TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Thylakoid protein phosphorylation is suppressed by 'free radical scavengers'. Correlation between PS II core protein degradation and thylakoid protein phosphorylation

Recent work has shown that the light-induced PS II core protein degradation, as monitored by immunostain reduction on Western blots, was stimulated even at low light during phosphorylation of thylakoid proteins in the presence of NaF, and that the thylakoid kinase inhibitor FSBA blocked completely the light- and ATP-stimulated degradation [Georgakopoulos and Argyroudi-Akoyunoglou (1997) Photosynth Res 53: 185–195]. To assess whether D1, D2 or both proteins are degraded, antibodies raised against D1/D2, or the D-E loop of D1 were used. Greatest immunostain reduction was observed with antibodies raised against D1/D2, immunostaining a 34 kDa protein on blots of 15% polyacrylamide-6 M urea gels, suggesting that the phosphorylation-induced degradation may be mainly directed against D2. To see how protein phosphorylation might be implicated in PS II core protein degradation we further tested the effect of free radical scavengers, on thylakoid protein phosphorylation. Active oxygen scavengers like n-propyl gallate, histidine, and imidazole, shown earlier to inhibit high light-induced D1 degradation, also suppressed the phosphorylation of thylakoid proteins; on the other hand, NaN3 and D-mannitol, known to stimulate light- induced D1 degradation did not suppress protein phosphorylation, whereas superoxide dismutase and catalase, known also to inhibit high light-induced D1 degradation, did not affect thylakoid protein phosphorylation. In addition, the ATP-induced degradation was also observed in the dark under conditions of kinase activation, and in the light under anaerobic conditions, that block light-induced degradation, whereas it was reduced in the absence of NaF, the phosphatase inhibitor. The results point to the involvement of a proteolytic system in PS II core protein degradation, which is active in its phosphorylated state.     

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of ¹³C-labeled glucose or ¹⁵N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of ¹³C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of ¹³C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (¹²Por¹²Phy), (ii) ¹³C-labeled chlorophyll (¹³Por¹³Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was ¹³C-labeled, whereas the other constituent of the molecule remained unlabeled (¹³Por¹²Phy and ¹²Por¹³Phy). The kinetics of ¹²Por¹²Phy disappearance, presumably due to chlorophyll de-esterification, and of ¹³Por¹²Phy, ¹²Por¹³Phy, and ¹³Por¹³Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of ¹²Por¹²Phy was three-fold faster in a photosystem I-less strain than in a photosystem II-less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de-esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of ¹³Por¹²Phy and ¹²Por¹³Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process.     

Immunoregulatory factors from a human macrophage-like cell line. II. A human T-cell lymphokine-induced suppressor factor for lymphocyte proliferation

It has been demonstrated that the human histiocytic lymphoma-derived cell line U937, which has monocytoid characteristics, responds to a concanavalin A-induced T-cell-derived suppressor supernatant (T-SFS) with the release of a factor markedly suppressing mitogen-stimulated proliferation of normal peripheral blood lymphocytes. The suppressor material is not dialyzable, appears within 2 hr of exposure of U937 cells to the T-SFS, persists for at least 24 hr, and has a Mr of approximately 40,000 by gel chromatography. The suppressor factor does not affect the proliferation of continuous T- and B-lymphoid cell lines, distinguishing it from the inhibitor of DNA synthesis also released by U937, but appears to be specific for a stage of activation of normal lymphocytes that is independent of (a) utilization of interleukin-2 and (b) inhibition of production of interleukin-2.     

Rapid isolation of photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography

With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10–25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC I-680) and Lhca1+4 (LHC I-730). Moreover, other highly enriched subfractions of LHC I such as Lhca2, Lhca3 and Lhca1+2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 moles O₂ mg Chl^–1 h^–1) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.Abbreviations a.u. arbitrary units - DCIP 2.6-dichlorophenol indophenol - LHC light harvesting complex     

N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with (¹⁵NH₄)₂SO₄ and Na¹⁵NO₃. Pigments extracted from cells were separated by HPLC and incorporation of the ¹⁵N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time (τ) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 μmol photons m⁻² s⁻¹ was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation (τ ∼200-400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4-5 fold (τ ∼50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.     

The suppression of experimental allergic encephalomyelitis in Lewis rats by treatment with myelin basic protein-cell conjugates

Pretreatment of Lewis rats with guinea pig (GP) myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) suppressed the subsequent induction of experimental allegic encephalomyelitis (EAE) with GP-MBP in Freund's complete adjuvant. The degree of suppression correlated positively with the amount of antigen coupled to the SL. GP-MBP coupled to syngeneic red blood cells (RBC) also resulted in suppression of EAE and the extent of the suppression was related to the dose of cells. These regimens of pretreatment also resulted in a decrease in the in vitro lymphocyte proliferative response to GP-MBP and in the extent of perivascular cuffing in the spinal cord. No decrease in the anti-MBP antibody response was detected in rats pretreated with either GP-MBP-SL or GP-MBP-RBC conjugates. Transfer of lymph node cells from rats pretreated with GP-MBP-RBC resulted in a decrease in disease severity in recipients. It is concluded that prior administration of MBP-cell conjugates is an effective way of suppressing the symptoms of EAE.     

Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor

Photosystem I (PSI), which consists of a core complex and light-harvesting complex I (LHCI), is an important multisubunit pigment-protein complex located in the photosynthetic membranes of cyanobacteria, algae and plants. In the present study, we described a rapid method for isolation and purification of PSI and its subfractions. For purification of PSI, crude PSI was first prepared by differential centrifugation, which was applicable on a large scale at low cost. Then PSI was purified by sucrose gradient ultracentrifugation in a vertical rotor to reduce the centrifugation time from more than 20 h when using a swinging bucket rotor to only 3 h. Similarly, for subfractionation of PSI into the core complex and light-harvesting complex I, sucrose gradient ultracentrifugation in a vertical rotor was also used and it took only 4 h to obtain the PSI core, LHCI-680, and LHCI-730 at the same time. The resulting preparations were characterized by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectroscopy, and 77 K fluorescence spectroscopy. In addition, their pigment composition was analyzed by high-performance liquid chromatography and the results showed that each Lhca could bind 1.5-1.6 luteins, 1.0 Violaxanthins, and 0.8-1.1 beta-carotenes on average, demonstrating that fewer carotenoids were released than with the slower traditional centrifugation. These results showed that the rapid isolation procedure, based on differential centrifugation and sucrose gradient ultracentrifugation in a vertical rotor, was efficient, and it should significantly facilitate preparation and studies of plant PSI. Moreover, the vertical rotor, rather than the swinging bucket rotor, may be a good choice for isolation of some other proteins.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.