Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes.

The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.     

RNA polymerase subunit homology among cyanobacteria, other eubacteria and archaebacteria.

RNA polymerase purified from vegetative cells of the cyanobacterium Anabaena sp. strain PCC 7120 contains a dissociable sigma factor and a core of five subunits: the beta', beta, and two alpha subunits characteristic of all eubacteria and an additional 66,000-molecular-weight polypeptide called gamma. Fifteen of fifteen strains of unicellular and filamentous cyanobacteria tested contained a serologically related gamma protein. Antiserum to gamma reacted with Escherichia coli beta' and the A subunit of RNA polymerase of the archaebacterium Sulfolobus acidocaldarius. Thus the evolution of the RNA polymerase beta' subunit has followed different paths in three groups of procaryotes: cyanobacteria, other eubacteria, and archaebacteria.     

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene.

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.     

Multiple rpoD-related genes of cyanobacteria.

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoD1, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal sigma factors of eubacterial strains. The rpoD1 gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

Chemoheterotrophic Growth of the Cyanobacterium Anabaena sp. Strain PCC 7120 Dependent on a Functional Cytochrome c Oxidase

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.     

Amino acid transport systems required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120.

Uptake of 16 amino acids by the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was characterized with regard to kinetic parameters of transport, intracellular accumulation of the transported amino acids, and sensitivity of the transport process to energy metabolism inhibitors. Mutants resistant to certain toxic analogs of some amino acids were isolated that were impaired in amino acid transport. Results obtained in this study, together with those reported previously (A. Herrero and E. Flores, J. Biol. Chem. 265:3931-3935, 1990), suggest that there are at least five amino acid transport systems in strain PCC 7120: one high-affinity, active system for basic amino acids; one low-affinity, passive system for basic amino acids; two high-affinity, active systems with overlapping, but not identical, specificities for neutral amino acids; and one putative system for acidic amino acids. Some of the amino acid transport mutants were impaired in diazotrophic growth. These mutants were unable to develop a normal percentage of heterocysts and normal nitrogenase activity in response to nitrogen stepdown. Putative roles for the amino acid transport systems in uptake of extracellular amino acids, recapture of amino acids that have leaked from the cells, and intercellular transfer of amino acids in the filaments of Anabaena sp. strain PCC 7120 are discussed.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.     

Evidence that sigma factors are components of chloroplast RNA polymerase.

Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae.     

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.     

Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti.

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhizobium meliloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins. Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins. The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.     

The rpoD1 gene of Synechococcus sp. strain PCC 7942 encodes the principal sigma factor of RNA polymerase

RNA polymerase was purified from the unicellular cyanobacterium, Synechococcus sp. strain PCC 7942, and found to be associated with a 52 kilodalton (kDa) polypeptide. The determined N-terminal sequence of the polypeptide was identical to the predicted amino-acid sequence of the rpoD1 gene product. Furthermore, the rpoD1 gene is suggested to be indispensable for viability by the inability to disrupt the gene. These results indicate that the rpoD1 gene product is the principal sigma factor of RNA polymerase.     

Molecular cloning and sequencing of the sodB gene from a heterocystous cyanobacterium Anabaena sp. PCC 7120

Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition.     

异形胞分化蓝细菌dnaA温度敏感型突变体的构建

以能分化异形胞的蓝细菌(Anabaenasp.PCC7120)为材料,采用重组PCR在体外对控制DNA复制起始的dnaA基因进行定点突变后克隆到整合质粒中,再通过三亲本杂交将整合质粒转移到Anabaena PCC7120中,以分离和筛选温度敏感型突变体。结果成功获得Anabaena PCC 7120 dnaA高温敏感性突变体。研究表明,利用重组PCR技术可在体外实现对Anabaena PCC 7120的dnaA的定点突变,并可通过同源重组双交换成功实行整合质粒中突变基因对野生型基因的置换,使突变基因插入到细胞染色体中,进而成功构建温度敏感型突变菌株。