SpoIVB is essential for intercompartmental signalling in the σK‐checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro‐σK to its mature and active form σK. We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self‐cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro‐σK processing and a yet, uncharacterized, second function which is essential for the formation of heat‐resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.

Lhcb and other nuclear genes for chloroplastic proteins are regulated by several signals. Among them, light and retrograde signals from the plastid itself appear to act through closely related mechanisms. To investigate this interaction, we analysed an Arabidopsis mutant, hy1, deficient in plastidic heme oxygenase. hy1 is defective in phytochrome chromophore biosynthesis, which has other indirect effects on tetrapyrrole metabolism. We generated double mutants between hy1-6.2, genetically a null mutation, and three known gun (genome uncoupled) mutants, defective in retrograde plastid signalling. Recent molecular evidence shows GUN5 to be involved in tetrapyrrole metabolism (N. Mochizuki and J. Chory, manuscript in preparation). We observed hy1gun4 to be semi-albino plants, and hy1gun5 albino lethal, in a high-light-sensitive manner. Both double mutants showed defective greening and chloroplast development, and expressed Lhcb at reduced levels specifically in high light. Their degree of 'genome uncoupling' (Lhcb expression in the absence of functional chloroplasts) was similar to that observed in single mutants. These results can be interpreted as a metabolic (rather than genetic) interaction between HY1 and GUN4 or GUN5, and this in turn supports the involvement of tetrapyrroles as plastid signals. The tetrapyrrole precursor 5-aminolevulinic acid (ALA) inhibited Lhcb expression in hy1. Surprisingly, ALA also rescued photomorphogenesis of hy1. We speculate that either one tetrapyrrole intermediate, which can accumulate anomalously in hy1, or an altered ratio between two intermediates, plays a role as a repressor of Lhcb expression. gun1 did not exacerbate the plastid or Lhcb expression phenotype of hy1. This can be interpreted as a role for gun1 strictly on the same pathway as hy1 or, more likely, as evidence for the existence of at least one separate, non-tetrapyrrole related plastid signal.

No abstract

SummaryThe SENSITIVE TO FREEZING2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.

A cytochrome b/cl complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-30 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol -cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.Respiratory as well as photosynthetic electron transport, in mitochondria [l] (Krinner et al., unpublished). Compared to the mitochondria1 complexes the latter two represent relatively simple polypeptide structures. Preparations from anoxygenic photosynthetic bacteria have not been reported. They would be desirable not only for comparative aspects, but also as a biochemical complimentation to the detailed flash spectrophotometric studies, especially on membranes from Rhodospirillaceae [3 -51.By adotping the procedure for the purification of the cytochrome flb6 complex from spinach chloroplasts [2] we succeeded in the isolation of an active cytochrome b/cl complex from Rhodopseudomonas sphaeroides GA, which is presented here. The procedure is based on the use of octylglucoside/ cholate mixtures to solubilize the complex, which has been also successfully employed to isolate coupling factor ATPases [12,13]. MATERIALS AND METHODS Preparation of Ubiquinol-Cytochrome c2 OxidoreductaseCells of Rhodopseudomonas sphaeroides GA were grown and harvested, and chromatophores were prepared thereDedicated to the memory of Assunta Baccarini-Melandri who initiated this work. Other MaterialsUbiquinols were kindly donated by Hoffmann-LaRoche, Basel. Cytochrome c2 from R. sphaeroides was prepared according to Bartsch [15], and cytochrome c from Saccharomyces cerevisiae was obtained from Sigma. All other sources for materials are given in [2]. AssaysActivity of ubiquinol -cytochrome c2 oxidoreductase was measured in an Aminco DW-2 spectrophotometer as described