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.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.     

Inhibition of H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase by strophanthidin.

Studies of the effect of strophanthidin on H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H(+)-transporting ATPase, Ca(2+)-transporting ATPase, as well as H+/K(+)-transporting ATPase and Na+/K(+)-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca(2+)-transporting ATPase and Na+/K(+)-transporting ATPase.     

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.     

Calcium carbonate formation by Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807

Precipitation of CaCO3 catalyzed by the growth and physiology of cyanobacteria in the genus Synechococcus represents a potential mechanism for sequestration of atmospheric CO2 produced during the burning of coal for power generation. Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807 were tested in microcosm experiments for their ability to calcify when exposed to a fixed calcium concentration of 3.4 mM and dissolved inorganic carbon concentrations of 0.5, 1.25 and 2.5 mM. Synechococcus sp. strain PCC 8806 removed calcium continuously over the duration of the experiment producing approximately 18.6 mg of solid phase calcium. Calcium removal occurred over a two-day time period when Synechococcus sp. strain PCC 8807 was tested and only 8.9 mg of solid phase calcium was produced. Creation of an alkaline growth environment catalyzed by the physiology of the cyanobacteria appeared to be the primary factor responsible for CaCO3 precipitation in these experiments.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942.

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.     

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.     

聚球藻PCC 7942氢酶的分离纯化及特性研究

报道了室温、空气环境下聚球藻Synechococcus sp.PCC7942氢酶的分离纯化.经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46U·mg^-1蛋白.纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa.该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH8.0.     

Iron stress responses in the cyanobacterium Synechococcus sp. PCC7942

In the present study, we describe the sequential events by which the cyanobacterium Synechococcus sp. PCC 7942 adapts to iron deficiency. In doing so, we have tried to elucidate both short and long-term acclimation to low iron stress in order to understand how the photosynthetic apparatus adjusts to low iron conditions. Our results show that after an initial step, where CP43' is induced and where ferredoxin is partly replaced by flavodoxin, the photosynthetic unit starts to undergo major rearrangements. All measured components of Photosystem I (PSI), PSII and cytochrome (Cyt) ƒ decrease relative to chlorophyll (Chl) a . The photochemical efficiencies of the two photosystems also decline during this phase of acclimation. The well-known drop in phycobilisome content measured as phycocyanin (PC)/Chl was not due to an increased degradation, but rather to a decreased rate of synthesis. The largest effects of iron deficiency were observed on PSI, the most iron-rich structure of the photosynthetic apparatus. In the light of the recent discovery of an iron deficiency induced CP43' ring around PSI a possible dual function of this protein as both an antenna and a quencher is discussed. We also describe the time course of a blue shift in the low temperature Chl emission peak around 715 nm, which originates in PSI. The shift might reflect the disassembly and/or degradation of PSI during iron deficiency and, as a consequence, PSI might under these conditions be found predominantly in a monomeric form. We suggest that the observed functional and compositional alterations represent cellular acclimation enabling growth and development under iron deficiency, and that growth ceases when the acclimation capacity is exhausted. However, the cells remain viable even after growth has ceased, since they resumed growth once iron was added back to the culture.     

The photoreduction of H(2)O(2) by Synechococcus sp. PCC 7942 and UTEX 625

It has been claimed that the sole H(2)O(2)-scavenging system in the cyanobacterium Synechococcus sp. PCC 7942 is a cytosolic catalase-peroxidase. We have measured in vivo activity of a light-dependent peroxidase in Synechococcus sp. PCC 7942 and UTEX 625. The addition of small amounts of H(2)O(2) (2.5 microM) to illuminated cells caused photochemical quenching (qP) of chlorophyll fluorescence that was relieved as the H(2)O(2) was consumed. The qP was maximal at about 50 microM H(2)O(2) with a Michaelis constant of about 7 microM. The H(2)O(2)-dependent qP strongly indicates that photoreduction can be involved in H(2)O(2) decomposition. Catalase-peroxidase activity was found to be almost completely inhibited by 10 microM NH(2)OH with no inhibition of the H(2)O(2)-dependent qP, which actually increased, presumably due to the light-dependent reaction now being the only route for H(2)O(2)-decomposition. When (18)O-labeled H(2)O(2) was presented to cells in the light there was an evolution of (16)O(2), indicative of H(2)(16)O oxidation by PS 2 and formation of photoreductant. In the dark (18)O(2) was evolved from added H(2)(18)O(2) as expected for decomposition by the catalase-peroxidase. This evolution was completely blocked by NH(2)OH, whereas the light-dependent evolution of (16)O(2) during H(2)(18)O(2) decomposition was unaffected.