SummaryThe Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, b 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were signi®cantly increased, when expression of this gene was controlled by the HSP70A±RBCS2 promoter fusion as compared to the RBCS2 promoter alone.

Coordination between the activities of organelles and the nucleus requires the exchange of signals. Using Chlamydomonas, we provide evidence that plastidderived chlorophyll precursors may replace light in the induction of two nuclear heat-shock genes (HSP70A and HSP70B) and thus qualify as plastidic signal. Mutants defective in the synthesis of Mg-protoporphyrin IX were no longer inducible by light. Feeding of Mg-protoporphyrin IX or its dimethyl ester to wild-type or mutant cells in the dark resulted in induction. The analysis of HSP70A promoter mutants that do or do not respond to light revealed that these chlorophyll precursors specifically activate the light signaling pathway. Activation of gene expression was not observed when protoporphyrin IX, protochlorophyllide, or chlorophyllide were added. A specific interaction of defined chlorophyll precursors with factor(s) that regulate nuclear gene expression is suggested.

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.

Blue light as an environmental cue plays a pivotal role in controlling the progression of the sexual life cycle in the green alga Chlamydomonas reinhardtii. Phototropin was considered a prime candidate for the blue-light receptor involved. By using the RNA interference method, knockdown strains with reduced phototropin levels were isolated. Those with severely reduced levels of this photoreceptor were partially impaired in three steps of the life cycle: in gametogenesis, the maintenance of mating ability, and the germination of zygotes. These observations suggest that phototropin is the principal sensory molecule used by this alga for the control of its life cycle by light.

Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light.

The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G؉C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B. japonicum. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by ؊24/؊12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.Nodulation (nod) genes and nitrogen fixation (nif) genes are the key determinants in the interaction between rhizobia and their host plants (14, 47). However, other loci influence the efficiency of the interaction or change the host range. Sequencing of the symbiotic plasmid of Rhizobium sp. strain NGR234 revealed a gene cluster that encodes a type III secretion system (22). Secreted proteins are encoded within the same cluster (95). The closely related Sinorhizobium fredii carries a type III secretion system as well (51, 61). Mutations within the secretion systems of the two strains influence symbiosis in a hostdependent manner. Plant and animal pathogens use related systems to target proteins to host cells (35), but such proteins have not been identified in rhizobia.During symbiosis, rhizobia exclusively rely on the carbon supply from the plant. Although bacteroids can utilize a wide range of carbon compounds, dicarboxylic acids are most likely the main carbon and energy source for bacteroids (45,83). The main argument is that several strains that have a defect in the dicarboxylic acid transport system show a Fix Ϫ phenotype (7,17,19,79,94) or are at least strongly impaired in nitrogen fixation (37).In our earlier work, we established a correlated physical and genetic map of the Bradyrhizobium japonicum genome (28,53) and discovered that all known nod and nif genes were clustered within a chromosomal region of about 400 kb. Furthermore, we found that the GϩC content of these genes was 58 mol% (76), considerably lower than the 61 to 65 mol% reported for the whole genome (43). Therefore, we concluded that the symbiotic genes have integrated into the chromosome after horizontal gene transfer from a different strain. In the absence of genomi...

SummaryWe have shown previously that the HSP70A (A) promoter, when fused upstream of other promoters, signi®cantly improves their performance in driving transgene expression in Chlamydomonas. Here, we employed the bacterial resistance gene ble, driven by the RBCS2 (R) promoter or an AR promoter fusion, to determine, by which mechanism(s) the A promoter may exert its enhancing effect. We observed that transformation rates of AR-ble constructs were signi®cantly higher than those of R-ble constructs. However, ble mRNA levels in pools of transformants generated with either construct type were the same. Co-transformation experiments revealed that the R-ble transgene was silenced in 80% of the transformants, whereas this fraction was reduced to 36% in transformants harbouring the AR-ble transgene. We conclude that the A promoter acts by decreasing the probability that a transgene becomes transcriptionally silenced. We mapped two elements within the A promoter that are responsible for this effect. The core of the ®rst element appears to be located between nucleotides ± 7 and + 67 relative to the HSP70A transcriptional start site. Its activity is strongly dependent on its spatial setting with respect to the R promoter and is increased by upstream sequences (± 196 to ± 8). The second element is independent of the ®rst and is located to the region from ± 754 to ± 197. Its activity is spacing-independent and additive to the ®rst element.

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