Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system.

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES-2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose-mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD-like tlr0903 (xcsB) and endoglucanase-like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose-mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.

Plastidic type I signal peptidase 1 is a redox-dependent thylakoidal processing peptidase.

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol-disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N-terminal targeting signal, which is removed in the lumen by a membrane-bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at -3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non-reducing conditions than under reducing conditions on SDS-PAGE. These results underpin the notion that Plsp1 is a redox-dependent signal peptidase in the thylakoid lumen.

Multiple fates of non-mature lumenal proteins in thylakoids.

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N-terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid-transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling-lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1-null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid-transfer signal removal on protein targeting and accumulation by examining the localization of non-mature lumenal proteins in the Arabidopsis plsp1-null mutant and performing a protein import assay using pea chloroplasts. In plsp1-null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non-mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid-transfer signal removal did not disrupt cpSEC- and cpTAT-dependent translocation, but prevented release of proteins from the membrane. Interestingly, non-mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440-kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1-null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.

A deletion mutation in the spacing within the psaA core promoter enhances transcription in a cyanobacterium Synechocystis sp. PCC 6803.

Transcriptional regulation of PSI reaction center psaA is one of the important physiological responses to changing environments. We previously reported that the Rrf2-type transcriptional regulator Slr0846 activates transcription of psaA in Synechocystis sp. PCC 6803. In the Δslr0846 mutant, transcripts from two promoters, P1 and P2, were downshifted and, as a result, a lower Chl content and slower growth were observed. Here, we report spontaneous suppressors which recovered Chl accumulation and photoautotrophic growth. Sequencing of the whole promoter region revealed in some suppressors the same single nucleotide deletion in a 9 bp G stretch (-21 to -29 from the transcriptional start point of P1), which is located between the -35 and -10 elements of the P1 core promoter (hereafter the -G mutation). The transcripts from P1 were higher in abundance in this pseudorevertant than in the Δslr0846 mutant. When the promoter was fused to a reporter gene, the -G mutation conferred ~4 times higher expression than the wild-type promoter. It has been shown that the P1 promoter activity of psaA is regulated by a high light regulatory element 1 just upstream of -35. The -G mutated P1 promoter still retained the high light response. Thus, the -G mutation enhanced the expression level of psaA without a loss of the response to the high light conditions. This is the first study of the spontaneous mutation of a spacer length of a promoter for expression in cyanobacteria.

An Rrf2-type transcriptional regulator is required for expression of psaAB genes in the cyanobacterium Synechocystis sp. PCC 6803.

Photosynthetic organisms must regulate photosystem stoichiometry (photosystem I-to-photosystem II ratio) under various light conditions. Transcriptional regulation of the psaAB genes is a critical process for this photoacclimation in cyanobacteria. In the course of our screening of transcriptional regulators in the cyanobacterium Synechocystis sp. PCC 6803, we found that chlorophyll accumulation was impaired in an Rrf2-type regulator Slr0846 mutant. DNA microarray and primer extension analyses showed that the expression of psaAB genes was markedly decreased in the mutant. Consistently, the mutant exhibited lower photosystem I-to-photosystem II ratio under normal light conditions, suggestive of decreased accumulation of the photosystem I reaction center. Gel-shift assay confirmed that the Slr0846 protein bound to a far upstream promoter region of psaAB. These phenotypes of the mutant varied substantially with light conditions. These results suggest that Slr0846 is a novel transcriptional regulator for optimal expression of psaAB.

slr1923 of Synechocystis sp. PCC6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide).

The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F(420)H(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex I. In this study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than the wild type under 100 microE m(-2) s(-1) but did not grow under high light intensity (300 microE m(-2) s(-1)). The cellular content of chlorophyll was lower in the mutant, and the absorption spectrum showed a shift in the absorption peak of the Soret band to a longer wavelength by about 10 nm compared with the wild type. It was found, by high-performance liquid chromatography analysis, that the retention time of chlorophyll of the mutant is shorter than that of the wild type and that the peak wavelength of the Soret band was also shifted to a longer wavelength by 11 nm. Proton nuclear magnetic resonance analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with a vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl)chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that the Slr1923 protein is essential for the conversion from divinylchlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).

Upregulation of PG synthesis on sulfur-starvation for PS I in Chlamydomonas.

Sulfur(S)-starvation was previously shown to induce the degradation of an acidic lipid in chloroplasts, sulfoquinovosyl diacylglycerol (SQDG), to yield a major internal S-source in a green alga, Chlamydomonas reinhardtii. We here found that the synthesis of phosphatidylglycerol (PG), the other acidic lipid in chloroplasts, is activated to elevate its content up to a level that just compensates for the loss of SQDG. Similar activation of PG synthesis was also observed in an SQDG-deficient mutant under S-replete conditions, which led us to propose that upregulation of PG synthesis under S-starved conditions occurs through direct sensing of SQDG-loss, but not of S-starvation. Moreover, thylakoid membranes isolated from S-starved cells were reduced in photosystem I activity on treatment with phospholipase A(2) that specifically broke down PG, which suggested a critical role of PG that is increased under S-starved conditions in the maintenance of the photosystem I activity.