HetR plays a key role in regulation of heterocyst differentiation. When the Cys-48 residue of the HetR from Anabaena sp. PCC 7120 was replaced with an Ala residue, the mutant HetR (HetR C48A) could not dimerize, indicating that HetR forms a homodimer through a disulfide bond. The Anabaena strain C48, containing the hetRc48a gene, could not produce HetR homodimer and failed to form heterocyst. We show that HetR is a DNA-binding protein and that its homodimerization is required for the DNA binding. HetR binds the promoter regions of hetR, hepA, and patS, suggesting a direct control of the expression of these genes by HetR. We present evidence that shows that the up-regulation of patS and hetR depends on DNA binding by HetR dimer. The pentapeptide RGSGR, which is present at the C terminus of PatS and blocks heterocyst formation, inhibits the DNA binding of HetR and prevents hetR up-regulation. The heterocystous cyanobacteria such as Anabaena sp. PCC strain 7120 contain specialized cells called heterocysts for nitrogen fixation when they are grown in the absence of combined nitrogen (1, 2). Many structural and metabolic changes occur during heterocyst differentiation (2-5). In cyanobacteria with long filaments, the spacing of heterocysts along the filaments is often regular, so there is a pattern formation (1). According to fossil records, the heterocyst pattern was one of the earliest pattern forms in evolution (6, 7).The pattern formation of cyanobacteria depends on cell-cell communication and molecular interactions (1, 8). Several important genes, such as ntcA (9, 10), hetR (11), hetC (12), hetF (13), hetN (14, 15), and patS (16, 17), play important roles in heterocyst differentiation and pattern formation. Recent evidence suggests that ␣-ketoglutarate could play an important role in the initiation of heterocyst differentiation (18).hetR is the master gene in controlling heterocyst differentiation and pattern formation (3,4,19), and it may also control other cellular processes in nonheterocystous cyanobacteria (20). By putting hetR under the control of the copper-inducible promoter PpetE, it was shown that heterocyst frequency was controlled by the expression level of hetR (19). hetR is autoregulatory (21) and regulates the expression of some other genes involved in heterocyst differentiation (8,21). HetR is a Ser-type protease required for heterocyst differentiation (22, 23). The mechanism for regulation of heterocyst differentiation by HetR is not clearly understood. The study by Buikema and Haselkorn (19) raised a very interesting question: Why does HetR as a protease function in a dose-dependent fashion?patS encodes a small peptide, and a diffusible shorter peptide could be generated from the full gene product (16,17). Like hetR, the expression of patS is localized primarily in proheterocysts and heterocysts (16). However, little is known about the regulation of the patS expression and the molecular mechanism for inhibition of heterocyst differentiation by PatS.In this communication, we report that HetR funct...
The cyanobacteria and red algae are two important groups of photosynthetic organisms that share a light-harvesting antenna known as the phycobilisome (PBS) [1][2][3][4] . PBSs are among the largest protein complexes in the living world and consist of phycobiliproteins (PBPs), including phycocyanin, phycoerythrin and allophycocyanin (APC), and linker proteins . Two subunits of PBPs, the α -and β -subunits, form an α β heterodimer that is conventionally called an (α β ) monomer. The monomer is then assembled into an (α β ) 3 trimer, the basic unit of PBS hierarchical assembly. The trimers of various PBPs are organized into a highly ordered supramolecular complex with the help of the linker proteins 1,5,6 . Four morphological types of PBS 1 are known: hemidiscoidal 7 , hemiellipsoidal 8 , block-type 9 and bundle-type 10. The hemidiscoidal PBS contains a central core surrounded by peripheral rods 1,11,12 . The chromophores in hemidiscoidal PBS are arranged in such a way that a photon absorbed by a chromophore in the peripheral rods is rapidly funnelled to chromophores in the core 13 and eventually to the terminal emitters (the core-membrane linker protein (L CM ) [14][15][16] or allophycocyanin D (ApcD) 17,18 ). The terminal emitters then transfer the energy to photosynthetic reaction centres 16,[19][20][21][22][23] . Currently, the mechanism of PBS assembly is poorly understood and the energy transfer routes within PBSs are not well defined. Although 3D structures of some individual PBPs have been reported (reviewed in refs 2, 3), the structures of most linker proteins are unknown and the complete structure of a PBS has not been published, to our knowledge. Here we report the cryo-electron microscopy (cryo-EM) structure of a PBS from the red alga Griffithsia pacifica at a resolution of 3.5 Å, which reveals details of the PBS architecture. Overall structureThe PBS from G. pacifica was purified and its intactness confirmed by its protein composition and spectroscopic features (Extended Data Fig. 1a-f). We reconstructed a 3D structure of the intact PBS by single particle cryo-EM with an overall resolution of 3.5 Å (Extended Data Table 1). Applying individual local masks improved the resolutions of local maps to 3.4-4.3 Å (Extended Data Fig. 2g). The PBS is one of the largest supramolecular complexes that has been reported, with a calculated molecular mass of approximately 16.8 megadaltons. The overall appearance is block-type 9 with twofold symmetry oriented perpendicularly to the thylakoid membranes (Fig. 1a-c and Extended Data Figs 1g-l, 3a-d). This PBS is larger than the hemiellipsoidal PBS isolated from Porphyridium cruentum8 and has dimensions of approximately 680 Å length, 390 Å height, and 450 Å thickness (Fig. 1a-c).The PBS contains a triangular core with the top cylinder B (formed by two APC trimers) sitting above two basal cylinders A and A′ (each formed by three APC trimers) surrounded by peripheral rods arranged in a staggered fashion (Fig. 1a-c and Extended Data Fig. 3c-e). In addition to the core and ...
The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechoccus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ [FA/FB]- back-reaction. ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC. ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [FA/FB]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-180], the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD.
1. Accurate assessments of fish species diversity and community composition are essential for understanding fish ecology and conservation management. Environmental DNA (eDNA) metabarcoding has become an integrated method for monitoring fish species. The accuracy and efficacy of eDNA metabarcoding rely heavily on the choice of primers used for PCR amplification. A wide selection of metabarcoding primers for fish has been developed; however, there exists no comprehensive and comparative evaluation of their amplification or taxonomic classification of a rich diversity of fish species, which hinders informed decisions regarding their suitability for different study systems. 2. Here we reviewed the literature and compiled a list of 22 primer sets for eDNAbased metabarcoding analysis of teleost fish, the performance of which was compared using in silico PCR, followed by in vitro metabarcoding analysis using eDNA from waterbodies in Beijing, which harbour a high number of freshwater fish species. 3. We found that the primers showed considerable differences in the amplified taxonomic ranges and proportions, fish taxa richness, species discrimination power and fish community compositions, both in silico and in vitro. The number of fish taxa detected from eDNA by the primer sets varied from 0 to 66. Primers targeting the 12S rRNA gene generally detected greater fish diversity than those targeting the 16S rRNA or COI genes, while primers targeting the cytochrome b gene amplified the fewest fish taxa in vitro. 4. Regarding target genes, 12S primers generally outperformed other primers in terms of amplified fish diversity. The results of in silico PCR and in vitro tests were not always in agreement, suggesting that primer choice for biodiversity surveys should not be based solely on in silico evaluation. The use of different primers can qualitatively and quantitatively affect the detected biodiversity and these effects should be considered in experimental design and data interpretation. These results will assist with primer selection for eDNA-based fish surveys, and consequently support conservation of freshwater biodiversity.
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