Alberto Vianelli

Why genes overlap in viruses

The genomes of most virus species have overlapping genes—two or more proteins coded for by the same nucleotide sequence. Several explanations have been proposed for the evolution of this phenomenon, and we test these by comparing the amount of gene overlap in all known virus species. We conclude that gene overlap is unlikely to have evolved as a way of compressing the genome in response to the harmful effect of mutation because RNA viruses, despite having generally higher mutation rates, have less gene overlap on average than DNA viruses of comparable genome length. However, we do find a negative relationship between overlap proportion and genome length among viruses with icosahedral capsids, but not among those with other capsid types that we consider easier to enlarge in size. Our interpretation is that a physical constraint on genome length by the capsid has led to gene overlap evolving as a mechanism for producing more proteins from the same genome length. We consider that these patterns cannot be explained by other factors, namely the possible roles of overlap in transcription regulation, generating more divergent proteins and the relationship between gene length and genome length.

Bacteriophage T4 Self-Assembly: Localization of gp3 and Its Role in Determining Tail Length

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

A study of Photosystem II fluorescence emission in terms of the antenna chlorophyll-protein complexes

Abstract Fluorescence emission spectra for the seven chlorophyll-protein complexes comprising the antenna system of Photosystem II (PS II) have been measured. All four outer antenna complexes (LHC II, CP24, CP26, CP29) have relatively greater emission near 648 nm and 680 nm with respect to the inner antenna complexes (CP43, CP47, D1/D2/cyt b-559). The emission spectra for both outer and inner antenna were calculated from the measured emission spectra of the single chlorophyll-protein complexes, using as weighting factors the excited state population at thermodynamic equilibrium in the various chlorophyll-protein complexes suggested by Jennings et al. (Jennings, R.C., Bassi, R., Garlaschi, F.M., Dainese, P. and Zucchelli, G. (1993) Biochemistry 32, 3203–3210). Subsequently, the overall emission spectra for the total PS II antenna (i.e., outer plus inner antenna) were calculated for situations in which varying excited state levels were assumed for the inner and outer antenna. In an attempt to determine the steady-state distribution of excited states between outer and inner antenna these calculated fluorescence spectra were compared with those measured for (a), PS II particles prepared from maize and (b), chloroplasts of wild-type barley and the chlorina F2 mutant. From this comparison it is concluded that at steady-state fluorescence emission, between 28% and 38% of the excited states in PS II are associated with the inner antenna and between 62% and 72% with the outer antenna. These results suggest that the PS II antenna is organised as a very shallow energy funnel. This antenna organisation is discussed in terms of the generation of non-photochemical quenching mechanisms which are designed to protect PS II from high light stress.

Influence of thylakoid protein phosphorylation on photosystem I photochemistry

The influence of the phosphorylation of thylakoid proteins on photosystem I photochemistry has been measured under conditions of linear dependence of the rate of electron transport to NADP on light intensity. It was found that the phosphorylation by ATP of light harvesting chlorophyll protein complex (LHC) II and other polypeptides stimulates the rate up to ∼ 40%; the stimulation is larger when the wavelength of actinic light corresponds to the main absorption of LHC II.

Bacteriophage T4 Self-Assembly: In Vitro Reconstitution of Recombinant gp2 into Infectious Phage

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in the recBCD(+) host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2(+) phage infection (H(+)) with tails from a gp2(+) or gp2(-) phage infection (T(+) or T(-)), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H(-)) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV(+) cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2(+) phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.

Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz.

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c.+ [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316–325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a.+ [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973–6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl ain vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

The relation between the minor chlorophyll spectral forms and fluorescence quenching in aggregated light harvesting chlorophyll ab complex II

Abstract The hypothesis that fluorescence quenching in aggregated light harvesting chlorophyll a b protein complex II is associated with the formation of minor spectral forms absorbing near 655 nm and between 680 nm-690 nm is examined. Using an homogeneous LHCII preparation, steady-state absorption changes measured at room temperature are quantitatively compared with the associated steady state fluorescence changes by means of the Stepanov relation. It is demonstrated that upon LHCII aggregation, the relative fluorescence yield is constant for chlorophyll forms absorbing between 650 nm and 690 nm. This indicates that the minor chlorophyll forms formed upon LHCII aggregation are not quenching species.

A comparison of the light-induced, non-reversible fluorescence quenching in Photosystem II with quenching due to open reaction centres in terms of the chlorophyll emission spectral forms

The recent demonstration that the room temperature fluorescence emission spectrum of Photosystem II may be described as a linear combination of Gaussian bands associated with the well-known chlorophyll spectral forms (Zucchelli, G., Jennings, R.C., Garlaschi, F.M. (1992) Biochim. Biophys. Acta, 1099, 163–169) has permitted analysis of a number of fluorescence quenching phenomena in terms of the chlorophyll spectral forms. In this context we have analysed the fluorescence quenching due to open reaction centres and also the light-induced, non-photochemical, non-reversible quenching in isolated thylakoids, a Photosystem II membrane preparation and the major light-harvesting chlorophyll a/b protein complex (LHCII). Whereas open reaction centres quench the emission associated with the spectral form chl688684 with greatest efficiency, thus indicating that it is this spectral form which is the most efficient in transferring energy to reaction centres, all the non-photochemical phenomena investigated show maximal quenching for both chl680678 and chl687684. It is concluded that the light-induced, non-photochemical, non-reversible quenching involves the generation of quenching centres within the Photosystem II antenna, possibly at the level of LHCII.

Regulation by localized protons of Photosystem II photochemical efficiency

Abstract In a previous report (Ehrenheim et al. (1991) Biochim. Biophys. Acta 1059, 106–110) it was shown that membrane-localized protons inhibit energy utilization at the Photosystem II reaction centre. We report here that the stimulation of PS II energy utilization due to the removal of such protons by nigericin is dependent on the wavelength of the actinic modulated light. Maximal fluorescence is not affected by nigericin. The effectiveness of the different uncouplers is related to their lipophilicity. These observations led us to conclude that electron-transport-dependent localization of protons in the membrane lowers the efficiency of energy transfer to the PS II reaction centre. It is also shown that the effect of uncouplers on PS II is not related to inhibition of electron recycling around PS II or cation-dependent energy spillover.

Optically detected magnetic resonance of intact membranes from Chloroflexus aurantiacus. Evidence for exciton interaction between the RC and the B808–866 complex

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808–866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808–866 antenna complex. This evidence was provided by the comparison of the T−S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.