Random genome deletion methods applicable to prokaryotes.

Through their enabling of simultaneous identification of multiple non-essential genes in a genome, large-segment genome deletion methods are an increasingly popular approach to minimize and tailor microbial genomes for specific functions. At present, difficulties in identifying target regions for deletion are a result of inadequate knowledge to define gene essentiality. Furthermore, with the majority of predicted open reading frames of completely sequenced genomes still annotated as putative genes, essential or important genes are found scattered throughout the genomes, limiting the size of non-essential segments that can be safely deleted in a single sweep.

Regulation of the expression of phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) genes in Corynebacterium glutamicum R.

The phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses the transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. In Corynebacterium glutamicum R, the genes ptsH and ptsI encode general components of the PTS, and genes ptsF, ptsS and ptsG each encode fructose-, sucrose- and glucose-specific components of the PTS, respectively. In this study, we examined the mRNA levels of the pts genes in the presence or absence of PTS sugars.

DivS, a novel SOS-inducible cell-division suppressor in Corynebacterium glutamicum.

DNA damage-induced SOS response elicits the induction of cell-division suppressor as well as DNA repair genes. In Gram-positive bacteria, cell-division suppressor genes, so far characterized from Bacillus subtilis (yneA) and Mycobacterium tuberculosis (rv2719c), share limited homology, but are both located in the vicinity of lexA on their respective genomes. Using this proximity to lexA, Corynebacterium glutamicum R divS (cgR1759) was identified as an SOS-inducible cell-division suppressor in this study.

Site-directed integration system using a combination of mutant lox sites for Corynebacterium glutamicum.

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an optimal C. glutamicum genome, a precise site-directed gene integration method was developed by using a pair of mutant lox sites, one a right element (RE) mutant lox site and the other a left element (LE) mutant lox site.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions.

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA).

Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions.

A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions.

Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R.

The complete genome sequence of Corynebacterium glutamicum strain R was determined to allow its comparative analysis with other corynebacteria. The biology of corynebacteria was explored by refining the definition of the subset of genes that constitutes the corynebacterial core as well as those characteristic of saprophytic and pathogenic ecological niches. In addition, the relative scarcity of corynebacterial sigma factors and the plasticity of their two-component system machinery reflect their relatively exacting nutritional requirements and reduced membrane-associated and secreted proteins.

Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum.

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2-186 kb). A total of 393.

Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants--stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco.

We tested the hypothesis that ferredoxin (Fd) limits the activity of cyclic electron flow around PSI (CEF-PSI) in vivo and that the relief of this limitation promotes the non-photochemical quenching (NPQ) of Chl fluorescence. In transplastomic tobacco (Nicotiana tabacum cv Xanthi) expressing Fd from Arabidopsis (Arabidopsis thaliana) in its chloroplasts, the minimum yield (F(o)) of Chl fluorescence was higher than in the wild type. F(o) was suppressed to the wild-type level upon illumination with far-red light, implying that the transfer of electrons by Fd-quinone oxidoreductase (FQR) from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants.

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts.

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba.

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach.

Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita APX maintains the electron flux through the water-water cycle in transplastomic tobacco plants.

We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light (>640 nm), 250 micromol photons m(-2) s(-1) in the absence of HCO3-] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities.

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence.

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C.

Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions.

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C.

Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves.

We tested the hypothesis that plants grown under high light intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI) compared with plants grown under low light (LL-plants). To evaluate the activity of CEF-PSI, the relationships between photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants which had been grown under different light intensities (150 and 1,100 micromol photons m(-2) s(-1), respectively) and with different amounts of nutrients supplied. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow.

Multiple large segment deletion method for Corynebacterium glutamicum.

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome.

Cre/loxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum.

Genome rearrangement is an increasingly important technique to facilitate the understanding of genome functions. A Cre/loxP-mediated deletion system for large-scale genome rearrangements in Corynebacterium glutamicum was developed. By comparative analysis of C.

A new insertion sequence, IS14999, from Corynebacterium glutamicum.

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence.

Enhancement of cyclic electron flow around PSI at high light and its contribution to the induction of non-photochemical quenching of chl fluorescence in intact leaves of tobacco plants.

Non-photochemical quenching (NPQ) of Chl fluorescence is a mechanism for dissipating excess photon energy and is dependent on the formation of a DeltapH across the thylakoid membranes. The role of cyclic electron flow around photosystem I (PSI) (CEF-PSI) in the formation of this DeltapH was elucidated by studying the relationships between O2-evolution rate [V(O2)], quantum yield of both PSII and PSI [Phi(PSII) and Phi(PSI)], and Chl fluorescence parameters measured simultaneously in intact leaves of tobacco plants in CO2-saturated air. Although increases in light intensity raised V(O2) and the relative electron fluxes through both PSII and PSI [Phi(PSII) x PFD and Phi(PSI) x PFD] only Phi(PSI) x PFD continued to increase after V(O2) and Phi(PSII) x PFD became light saturated.

Inhibitor-associated transposition events in Corynebacterium glutamicum.

In up to 100% of all bacteria grown in the presence of initially inhibitory concentrations of five diverse inhibitors, an extra copy of the resident insertion element IS 31831 was found in specific chromosomal regions, the sites of which apparently depended on the inhibitor used. Thus, in nine out of nine independently isolated cyanide-associated transpositions, the acquired copy was located within an ORF encoding a protein related to the hypothetical but conserved protein YeiH of Escherichia coli. A putative Sox box upstream of the yeiH gene implicates superoxide as a potential regulator of the gene, a possibility further supported by the finding that superoxide dismutase (SodA) is overexpressed in cells cultured in cyanide-containing medium.

Ab initio study on the structures of fluorinated formates and hydrogen abstraction reaction with OH radical.

The conformational potential energy surfaces for mono- and difluoromethyl formate have been determined by using a modified G2(MP2) level of calculations. The structures and vibrational frequencies for the conformers of mono- and difluoromethyl formate have been reported. The hydrogen abstraction reaction channels between these two formates and OH radicals have been studied at the same level of theory.

Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants.

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured.

Structure of the urease operon of Corynebacterium glutamicum.

Urease activity of Corynebacterium glutamicum results in a rapid pH increase upon addition of urea to the growth medium. The urease operon C. glutamicum was isolated of and sequenced.

A periplasmic, alpha-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake.

Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified >2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to alpha-type CAs.

The role of structural intersubunit microheterogeneity in the regulation of the activity in hysteresis of ribulose 1, 5-bisphosphate carboxylase/oxygenase.

Many enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits.