Ploidy in cyanobacteria

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.     

Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

Sucrose may play an additional role to that of an osmolyte in Synechocystis sp. PCC 6803 salt-shocked cells.

The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: D-fructose-6-phosphate 2-alpha-D-glucosyltransferase; EC 2.4.1.14), the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.     

Tocopherols protect Synechocystis sp. strain PCC 6803 from lipid peroxidation

Tocopherols (vitamin E) are lipid-soluble antioxidants synthesized only by photosynthetic eukaryotes and some cyanobacteria, and have been assumed to play important roles in protecting photosynthetic membranes from oxidative stress. To test this hypothesis, tocopherol-deficient mutants of Synechocystis sp. strain PCC 6803 (slr1736 and slr1737 mutants) were challenged with a series of reactive oxygen species-generating and lipid peroxidation-inducing chemicals in combination with high-light (HL) intensity stress. The tocopherol-deficient mutants and wild type were indistinguishable in their growth responses to HL in the presence and absence of superoxide and singlet oxygen-generating chemicals. However, the mutants showed enhanced sensitivity to linoleic or linolenic acid treatments in combination with HL, consistent with tocopherols playing a crucial role in protecting Synechocystis sp. strain PCC 6803 cells from lipid peroxidation. The tocopherol-deficient mutants were also more susceptible to HL treatment in the presence of sublethal levels of norflurazon, an inhibitor of carotenoid synthesis, suggesting carotenoids and tocopherols functionally interact or have complementary or overlapping roles in protecting Synechocystis sp. strain PCC 6803 from lipid peroxidation and HL stress.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.     

The rnb gene of Synechocystis PCC6803 encodes a RNA hydrolase displaying RNase II and not RNase R enzymatic properties

Cyanobacteria are photosynthetic prokaryotic organisms that share characteristics with bacteria and chloroplasts regarding mRNA degradation. Synechocystis sp. PCC6803 is a model organism for cyanobacteria, but not much is known about the mechanism of RNA degradation. Only one member of the RNase II-family is present in the genome of Synechocystis sp PCC6803. This protein was shown to be essential for its viability, which indicates that it may have a crucial role in the metabolism of Synechocystis RNA. The aim of this work was to characterize the activity of the RNase II/R homologue present in Synechocystis sp. PCC6803. The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates. When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics. This is the first reported case where when only one member of the RNase II/R family exists it displays RNase II and not RNase R characteristics.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

Sequence, regulation, and functions of fis in Salmonella typhimurium.

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.     

Identification of an atypical membrane protein involved in the formation of protein disulfide bonds in oxygenic photosynthetic organisms

The evolution of oxygenic photosynthesis in cyanobacteria nearly three billion years ago provided abundant reducing power and facilitated the elaboration of numerous oxygen-dependent reactions in our biosphere. Cyanobacteria contain an internal thylakoid membrane system, the site of photosynthesis, and a typical Gram-negative envelope membrane system. Like other organisms, the extracytoplasmic space in cyanobacteria houses numerous cysteine-containing proteins. However, the existence of a biochemical system for disulfide bond formation in cyanobacteria remains to be determined. Extracytoplasmic disulfide bond formation in non-photosynthetic organisms is catalyzed by coordinated interaction between two proteins, a disulfide carrier and a disulfide generator. Here we describe a novel gene, SyndsbAB, required for disulfide bond formation in the extracytoplasmic space of cyanobacteria. The SynDsbAB orthologs are present in most cyanobacteria and chloroplasts of higher plants with fully sequenced genomes. The SynDsbAB protein contains two distinct catalytic domains that display significant similarity to proteins involved in disulfide bond formation in Escherichia coli and eukaryotes. Importantly, SyndsbAB complements E. coli strains defective in disulfide bond formation. In addition, the activity of E. coli alkaline phosphatase localized to the periplasm of Synechocystis 6803 is dependent on the function of SynDsbAB. Deletion of SyndsbAB in Synechocystis 6803 causes significant growth impairment under photoautotrophic conditions and results in hyper-sensitivity to dithiothreitol, a reductant, whereas diamide, an oxidant had no effect on the growth of the mutant strains. We conclude that SynDsbAB is a critical protein for disulfide bond formation in oxygenic photosynthetic organisms and required for their optimal photoautotrophic growth.     

Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase-coupled transition of E. coli phenotype is discontinuous.     

Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis.     

Transition from exponential to linear photoautotrophic growth changes the physiology of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Phototrophic microorganisms like cyanobacteria show growth curves in batch culture that differ from the corresponding growth curves of chemotrophic bacteria. Instead of the usual three phases, i.e., lag-, log-, and stationary phase, phototrophs display four distinct phases. The extra growth phase is a phase of linear, light-limited growth that follows the exponential phase and is often ignored as being different. Results of this study demonstrate marked growth phase-dependent alterations in the photophysiology of the cyanobacterium Synechocystis sp. PCC 6803 between cells harvested from the exponential and the linear growth phase. Notable differences are a gradual shift in the energy transfer of the light-harvesting phycobilisomes to the photosystems and a distinct change in the redox state of the plastoquinone pool. These differences will likely affect the result of physiological studies and the efficiency of product formation of Synechocystis in biotechnological applications. Our study also demonstrates that the length of the period of exponential growth can be extended by a gradually stronger incident light intensity that matches the increase of the cell density of the culture.     

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

阻断集胞藻6803 PHB合成途径提高胞内NADPH含量

蓝藻是探索利用太阳能生产化学品的重要微生物,但产量低限制了蓝藻化学品的工业应用。提高宿主还原力水平是提高微生物合成化学品产量的重要手段。为提高集胞藻细胞内NADPH含量,利用同源重组方法,获得敲除聚羟基丁酸酯PHB合酶编码基因phaC和phaE的集胞藻Synechocystis sp. PCC 6803突变体S.DphaC&E。PCR结果证明突变体S.DphaC&E基因组中phaC和phaE已完全被氯霉素抗性基因取代。生长曲线结果显示S.DphaC&E的生长与野生型无明显差异,说明敲除phaC和phaE对     

Effect of plant growth regulators on the cellular growth and levels of intracellular protein,starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 M 2,4-dichlorophenoxyacetic acid, 0.5 M 6-benzylaminopurine and 0.5 M Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.