Carbon cycling: the prokaryotic contribution.

Although the debate continues, the concept of global warming as a consequence of the increased production of 'greenhouse gases' via human activities is now widely accepted. The role of microbes, especially the prokaryotes, in the formation, trapping and retention of 'greenhouse gases' has, for the most part, been overlooked. The future requires that we pay close attention to these organisms for possible solutions to adverse global changes.

Spectroscopic evidence for amyloid-like interfacial self-assembly of hydrophobin Sc3.

Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecular structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Secondary-structure prediction indicated that hydrophobin Sc3 is an all-beta protein. Many amyloidogenic proteins self-assemble into insoluble amyloid fibrils while undergoing a change to an all-beta conformation. In this study we show that two dyes, thioflavin T, and Congo red, which are widely used for specific detection of stacked beta sheets, interact with Sc3 assemblies in the same way as with the amyloid beta-sheet fibrils. We conclude that Sc3, and probably other hydrophobins too, self-assemble at interfaces in the same manner as amyloidogenic proteins, i.e., through beta-sheet stacking.

Sc3p hydrophobin organization in aqueous media and assembly onto surfaces as mediated by the associated polysaccharide schizophyllan.

Sc3p is the major hydrophobin produced and excreted by the wood-rotting fungus Schizophyllum commune when grown as a monokaryon in culture. In addition to Sc3p, several lower molecular weight proteins of unknown function and a high molecular weight polysaccharide, schizophyllan, are present in this fungal culture medium. Previous studies in our laboratory indicated that schizophyllan and hydrophobin, as produced in the culture supernatant, work in concert to ultimately produce stable films on hydrophilic substrates. We report a new procedure for isolation of hydrophobin that does not require treatment with strong organic acids. This procedure allows a systematic evaluation of the importance of the individual hydrophobin and schizophyllan components in solution stabilization and film formation from a conformational state more representative of that found during fungal production. Experimental results indicate that schizophyllan stabilizes small hydrophobin multimers in aqueous solution. In addition, schizophyllan is necessary for subsequent hydrophobin assembly into films on hydrophilic surfaces. By contrast, hydrophobin alone is sufficient for thin film assembly onto a hydrophobic surface; however, hydrophobin aggregation at high protein concentration in the absence of schizophyllan may limit the efficiency of film formation. A model has been proposed in which the polysaccharide acts as a hydrophilic stabilizer of hydrophobin in solution preventing self-aggregation. The schizophyllan-stabilized complex may also allow conformational mobility for lateral reorganization during deposition and film coalescence.

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro.

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15-20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per microg of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.

Expression of the copper metallothionein CUPI from Saccharomyces cerevisiae in the cyanobacterium Synechococcus R2-PIM8(smtA).

The coding sequence for Saccharomyces cerevisiae copper metallothionein (CUPI), the protein responsible for enhanced sequestration of Cu2+ in yeast, was placed under the control of an inducible synthetic Escherichia coli promoter in the cyanobacterial vector pTrcIK. Strain R2-PIM8(smtA) of Synechococcus sp. PCC 7942 was transformed with the resulting construct pMcK2, and the yeast CUPI gene was integrated into its genome via homologous recombination. The pMcK2 plasmid directed the synthesis of a protein product of the expected size in an in vitro E. coli transcription/translation system. In the transgenic cyanobacteria, the integrated CUPI gene was transcribed and produced a protein product with the expected metallothionein characteristics, as determined by 109Cd2+ binding assays. At this level of expression, the yeast metallothionein, although functional, did not increase the tolerance range of the transgenic Synechococcus to Cu2+ or Cd2+ beyond that of the untransformed R2-PIM8(smtA).

Partial purification and characterization of the DNA polymerase from the cyanelles of Cyanophora paradoxa.

A DNA polymerase was partially purified and characterized from the photosynthetic organelles (cyanelles) of the protist, Cyanophora paradoxa. While cyanelles have several cyanobacterial features, such as a lysozyme-sensitive cell wall, unstacked thylakoids and light harvesting phycobilisomes, their genome size and structure resemble those of chloroplasts, suggesting that cyanelles occupy a unique intermediate position between chloroplasts and their phylogenetic ancestors, the cyanobacteria. When comparing the biochemical characteristics of the cyanelle DNA polymerase to those of its counterparts from higher plant chloroplasts and from a cyanobacterium, it is clear that the cyanelle enzyme resembles chloroplast DNA polymerases which are eukaryotic gamma-type enzymes.

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 repair 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.

Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease.

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis.

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.

Purification and characterization of a gamma-like DNA polymerase from Chenopodium album L.

A DNA polymerase activity from mitochondria of the dicotyledonous angiosperm Chenopodium album L. was purified almost 9000 fold by successive column chromatography steps on DEAE cellulose, heparin agarose and ssDNA cellulose. The enzyme was characterized as a gamma-class polymerase, based on its resistance to inhibitors of the nuclear DNA polymerase alpha and its preference for poly(rA).(dT)12-18 over activated DNA in vitro. The molecular weight was estimated to be 80,000-90,000. A 3' to 5' exonuclease activity was found to be tightly associated with the DNA polymerase activity through all purification steps. This is the first report of an association between a DNA polymerase and an exonuclease activity in plant mitochondria.

In situ assay of ribulose-1,5-bisphosphate carboxylase/oxygenase in Thiobacillus neapolitanus.

Cells permeabilized with chloroform yielded ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activities nearly equal to those of cell extracts, thus indicating that both cytoplasmic and carboxysomal RuBisCO are functional in situ. The carboxysomal and cytoplasmic RuBisCO both form the CO2-Mg2(+)-enzyme ternary complex, as evidenced by stabilization with 2-C-carboxy-D-arabinitol-1,5-bisphosphate (CABP), a potent competitive inhibitor of RuBisCO. The data are consistent with the hypothesis that the carboxysome is functional in carbon dioxide fixation.

Purification and characterization of a DNA polymerase from the cyanobacterium Anacystis nidulans R2.

A DNA polymerase has been highly purified from Anacystis nidulans R2. Electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels revealed that the final fraction contains three bands of Mr 107,000, 93,000, and 51,000, respectively. Analysis of purified DNA polymerase activity in situ indicates that of the three polypeptides the Mr 107,000 species has the catalytic activities. The native molecular weight of the enzyme was estimated by glycerol gradient sedimentation to be 100,000. The enzyme has an absolute requirement for a divalent cation. Mg2+ can be replaced with Mn2+, but the DNA polymerase is less active. Potassium chloride stimulates the enzyme, while potassium phosphate has no apparent effect. The enzyme is active over a pH range from 7.5 to 9.5 in 50mM Tris-HCl buffer. The ability of the cyanobacterial DNA polymerase to use activated DNA as a template, its associated 3'----5' and 5'----3' exonuclease activities, as well as its resistance to N-ethylmaleimide, dideoxynucleotides, arabinosyl-CTP and aphidicolin suggest a similarity between this enzyme and E. coli DNA polymerase I. This is the first characterization of a DNA polymerase from a cyanobacterium.

Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max.

A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg2+ or Mn2+. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.

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 alpha. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases gamma. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.

An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants.

DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome.

Sequence analysis on microcomputers.

Overall, each of the program packages performed their tasks satisfactorily. For analyses where there was a well-defined answer, such as a search for a restriction site, there were few significant differences between the program sets. However, for tasks in which a degree of flexibility is desirable, such as homology or similarity determinations and database searches, DNASTAR consistently afforded the user more options in conducting the required analysis than did the other two packages. However, for laboratories where sequence analysis is not a major effort and the expense of a full sequence analysis workstation cannot be justified, MicroGenie and IBI-Pustell offer a satisfactory alternative. MicroGenie is a polished program system. Many may find that its user interface is more "user friendly" than the standard menu-driven interfaces. Its system of filing sequences under individual passwords facilitates use by more than one person. MicroGenie uses a hardware device for software protection that occupies a card slot in the computer on which it is used. Although I am sympathetic to the problem of software piracy, I feel that a less drastic solution is in order for a program likely to be sharing limited computer space with other software packages. The IBI-Pustell package performs the required analysis functions as accurately and quickly as MicroGenie but it lacks the clearness and ease of use. The menu system seems disjointed, and new or infrequent users often find themselves at apparent "dead-end menus" where the only clear alternative is to restart the entire program package. It is suggested from published accounts that the user interface is going to be upgraded and perhaps when that version is available, use of the system will be improved. The documentation accompanying each package was relatively clear as to how to run the programs, but all three packages assumed that the user was familiar with the computational techniques employed. MicroGenie and IBI-Pustell further complicated their documentation by mixing instructions for the version based on floppy disk operation with that for the hard disk version.(ABSTRACT TRUNCATED AT 400 WORDS)

Organellar DNA synthesis in permeabilized soybean cells.

Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L-α-lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase α inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.

Heterotrophic carbon metabolism by Beggiatoa alba.

The assimilation and metabolism of CO(2) and acetate by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(2) for growth, the addition of excess CO(2) (as NaHCO(3)) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to (14)CO(2) by B. alba. The apparent V(max) values for combined assimilation and oxidation of [2-(14)C]acetate by B. alba were 126 to 202 nmol min(-1) mg of protein(-1) under differing growth conditions. The V(max) values for CO(2) assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min(-1) mg of protein(-1), respectively. The low V(max) values for CO(2) assimilation, coupled with the high V(max) values for acetate oxidation, suggested that the required CO(2) was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by B. alba for the fixation of CO(2). From 61 to 73% of the [(14)C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-(14)C]acetate was incorporated into poly-beta-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-beta-hydroxybutyric acid. Succinate and CO(2) were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-beta-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-(14)C]acetate assimilation. Glutamate and aspartate were the first stable (14)CO(2) fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable (14)CO(2) fixation products over a 30-min period. The CO(2) fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO(2) fixation mechanisms are absent from B. alba B18LD and that the CO(2) and acetate metabolism pathways are probably linked.

Icosahedral inclusions (carboxysomes) of Nitrobacter agilis.

The icosahedral bodies of Nitrobacter agilis are about 120 nm in diameter and, as viewed by electron microscopy, consist of an outer shell enclosing 10-nm particles. The inner 10-nm particle is the enzyme D-ribulose 1,5-bisphosphate carboxylase. The bodies isolated from cells incubated 1 month without nitrite had a specific activity for the enzyme of 0.54 mu mol of CO2 fixed per min per mg of protein.