur clusters in vivo. The two redox levels of the [2Fe-2S](2+/+) metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and M?ssbauer spectroscopies. A full-spin Hamiltonian analysis is given for the M?ssbauer spectra. In aggregate, the spectroscopic data reveal differences with both the plant-type and mammalian-type ferredoxins, in keeping with the sequence comparisons. The midpoint potential of the [2Fe-2S](2+/+) couple, at -375 mV versus the normal hydrogen electrode, is more negative than those of mammalian-type ferredoxins and at the upper end of the range covered by plant-type ferredoxins. A. aeolicus ferredoxin contains two cysteines in addition to the four that are committed as ligands of the [2Fe-2S] cluster. These two residues have been shown by chemical modification and site-directed mutagenesis to form a disulfide bridge in the native protein. While that cystine unit plays a significant role in the exceptional thermostability of A. aeolicus ferredoxin (T(m) = 121 degrees C at pH 7 versus T(m) = 113 degrees C in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the [2Fe-2S](2+/+) chromophore. This observation is consistent with the large distance (ca. 20 A) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.     

Simultaneous measurements on the effect of oxygen concentration on water vapor and carbon dioxide exchange in leaves

Summary Transpiration rate was unaffected by O₂ concentration in the range 1–80% when illuminated leaves ofAtriplex patula andA. rosea were kept at low CO₂ concentrations. Moreover, the marked increase in the rate of light-saturated photosynthesis that takes place in many plant species when O₂ concentration is reduced from 21% to a few percent was not accompanied by any change in transpiration rate inA. patula andSolanum dulcamara. The results indicate that higher plant photosynthesis in normal air and saturating light is not determined by physical barriers to gas diffusion alone but that it is markedly limited by biochemical processes.C.I.W.-D.P.B.Publ. No. 454.     

Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light

Summary Ultraviolet light was demonstrated to stimulate flavone glycoside synthesis in Petroselinum cell suspension cultures. The data presented suggest the involvement of phytochrome in this response: Flavone glycoside formation resulting from 1 h of ultraviolet irradiation was increased by subsequent continuous far-red light irradiation. However, the ultraviolet effect was reduced by a subsequent irradiation with 10 min of far-red. This far-red effect was fully reversed by a sub-sequent irradiation with 10 min of red. Red and far-red irradiations were ineffective without ultraviolet preirradiation. It is concluded that in this system ultraviolet irradiation is required in order to change the cells in such a way as to allow a physiological effectiveness of the phytochrome system.     

Construction of dense linkage maps “on the fly” using early generation wheat breeding populations

In plant species, construction of framework linkage maps to facilitate quantitative trait loci mapping and molecular breeding has been confined to experimental mapping populations. However, development and evaluation of these populations is detached from breeding efforts for cultivar development. In this study, we demonstrate that dense and reliable linkage maps can be constructed using extant breeding populations derived from a large number of crosses, thus eliminating the need for extraneous population development. Using 565 segregating F₁ progeny from 28 four-way cross breeding populations, a linkage map of the hexaploid wheat genome consisting of 3,785 single nucleotide polymorphism (SNP) loci and 22 simple sequence repeat loci was developed. Map estimation was facilitated by application of mapping algorithms for general pedigrees implemented in the software package CRI-MAP. The developed linkage maps showed high rank-order concordance with a SNP consensus map developed from seven mapping studies. Therefore, the linkage mapping methodology presented here represents a resource efficient approach for plant breeding programs that enables development of dense linkage maps “on the fly” to support molecular breeding efforts.     

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.     

Photoprotection, photosynthesis and growth of tropical tree seedlings under near-ambient and strongly reduced solar ultraviolet-B radiation

Seedlings of two late-successional tropical rainforest tree species, Tetragastris panamensis (Engler) O. Kuntze and Calophyllum longifolium (Willd.), were field grown for 3-4 months at an open site near Panama City (9 degrees N), Panama, under plastic films that either transmitted or excluded most solar UV-B radiation. Experiments were designed to test whether leaves developing under bright sunlight with strongly reduced UV-B are capable of acclimating to near-ambient UV-B conditions. Leaves of T. panamensis that developed under near-ambient UV-B contained higher amounts of UV-absorbing substances than leaves of seedlings grown under reduced UV-B. Photosynthetic pigment composition, content of alpha-tocopherol, CO(2) assimilation, potential photosystem II (PSII) efficiency (evaluated by F(v)/F(m) ratios) and growth of T. panamensis and C. longifolium did not differ between seedlings developed under near-ambient and reduced solar UV-B. When seedlings were transferred from the reduced UV-B treatment to the near-ambient UV-B treatment, a pronounced inhibition of photosynthetic capacity was observed initially in both species. UV-B-mediated inhibition of photosynthetic capacity nearly fully recovered within 1 week of the transfer in C. longifolium, whereas in T. panamensis an about 35% reduced capacity of CO(2) uptake was maintained. A marked increase in UV-absorbing substances was observed in foliage of transferred T. panamensis seedlings. Both species exhibited enhanced mid-day photoinhibition of PSII immediately after being transferred from the reduced UV-B to the near-ambient UV-B treatment. This effect was fully reversible within 1d in T. panamensis and within a few days in C. longifolium. The data show that leaves of these tropical tree seedlings, when developing in full-spectrum sunlight, are effectively protected against high solar UV-B radiation. In contrast, leaves developing under conditions of low UV-B lacked sufficient UV protection. They experienced a decline in photosynthetic competence when suddenly exposed to near-ambient UV-B levels, but exhibited pronounced acclimative responses.     

Replication of poliovirus requires binding of the poly(rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site

The 5' nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CD(pro). The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C(93-95) and C(98-100)) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C(93-95)) resulted in slower viral growth, whereas the C(98-100)A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C(94-95)) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5'-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CD(pro) to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.     

Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase

The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene. Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and M?ssbauer spectroscopies. BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron site. Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization. Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number. At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and M?ssbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers. These results are in agreement with our previous study of naphthalene 1,2-dioxygenase [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953], which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone. The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme. The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product. Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced. Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis. The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters. The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.     

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log₁₀TCID₅₀/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.