In situ enzyme immunodetection of surface or intracellular bacterial antigens using nitrocellulose sheets.

We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB.

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.

Factors regulating the slow electrogenic phase in green algae and higher plants.

The electrochromic rise in the millisecond range, corrected for the subsequent decay, has been studied in Chlorella cells and spinach chloroplasts. The half-time of the electrochromic rise in the millisecond range is independent of the redox states of the components in the electrogenic loop. It is also independent of pH in the absence of a pH gradient, but it is dependent upon the transmembrane electric field and the pH gradient. The limiting step of the overall process is thus the electrogenic reaction itself. The amplitude of the electrochromic rise in the millisecond range is controlled by two classes of factors: the redox state of its reactants and the free energy available in the overall process including the electrogenic reaction. This free energy depends upon delta pH and transmembrane electric field. A fast reduction of cytochrome f+ (and thus probably of the Rieske protein) by Photosystem II is still possible when the electrogenic reaction is impeded for energetic reasons. The kinetic behavior of the electrogenic reaction could be interpreted by the following reaction: UH2int + FeS+ + X2 + Hext in equilibrium U + 2H+int + FeS + X2H where FeS is the Rieske protein; X2 would be a new protein (observed in Bouges-Bocquet, B. (1980) FEBS Lett. 117, 54-58); UH2 which is likely a special quinone, would release its two protons inside the thylakoids and X2 would use protons from the external medium.

Electron and proton transfers from P-430 to ferredoxin-NADP-reductase in Chlorella cells.

After blocking Photosystem II on whole Chlorella cells, we measured the absorption changes between 0 degrees C and -10 degrees C. The absorption changes measured 2 mus after the beginning of a Xenon Flash are the sum of changes due to P(+)-700 and changes due to P(-)-430 (after the subtraction of the carotenoid triplet change and of the electrochromic effect). The reduction of P(-)-430 is not resolved by our technique. Its reoxidation presents a half-time around 1 mus at 0 degrees C and around 2 mus at -10 degrees C. The reduction and protonation of ferredoxin-NADP-reductase to its neutral semi-quinoid form FNRH present a half-time of about 3 mus at 0 degrees C and 6 mus at -10 degrees C. The presence of only one photoreducible ferredoxin-NADP-reductase per Photosystem I center is confirmed, but is an acceptor X' the differential extinction coefficients of which are weak or null from 420 nm to 480 nm. Tentative explanations which would reconcile these results with what was already known about ferredoxin are proposed.

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. I. Oxidation kinetics after a flash.

P-700, plastocyanin and cytochrome f redox kinetics were measured after one flash, using dark-adapted Chlorella in the presence of hydroxylamine and 3(3,4-dichlorophenyl)-1,1-dimethylurea. Plastocyanin becomes increasingly oxidized with a half-time of 70 microseconds, then undergoes reduction with a half-time of 7ms. Cytochrome f oxidation has a sigmoidal time-course and a half-time of 100 microseconds. Its redution exhibits a half-time of 4 ms. These results are interpreted in a linear scheme: (formula: see text). An equilibrium constant of 2 between cytochrome f and plastocyanin (PC), which contrasts with the large equilibrium constant between PC and P-700 is computed. The presence of cytochrome b6 in a cyclic path around Photosystem I is confirmed under these conditions.

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. II. Reduction kinetics and electric field increase in the 10 ms range.

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC+ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern. The reduction of PC+ and cytochrome f+ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267-284). Both need the presence of a compound R' in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R' as terminal reducant, PC+ and cytochrome f+ as terminal oxidants, would account for this field generation. Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.

Inhibition of oxygen evolution following illumination of Chlorella cells with far-red light.

The effect of brief far-red illumination on Chlorella pyrenoidosa cells has been investigated. An inhibition of oxygen evolution occurs 20 to 30 s after the end of the far-red illumination. This inhibition occurs in a step following the initial charge separation process by the System II centers. It is reversible in the light through a purely photochemical process.

Electron transport in photosystem I in spinach chloroplasts.

We have studied the recovery of the photochemical activity of Photosystem I after the charge separation induced by a flash under conditions where the secondary donors are in the reduced form. The rate-limiting steps are on the donor side. The first step is completed within 400 mus. The second step is much slower (half time approximately equal to 1 ms) and corresponds to the transfer of electrons from plastoquinone. Under our conditions, only one intermediate is involved in electron transfer between the centers and the plastoquinone pool. Electron exchange between the Sytem I centers has been demonstrated.