Thermo-desorption measurements during N-doped Ge-rich Ge2Sb2Te5crystallization.

Ge-rich Ge2Sb2Te5(GGST) is considered as one of the best candidates for industrial phase change memory production. GGST memory cells are generally embedded with Si or Ti nitride layers to prevent oxidation, as it leads to an undesired decrease of the GGST crystallization temperature. Furthermore, GGST films are usually doped with elements such as N, C, O, or Bi, aiming to delay GGST crystallization during the fabrication process as well as during memory cell operation. In this work, ultrahigh vacuum thermal desorption spectroscopy (TDS) was performed during isochronal annealing of a N-doped GGST film covered by a 10 nm-thick TiNxlayer. Desorption is observed before GGST crystallization, but the comparison between TDS andin situx-ray diffraction measurements shows that the main desorption peak, observed between 653 K and 703 K, occurs after GGST full crystallization. The most prominent desorbing species are Ar, N2, H2, and H. These results show that the TiNxpolycrystalline layer cannot prevent N atoms from leaving the GGST layer during annealing, suggesting a progressive change of the N-doped GGST chemical composition during thermal annealing and crystallization.

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.

Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic alpha-mannosyltransferases.

A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families. The sequences of several bacterial alpha-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA). These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods. However, a region spanning approx. 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified. These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases. This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features. It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases [Henrissat, B. (1991) Biochem. J. 280, 309-316].

Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum.

A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase enzyme, which is responsible for the transfer of an alpha-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial alpha-mannosyltransferases have a short COOH-terminal amino acid sequence in common.

Sequence-specific DNA modification in Acetobacter xylinum.

Two cryptic plasmids have been discovered in Acetobacter xylinum B42 and in its derivative PEA-1, a cellulose defective mutant. These two plasmids were designated pAX1 and pAX2 (50 and 105 kb in size, respectively). A restriction map was constructed for pAX1. Attempts to cure these plasmids were unsuccessful. Enzyme restriction analysis showed that these plasmids contain protected EcoRI and ApoI sites. Using Southern blot and hybridization techniques, the protection was extended to chromosomal DNA. Enzyme restriction analysis of several plasmids, from different origins and containing different incompatibility groups, isolated from strain PEA-1 also showed EcoRI and ApoI protection. The presence of modifications on specific sequences was not found in A. xylinum 8747. These results strongly suggest the presence of a modification system in A. xylinum B42 that recognizes the tetranucleotide 5'-AATT.

Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Cabañas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.