Elzbieta Kraszewska

Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates

A cDNA corresponding to the At3g26690 gene, which encodes a Nudix protein (AtNUDT13) with predicted mitochondrial localization, was isolated from an Arabidopsis thaliana library. The 202 amino acid AtNUDT13 polypeptide was overexpressed in Escherichia coli and purified to homogeneity. The preferred substrate for this hydrolase was diadenosine hexaphosphate (Ap6A), with Km and kcat/Km values of 0.61 mm and 16.0 × 103 m–1·s−1, respectively. Optimal activity was at alkaline pH (8.5) with Mg2+ (5 mm) as the cofactor. MS analysis revealed that the products of diadenosine hexaphosphate hydrolysis were ADP and adenosine tetraphosphate. Diadenosine pentaphosphate and adenosine tetraphosphate were additional substrates, but diadenosine tetraphosphate and diadenosine triphosphate, adenosine nucleotides, diphosphoinositol polyphosphate and phosphoribosyl pyrophosphate were not hydrolyzed. Chemical crosslinking and size exclusion chromatography demonstrated that the protein exists as a monomer in solution. Subcellular localization studies indicated that the AtNUDT13 protein is targeted to the mitochondria. This is the first description of a plant pyrophosphatase catalyzing the hydrolysis of long‐chain diadenosine polyphosphates: molecules with multiple biological activities.

Transient and stable transformation of wheat with DNA preparations delivered by a biolistic method

An optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PDS 1000/He to deliver foreign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo- derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.

Characterization of the early synthesized DNA in germinating Triticum aestivum embryos

Abstract A radioactive DNA preparation was isolated from the post-mitochondrial supernatant fraction of thymidine-[ 14 C] fed wheat embryos. The isolated s DNA preparation was similar to cytoplasmic non-mitochondrial DNA of other eukaryotic cells. The buoyant density and frequency of pyrimidine nucleotide clusters found for the s DNA were, essentially, the same as those found for the nuclear DNA. In contrast to DNA that can be leaked from nuclei or other DNA-containing organelles, the s DNA is firmly bound to a protein component. At an early germination stage (6–12 hr), the s DNA is the only newly-synthesized DNA fraction that can be isolated from the embryo homogenate. Considerable synthesis of nuclear and organellar DNA starts 18 hr after the beginning of germination, just prior to the first maximum of the cell divisions. It is concluded that wheat embryo cells contain cytoplasmic non-mitochondrial DNA and are able to resume its synthesis at an early germination stage, prior to the first post-dormant round of nuclear DNA replication.

NudC Nudix hydrolase from Pseudomonas syringae, but not its counterpart from Pseudomonas aeruginosa, is a novel regulator of intracellular redox balance required for growth, motility and biofilm formation

Nudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria.

Nudix‐type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa

The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5′‐end of triphosphorylated RNA transcripts. Trans‐complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down‐regulating production of virulence‐associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild‐type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

Tobacco BY-2 cells excise both 3-methyladenine and 7-methylguanine from methylated DNA

Two of the major products in DNA resulting from exposure to alkylating agents are 7-methylguanine and 3-methyladenine. N-methylpurine-DNA-glycosylase is required for excision of these lesions. Recently, the 3-methyladenine-DNA-glycosylase gene of Arabidopsis thaliana was cloned and shown to be involved only in repair of 3-methyladenine. In BY-2 tobacco cells, we showed an enzymatic activity which excised both 3-methyladenine and 7-methylguanine from methylated DNA.