CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.     

HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS

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.     

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120

Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.     

Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation

The HetR protein has long been recognized as a key player in the regulation of heterocyst development. HetR is known to possess autoproteolytic and DNA-binding activities. During a search for mutants of Anabaena sp. PCC 7120 that can overcome heterocyst suppression caused by overexpression of the patS gene, which encodes a negative regulator of differentiation, a bypass mutant strain, S2-45, was isolated that produced a defective pattern (Pat phenotype) of irregularly spaced single and multiple contiguous heterocysts (Mch phenotype) in combined nitrogen-free medium. Analysis of the S2-45 mutant revealed a R223W mutation in HetR, and reconstruction in the wild-type background showed that this mutation was responsible for the Mch phenotype and resistance not only to overexpressed patS, but also to overexpressed hetN, another negative regulator of differentiation. Ectopic overexpression of the hetRR223W allele in the hetRR223W background resulted in a conditionally lethal (complete differentiation) phenotype. Analysis of the heterocyst pattern in the hetRR223W mutant revealed that heterocysts differentiate essentially randomly along filaments, indicating that this mutation results in an active protein that is insensitive to the major signals governing heterocyst pattern formation. These data provide genetic evidence that, apart from being an essential activator of differentiation, HetR plays a central role in the signaling pathway that controls the heterocyst pattern.     

Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

In response to combined nitrogen starvation in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 is able to develop a particular cell type, called a heterocyst, specialized in molecular nitrogen fixation. Heterocysts are regularly intercalated among vegetative cells and represent 5-10% of all cells along each filament. In unicellular cyanobacteria, the key Krebs cycle intermediate, 2-oxoglutarate (2-OG), has been suggested as a nitrogen status signal, but in vivo evidence is still lacking. In this study we show that nitrogen starvation causes 2-OG to accumulate transiently within cells of Anabaena PCC 7120, reaching a maximal intracellular concentration of approximately 0.1 mM 1 h after combined nitrogen starvation. A nonmetabolizable fluorinated 2-OG derivative, 2,2-difluoropentanedioic acid (DFPA), was synthesized and used to demonstrate the signaling function of 2-OG in vivo. DFPA is shown to be a structural analogue of 2-OG and the process of its uptake and accumulation in vivo can be followed by (19)F magic angle spinning NMR because of the presence of the fluorine atom and its chemical stability. DFPA at a threshold concentration of 0.3 mM triggers heterocyst differentiation under repressing conditions. The multidisciplinary approaches using synthetic fluorinated analogues, magic angle spinning NMR for their analysis in vivo, and techniques of molecular biology provide a powerful means to identify the nature of the signals that remain unknown or poorly defined in many signaling pathways.     

A gene encoding a protein related to eukaryotic protein kinases from the filamentous heterocystous cyanobacterium Anabaena PCC 7120.

Protein kinases play essential roles in the development of eukaryotic cells. These enzymes display various degrees of sequence similarity in their catalytic domains. This conservation has allowed the identification of protein kinases in a variety of organisms, including the Gram-negative bacterium Myxococcus xanthus. In this study, sequences related to those encoding eukaryotic protein kinases were amplified by PCR from DNA of Anabaena PCC 7120, a filamentous cyanobacterium that differentiates cells specifically for nitrogen fixation, called heterocysts, under conditions of combined nitrogen limitation. Results from Southern hybridization and sequencing of PCR products suggest the presence of a family of similar protein kinases in this strain. One of the corresponding genes (pknA) was isolated from a gene library. The N-terminal region of its amino acid sequence shows significant similarity to the catalytic domains of eukaryotic-type protein kinases. Expression of this gene was found to be developmentally regulated. Inactivation of pknA led to colonies that appeared light green and rough in the absence of combined nitrogen. Mutant filaments produce fewer heterocysts than wild-type ones. These results suggest that pknA is required for both normal cellular growth and differentiation of Anabaena PCC 7120.     

Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes

In the filamentous blue-green alga Anabaena 7120, aerobic nitrogen fixation is linked to the differentiation of specialized cells called heterocysts. In order to study control of heterocyst development and nitrogen fixation in Anabaena, we have used cloned fragments of the Klebsiella pneumoniae nitrogen fixation (nif) genes as probes in DNA·DNA hybridizations with restriction endonuclease fragments of Anabaena DNA. Using this technique, we were able to identify and clone Anabaena nif genes, demonstrating the feasibility of using heterologous probes to identify genes for which no traditional genetic selection exists. From the patterns of hybridization observed, we deduced that although DNA sequence homology has been retained between some of the nif genes of these divergent organisms, the nif gene order has been rearranged.     

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.     

A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated

The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.1] are shown to be contained within a 7.5-kilobase-pair (kbp) HindIII fragment that has been cloned by plaque hybridization. The hybridization probe for the cyanobacterial gene was a recombinant plasmid containing the glnA gene from Escherichia coli K-12. Evidence that the cloned Anabaena fragment contains the glnA gene includes complementation of a glnA deletion mutant of E. coli and immunological identity of the enzyme produced by the cloned Anabaena fragment in E. coli with glutamine synthetase purified from Anabaena 7120. Heteroduplex analysis reveals 0.65 kbp of homology between the 7.5-kbp Anabaena 7120 fragment and an 11-kbp E. coli fragment that codes for E. coli glutamine synthetase. Studies of Anabaena glnA gene activity in E. coli suggest that the cyanobacterial gene is not repressible and that the Anabaena 7120 glutamine synthetase is not adenylylated in E. coli.     

The patA gene product, which contains a region similar to CheY of Escherichia coli, controls heterocyst pattern formation in the cyanobacterium Anabaena 7120.

In Anabaena 7120, heterocysts (cells specialized for nitrogen fixation) develop at the ends of filaments and at intervals within each filament. We have isolated a mutant Anabaena strain that develops heterocysts mostly at the ends of filaments. This mutant, PAT-1, grows poorly under nitrogen-fixing conditions. The wild-type gene that complements the mutation in PAT-1, called patA, was cloned and sequenced. The predicted PatA protein contains 379 amino acids distributed among three "domains" based on predictions of hydropathy and flexibility. The carboxyl-terminal domain is very similar to that of CheY and other response regulators in two-component regulatory systems in eubacteria. The patA mutation suppresses the multiheterocyst phenotype produced by extra copies of the wild-type hetR gene described previously, suggesting that PatA and HetR are components of the same environment-sensing regulatory circuit in Anabaena.     

高效灭蚊幼转基因蓝藻的研究——Ⅰ.蚊虫孳生地蓝藻的分离及对蚊幼虫适口性的观察

本文报告了两种蚊虫孳生地分布的蓝藻Synechocystks sp和Oscillatoria sp.的分离,并发现Anabaena sp.7120株可在城市污水中生长。以上三种蓝藻在自然水和培养条件下生活状态各不相同,但皆可作为蚊幼虫食物。其中,Oscillatoria sp.藻丝长而交织生长,藻体集结成块,仍然可为中华按蚊(Anopheles sinensis)幼虫取食。以含有大量固体悬浮颗粒及细菌、有机质的生活污水直接培养Anabaena sp. 7120,可为淡色库蚊(Culex pipien pallenns)幼虫有效取食,初步证明蓝藻在自然水中作为蚊幼虫食物的可能性。     

Molecular characterization of the terminal energy acceptor of cyanobacterial phycobilisomes.

Cyanobacteria harvest light energy through multimolecular structures, the phycobilisomes, regularly arrayed at the surface of the photosynthetic membranes. Phycobilisomes consist of a central core from which rods radiate. A large polypeptide (LCM, 75-120 kDa) is postulated to act both as terminal energy acceptor and as a linker polypeptide that stabilizes the phycobilisome architecture. We report here the characterization of the gene (apcE) that encodes this LCM polypeptide in Calothrix sp. PCC 7601. It is located upstream from the genes encoding the major components of the phycobilisome core (allophycocyanin) and is part of the same operon. The deduced amino acid sequence shows that the N-terminal region of LCM shares homology with the other phycobiliprotein subunits and thus constitutes the chromoprotein domain. The other part of the molecule is made up of four repeated domains that are highly homologous to the N-terminal regions of the phycocyanin rod linker polypeptides. The predicted secondary structure of the different domains of the LCM is discussed in relation to the different roles and properties of this large molecule.     

Artificial mobile DNA element constructed from the EcoRI endonuclease gene.

There exist several examples of mobile group I introns. These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes. Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless recipient DNA molecule, we reasoned that it should in principle be possible to construct artificially mobile DNA sequences. We have constructed an artificial mobile element from the gene for the restriction enzyme EcoRI that is capable of site-specific insertion at rates near those of authentic mobile introns. The generality of the mobility mechanism may enable high-efficiency targeted gene replacements or disruptions in a variety of organisms.     

Anabaena sensory rhodopsin is a light-driven unidirectional rotor

The implementation of multiconfigurational quantum chemistry methods into a quantum-mechanics/molecular-mechanics protocol has allowed the construction of a realistic computer model for the sensory rhodopsin of the cyanobacterium Anabaena PCC 7120. The model, which reproduces the absorption spectra of both the all-trans and 13-cis forms of the protein and their associated K and L intermediates, is employed to investigate the light-driven steps of the photochromic cycle exhibited by the protein. It is found that the photoisomerizations of the all-trans and 13-cis retinal chromophores occur through unidirectional, counterclockwise 180° rotations of the =C14-C15= moiety with respect to the Lys210-linked end of the chromophore axis. Thus, the sequential interconversions of the all-trans and 13-cis forms during a single photochromic cycle yield a complete (360°) unidirectional rotation of the =C14-C15= moiety. This finding implies that Anabaena sensory rhodopsin is a biological realization of a light-driven molecular rotor.     

Tissue- and site-specific DNA recombination in transgenic mice.

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries.