Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Chloroplast and mitochondrial DNA polymerases from cultured soybean cells

DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT_12-18 over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase α. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases γ. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.     

Protein-based organelles in bacteria: carboxysomes and related microcompartments

Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.     

Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species

Background

The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO₂ fixation via the Calvin-Benson-Bassham cycle.

Methodology/Principal Findings

To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition.

Conclusions/Significance

These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.     

Attachment of ampicillin to expanded poly(tetrafluoroethylene): surface reactions leading to inhibition of microbial growth

The broad spectrum antibiotic, ampicillin (AM), was reacted to expanded poly (tetrafluoroethylene) (ePTFE) surfaces and resulted in the formation of antimicrobial surfaces effective against gram-positive, Staphylococcus aureus, Bacillus thuringiensis, and Enterococcus faecalis, and gram-negative, Escherichia coli, Pseudomonas putida, and Salmonella enterica bacteria. These ePTFE surface modifications were accomplished by utilization of microwave maleic anhydride (MA) plasma reactions leading to the formation of acid groups, followed by amidation reactions of heterofunctional NH 2/COOH-terminated polyethylene glycol (PEG). The final step, the attachment of AM to the PEG spacer, was achieved by amidation reactions between COOH-terminated PEG and NH 2 groups of AM. This approach protects the COOH-AM functionality and diminishes the possibility of hydrolysis of the antimicrobial active portion of AM. These studies also show that approximately 90% of AM molecules are still covalently attached to PEG-MA-ePTFE surfaces after exposure to the bacteria solutions. Even after a 24 h period, the AM volume concentration changes only from 2.25 to 2.04 microg/m3, and depending upon the bacteria type, the bacteria suspensions containing AM-PEG-MA-ePTFE specimens retain 85-99% of their initial optical density.     

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

Chloroplast DNA synthesis during the cell cycle in cultured cells of Nicotiana tabacum: inhibition by nalidixic acid and hydroxyurea

The effects of nalidixic acid and hydroxyurea on nuclear and chloroplast DNA formation in cultured cells of Nicotiana tabacum were investigated. At low concentrations (5 and 20 micrograms/ml) nalidixic acid, an inhibitor of DNA gyrase, exhibited a greater inhibitory effect on plastid DNA synthesis than on nuclear DNA formation. Since the plastid genome is a circular double-stranded DNA, this is consistent with the proven involvement of a DNA gyrase in the replication of closed circular duplex DNA genomes in procaryotic cells. At a high concentration of nalidixic acid (50 micrograms/ml), DNA synthesis in both the plastid and nuclear compartment was rapidly inhibited. Removal of the drug from the culture medium led to the resumption of DNA synthesis in 8 h. Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, also depresses nuclear as well as plastid DNA formation. Removal of hydroxyurea from the blocked cells leads to a burst of nuclear DNA synthesis, suggesting that the cells had been synchronized at the G1/S boundary. The recovery of plastid DNA synthesis occurs within the same time frame as that of nuclear DNA. However, whereas plastid DNA formation is then maintained at a constant rate, nuclear DNA synthesis reaches a peak and subsequently declines. These results indicate that the synthesis of plastid DNA is independent of the cell cycle events governing nuclear DNA formation in cultured plant cells.