Ionic-to-Electronic Conductivity Crossover in CdTe-AgI-As2Te3 Glasses: An 110mAg Tracer Diffusion Study.

Conductivity isotherms of (CdTe) x(AgI)0.5- x/2(As2Te3)0.5- x/2 glasses (0.0 ≤ x ≤ 0.15) reveal a nonmonotonic behavior with increasing CdTe content reminiscent of mixed cation effect in oxide and chalcogenide glasses. Nevertheless, the apparent similarity appears to be partly incorrect. Using 110mAg tracer diffusion measurements, we show that semiconducting CdTe additions produce a dual effect: (i) decreasing the Ag+ ion transport by a factor of ≈200 with a simultaneous increase of the diffusion activation energy and (ii) increasing the electronic conductivity by 1.5 orders of magnitude. Consequently, the conductivity minimum at x = 0.05 reflects an ionic-to-electronic transport crossover; the silver-ion transport number decreases by 3 orders of magnitude with increasing x.

Mercury Sulfide Dimorphism in Thioarsenate Glasses.

Crystalline mercury sulfide exists in two drastically different polymorphic forms in different domains of the P,T-diagram: red chain-like insulator α-HgS, stable below 344 °C, and black tetrahedral narrow-band semiconductor ß-HgS, stable at higher temperatures. Using pulsed neutron and high-energy X-ray diffraction, we show that these two mercury bonding patterns are present simultaneously in mercury thioarsenate glasses HgS-As2S3. The population and interconnectivity of chain-like and tetrahedral dimorphous forms determine both the structural features and fundamental glass properties (thermal, electronic, etc.). DFT simulations of mercury species and RMC modeling of high-resolution diffraction data provide additional details on local Hg environment and connectivity implying the (HgS2/2)m oligomeric chains (1 ≤ m ≤ 6) are acting as a network former while the HgS4/4-related mixed agglomerated units behave as a modifier.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY.

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl beta-glucoside and sucrose utilization respectively. Their RNA-binding activity is carried by the N-terminal domain (CAT), and is regulated by two similar C-terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl beta-glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT-PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP-mediated inhibition process was found to essentially involve His100 of LicT-PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by phosphorylated BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII-mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator.

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It belongs to a newly characterized family of bacterial regulators whose activity is controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). LicT contains an N-terminal RNA-binding domain (56 residues), and a PTS regulation domain (PRD, 221 residues) that is phosphorylated on conserved histidines in response to substrate availability. Replacement of both His207 and His269 with a negatively charged residue (aspartic acid) led to a highly active LicT variant that no longer responds to either induction or catabolite repression signals from the PTS. In contrast to wild type, the activated mutant form of the LicT regulatory domain crystallized easily and provided the first structure of a PRD, determined at 1.55 A resolution. The structure is a homodimer, each monomer containing two analogous alpha-helical domains. The phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners. The structure suggests important tertiary and quaternary rearrangements upon LicT activation, which could be communicated from the protein C-terminal end up to the RNA-binding domain.

Phosphotransfer functions mutated Bacillus subtilis HPr-like protein Crh carrying a histidine in the active site.

The Bacillus subtilis protein Crh exhibits strong similarity to HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr phosphorylated at His-15 can transfer its phosphoryl group to several EIIAs of the PTS for sugar transport and phosphorylation. In addition, it phosphorylates and activates transcriptional regulators containing PTS regulation domains (PRDs). In Gram-positive bacteria, it also controls the enzyme glycerol kinase. Since in Crh the active site His-15 of HPr is replaced with a glutamine, Crh was not able to carry out the catalytic and regulatory functions mediated by P approximately His-HPr. However, when Gln-15 of Crh was replaced with a histidine, Crh gained most of the catalytic and regulatory functions exerted by HPr. To allow CrhQ15H to efficiently phosphorylate and activate the PRD-containing antiterminator LicT, which controls the expression of the bgIS gene and the bgIPH operon, it was sufficient to express the crhQ15H allele under control of the spac promoter in monocopy. By contrast, to phosphorylate and activate glycerol kinase and to allow a ptsH deletion strain (devoid of HPr) to slowly grow on the non-PTS substrate glycerol and to efficiently utilize the PTS sugars glucose and mannitol, the crhQ15H allele had to be expressed from a multicopy plasmid.

Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis.

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.

Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system.

The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, the B. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity.

A ribonucleic antiterminator sequence (RAT) and a distant palindrome are both involved in sucrose induction of the Bacillus subtilis sacXY regulatory operon.

The Bacillus subtilis sacXY regulatory operon is involved in sucrose induction of the levansucrase sacB gene by an antitermination mechanism. In the presence of sucrose, the activated SacY antiterminator protein stabilizes the secondary structure of a ribonucleic antiterminator sequence (RAT) located in the leader region of the sacB transcript, and overlapping a rho-independent transcription terminator. Formation of the SacY-RAT complex prevents alternative formation of the terminator, allowing transcription of the downstream sequences. In the absence of sucrose, inhibition of SacY activity by SacX leads to termination of transcription. Expression of sacXY is also sucrose-inducible. This induction was previously shown to be mediated by SacY itself and/or SacT, another antiterminator involved in induction of genes belonging to a distinct sucrose pathway. These antiterminators are not activated at the same concentration of sucrose. We show here that sacXY induction occurs through activation of either SacY or SacT antiterminators, at their respective sucrose activation concentration. This result demonstrates a link between SacY- and SacT-mediated metabolic pathways. In addition, the sacXY leader region carries a RAT-like sequence, which however does not appear to overlap any apparent rho-independent transcription terminator. Site-directed mutagenesis experiments on this RAT-like sequence demonstrated its involvement in sucrose induction. Deletions generated in the sacXY leader region showed that a palindrome, located 100 nt downstream from the RAT-like sequence, also acts as a cis-acting element. Computer analysis of the leader RNA suggested that formation of the secondary structure of the RAT-like sequence and the palindrome could be mutually exclusive.

New beta-glucoside (bgl) genes in Bacillus subtilis: the bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog.

The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes. A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT. The nucleotide sequence of bglP showed a high degree of homology with the E. coli bglF gene (BglF is a beta-glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator). Complementation studies of an E. coli strain with a deletion of the bgl operon showed that BglP was a functional beta-glucoside permease. In B. subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion. Disruption of licT abolished the suppressor phenotype in a bglP mutant. LicT is a recently identified third B. subtilis antiterminator of the BglG/SacY family. These observations indicated that BglP was also a negative regulator of LicT. Both LicT and BglP seem to be involved in the induction by beta-glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho-beta-glucosidase. Other beta-glucoside genes homologous to bglP and bglH have been recently described in B. subtilis. Thus, B. subtilis possesses several sets of beta-glucoside genes, like E. coli, but these genes do not appear to be cryptic.

Dual effect of a Tn917 insertion into the Bacillus subtilis sacX gene.

The most common effect of transposon insertion is the inactivation of genes. However, in some cases, transposons can activate in cis the expression of genes in the neighbourhood of their integration site. We previously described an insertion of the transposon Tn917 into the Bacillus subtilis sacXY locus. sacX and sacY encode respectively a negative and a positive regulator involved in induction by sucrose of the exoenzyme levansucrase. Data in this paper show that the Tn917 insertion had two effects: it inactivated sacX and it increased the transcription of sacY. The latter effect involved one or several elements internal to the transposon.

Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system.

The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.

Induction of saccharolytic enzymes by sucrose in Bacillus subtilis: evidence for two partially interchangeable regulatory pathways.

Sucrose induces two saccharolytic enzymes in Bacillus subtilis, an intracellular sucrase and an extracellular levansucrase, encoded by sacA and sacB, respectively. It was previously shown that the sacY gene encodes a positive regulator involved in a sucrose-dependent antitermination upstream from the sacB coding sequence. We show here that the sacY product is not absolutely required for sacB induction: a weak but significant induction can be observed in strains harboring a sacY deletion. The sacY-independent induction was altered by mutations located in the sacP and sacT loci but was observed in both sacU+ and sacU32 genetic backgrounds. These results suggest that B. subtilis has two alternative systems allowing sacB induction by sucrose. Both systems also seem to be involved in sacA induction.

Enhanced polyploidy by glutaraldehyde in cultured HL60 leukemic cells.

Glutaraldehyde 10(-4) M weakened cell proliferation of HL60 cultured cells and enhanced the appearance of giant polyploid cells, up to 32.5% after 6 days. The size and structure of these cells, the quantitative changes in their DNA content with respect to diploid ones demonstrate their polyploid nature, which may be corrected by the occurrence of pluripolar mitoses. However the slowing down of cell proliferation is not enough to orient the cells towards differentiation. Maturation of polyploid cells may be stimulated by retinoic acid and dexamethasone as for diploid ones. Several possible mechanisms of polyploidy are discussed. Except the possibility that the cells may directly fuse, the mechanisms which are considered, may involve a preprophase inhibition, a mitotic arrest at metaphase or a reduction of asters which may result in a defect in cytokinesis, the latter followed by secondary fusion of nuclei.

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites.

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially alpha-amylase. Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.

Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli.

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.

[Genetic analysis of sacB, the structural gene of a secreted enzyme, levansucrase of Bacillus subtilis Marburg]. / Analyse génétique de sacB, gène de structure d'une enzyme secrétée, la lévane-saccharase de Bacillus subtilis Marburg.

The structural gene sacB encoding B. subtilis levansucrase, a secreted enzyme, expresses in E. coli. E. coli hosts of the sacB gene are poisoned by sucrose. This property allowed a powerful selection of mutants affected in the cloned gene. The plasmidic mutations were readily introduced in the B. subtilis chromosome. Using a collection of plasmids bearing various deletions extending in sacB we developed a technique of deletion mapping based on plasmid integration in the chromosome of B. subtilis. A generalization of this technique is discussed.

[Peroxidase deficiency in neutrophils in systemic mastocytosis (author's transl)]. / Déficit en peroxydase des granulocytes neutrophiles au cours d'une mastocytose diffuse.

A case associating a systemic mastocytosis and an acquired myeloperoxidase deficiency is reported. The myeloperoxydase deficiency is studied by cytochemical techniques in optical and electron microscopy and confirmed by biochemical measures. An important defect in bactericidal and candidacidal activity is demonstrated in vitro in P.N.M. The authors discuss the links between the two anomalies which might bring one more argument for the common origin of both granulocytes and mastocytes.