ALBERT

Notes: Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD-cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co-regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine–Dalgarno sequence present in the cnf1 coding sequence [fold-back inhibition (cnf1 fbi)]. The cnf1 fbi was not regulated by elements present in the igs. An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100-fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co-regulation.

Notes: The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life-threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination.

Notes: Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA, pgk, tpi, pgm and eno. They are transcribed in a hexacistronic operon together with cggR, the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN6TGTCN4CGGGACN6TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G+C, Gram-positive bacterial species.

Notes: We describe the isolation and characterization of ICL1 from the rice blast fungus Magnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle. ICL1 shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence by M. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection-related development and cuticle penetration in Δicl1 mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungus Candida albicans and the bacterial pathogen Mycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.

Notes: The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.

Notes: A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Notes: Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.

Notes: The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

Notes: The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.

Notes: SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The  rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase-like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini-linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.

Notes: Environmental signals can be transduced into intracellular responses by the action of MAP kinase cascades. Sequential phosphorylation results in the transient activation of a MAP kinase, which in turn activates certain transcription factors and thus a set of pathway-specific genes. Many steps in this cascade are conserved, and homologues have been discovered from yeast to human. We have characterized the MAPKK kinase, SteC, a homologue of Saccharomyces cerevisiae Ste11, in the filamentous fungus Aspergillus nidulans. The 886-amino-acid-long protein shares the highest similarity to Neurospora crassa Nrc-1. Deletion of the gene in A. nidulans results in a slower growth rate, the formation of more branched hyphae, altered conidiophore morphology, an inhibition of heterokaryon formation and a block of cleistothecium development. The gene is transcriptionally activated during asexual development and controls the phosphorylation of two putative MAP kinases.

Notes: PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA-deleted mutant (EGDe ΔprfA). Both strains were grown at 37°C in brain–heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178–lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA-deleted mutant (P14ΔprfA) and the EGDe strain revealed interesting strain-specific differences. Sequences strongly similar to a sigma B-dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A-dependent promoter. In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.

Notes: Mycothiol, MSH or 1d-myo-inosityl 2-(N-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside, is an unusual conjugate of N-acetylcysteine (AcCys) with 1d-myo-inosityl 2-acetamido-2-deoxy-α-d-glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced ≈ 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs.

Notes: The development of new methods, including genomics, which can even be applied to unculturable microorganisms, has significantly increased our knowledge about bacterial pathogenesis and symbiosis and, in consequence, is profoundly modifying our views on the evolution and the genetic and physiological basis of bacteria–host interactions. The presentations at this symposium revealed conceptual links between bacterial pathogenesis and symbiosis. The close co-operation of experts in both fields will result in significant synergy and new insights into basic mechanisms of bacteria–host interactions and their evolution. The meeting provided fascinating news about the genetic and metabolic consequences that the change in their lifestyle had for bacteria that developed from free-living to permanent host-associated organisms exemplified by intracellular pathogens or symbionts. In addition, surprising similarities but also striking differences between the strategies involved in the establishment of a symbiotic versus a parasitic lifestyle can be noted. In the long run, the characterization of such differences might lead to lifestyle prediction or to an evaluation of the pathogenic potential of newly isolated bacteria via the definition of genetic and/or metabolic signatures characteristic for pathogenic or symbiotic organisms. Moreover, it is expected that these investigations will lead to new strategies for the treatment or prevention of bacterial infections, or the avoidance of pathogen transmission.

Notes: In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP–IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.

Notes: Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1-carbapen-2-em-3-carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N-acyl homoserine lactone (N-AHL) quorum sensing, with synthesis of N-AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum-sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA–O, are transcribed as a single polycistronic message in an N-AHL-dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR-type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N-AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone-independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild-type backgrounds confers a hyper-Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high-affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper-production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006.

Notes: Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori -mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo- l -norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial γ-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant γ-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ-glutamyl transpeptidase plays a significant role in H. pylori -mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial γ-glutamyl transpeptidase function.

Notes: A yeast mutant was isolated that was resistant to Bax-induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c-releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co-regulation of apoptosis and autophagy in mammalian cells.

Notes: The localization of penicillin-binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)–PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot-like pattern and also at mid-cell during cell division. PBP2 disappeared from mid-cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid-cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid-cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.

Notes: The Salmonella ugd gene is required for the incorporation of 4-aminoarabinose in the lipopolysaccharide and resistance to the antibiotic polymyxin B. Transcription of the ugd gene is induced by Fe3+ via the PmrA–PmrB two-component system and by low Mg2+ in a process that requires the PhoP–PhoQ two-component system, the PhoP-activated PmrD protein and the PmrA–PmrB system. Here, we establish that mutation of the tolB gene promotes ugd transcription independently of both the PhoP–PhoQ and PmrA–PmrB systems. This activation is mediated by the RcsC–YojN–RcsB phosphorelay and the RcsA protein, suggesting a role for ugd in capsule synthesis. Binding sites for the RcsB, PmrA and PhoP proteins were identified in the ugd promoter. Although the PmrA–PmrB and RcsC–YojN–RcsB systems promoted ugd transcription independently of the PhoP–PhoQ system under different environmental conditions, ugd expression inside macrophages was strictly dependent on PhoP–PhoQ, suggesting that low Mg2+ is a cue for the intracellular environment.

Notes: Nostoc punctiforme is an example of a filamentous cyanobacterium that is capable of differentiating non-growing cells that constitute gliding filaments termed hormogonia. These gliding filaments serve in short distance dispersal and as infective units in establishing a symbiosis with plants, such as the bryophyte Anthoceros punctatus . Mutants of N . punctiforme exist which show elevated levels of initial infection of A . punctatus as a consequence of repeated cycles of hormogonium differentiation. Such mutations occur within the hrmA and hrmU genes. Further characterization of the hrm locus revealed several genes with an organizational and predicted protein sequence similarity to genes of heterotrophic bacteria that are involved in hexuronic acid metabolism. Genes in the N. punctiforme locus are transcribed in response to the presence of a plant extract containing hormogonium-repressing factors. A predicted transcriptional repressor encoded in the locus, HrmR , was shown herein to be a specific DNA binding protein that regulates the transcription of its own gene and that of hrmE , a nearby gene. The ability of HrmR to bind DNA was abolished upon addition of either galacturonate or lysate from specifically induced N . punctiforme cells, implying that the in vivo HrmR binding activity is modulated via an internal compound, most likely a sugar molecule.

Notes: Bacteria exhibit a bewildering range of behavioural responses and permutations of metabolic pathways for maximum exploitation of their environment. These are based on sensory perception of external and internal signals through batteries of surface and cytoplasmic receptors, evaluation of complex information flows and rapid decision making. Appreciation of the diversity of bacterial behaviour and adaptation capacities requires the study of a broad range of organisms and at this meeting we sampled more than 30 species with new findings which included the nature of gaseous receptors, advances in chemotaxis, subversion of host defences by pathogens, adaptation to high salt, community life and its obvious benefits, cell to cell communications and even the nature of bacterial circadian rhythms. With around 80 bacterial genomes now completed, and many more almost there, it was appropriate to complete the meeting with an introduction to Systems Biology and prospects for simulating the virtual cell. The versatility and seemingly ‘intelligent’ behaviour of bacteria will continue to fascinate, and this meeting on Bacterial Neural Networks fully reflected the excitement of this field.

Notes: We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (λmax 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18°C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix–helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.

Notes: Pseudomonas syringae pv. maculicola strain M6 (Psm M6) carries the avrRpm1 gene, encoding a type III effector, on a 40 kb plasmid, pFKN. We hypothesized that this plasmid might carry additional genes required for pathogenesis on plants. We report the sequence and features of pFKN. In addition to avrRpm1, pFKN carries an allele of another type III effector, termed avrPphE, and a gene of unknown function (ORF8), expression of which is induced in planta, suggesting a role in the plant–pathogen interaction. The region of pFKN carrying avrRpm1, avrPphE and ORF8 exhibits several features of pathogenicity islands (PAIs). Curing of pFKN (creating Psm M6C) caused a significant reduction in virulence on Arabidopsis leaves. However, complementation studies using Psm M6C demonstrated an obvious virulence function only for avrRpm1. pFKN can integrate and excise from the chromosome of Psm M6 at low frequency via homologous recombination between identical sequence segments located on the chromosome and on pFKN. These segments are part of two nearly identical transposons carrying avrPphE. The avrPphE transposon was also detected in other strains of P. s. pv. maculicola and in P. s. tomato strain DC3000. The avrPphE transposon was found inserted at different loci in different strains. The analysis of sequences surrounding the avrPphE transposon insertion site in the chromosome of Psm M6 indicates that pFKN integrates into a PAI that encodes type III effectors. The integration of pFKN into this chromosomal region may therefore be seen as an evolutionary process determining the formation of a new PAI in the chromosome of Psm M6.

Notes: A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human Vβ1+ and Vβ23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.

Notes: Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus.

Notes: Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so-called pheromone-based signal-transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln-regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA-responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activa-tion of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter-proximal se-quence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.

Notes: The wild-type strain YSH6000 of Shigella flexneri growing in minimal medium contains the modified nucleoside epoxy-Q (oQ) in a subset of tRNAs. This nucleoside is lacking in tRNA from a tgt mutant of this bacterium. When these bacteria are growing in minimal medium, the expression of virulence genes is 10-fold lower in the tgt mutant than in the wild type, although only a twofold reduction in the expression of these virulence factors is observed in broth. Such a strong media-dependant expression of virulence genes was not observed in the wild type. Accordingly, the level of the positive regulator of virulence, VirF, is much lower in the mutant than in the wild type. However, the transcription of the virF gene in minimal medium is the same in the wild type and in the tgt mutant. As the undermodification of tRNA is not affected by the quality of the growth medium, we conclude that such an environmental change in growth conditions partly restores virulence gene expression by counteracting poor translation of the virF mRNA mediated by an oQ-deficient tRNA. Virulence gene expression is partly restored in the tgt mutant by the addition of a mixture of arginine and methionine. Addition of the polyamine putrescine, synthesis of which is metabolically related to that of arginine and methionine, has a comparable stimulatory effect on virulence gene expression. These results not only suggest a role for amino acids and polyamines in the environmental regulation of virulence gene expression in S. flexneri, but also demonstrate a strong and specific involvement of tRNA modifications, and especially oQ, in the adaptation of virulence gene expression to the nutritional quality of the growth medium.

Notes: Deciphering the structure of pathogen populations is instrumental for the understanding of the epidemiology and history of infectious diseases and for their control. Although Mycobacterium tuberculosis is the most widespread infectious agent in humans, its actual population structure has remained hypothetical until now because: (i) its structural genes are poorly polymorphic; (ii) adequate samples and appropriate statistics for population genetic analysis have not been considered. To investigate this structure, we analysed the statistical associations (linkage disequilibrium) between 12 independent M. tuberculosis minisatellite-like loci by high-throughput genotyping within a model population of 209 isolates representative of the genetic diversity in an area with a very high incidence of tuberculosis. These loci contain variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs). Highly significant linkage disequilibrium was detected among the MIRU-VNTR loci in this model. This linkage disequilibrium was also evident when the MIRU-VNTR types were compared with the IS6110 restriction fragment length polymorphism types. These results support a predominant clonal evolution of M. tuberculosis.

Notes: Myxococcus xanthus cells respond to blue light by producing carotenoids. Light triggers a network of regulatory actions that lead to the transcriptional activation of the carotenoid genes. By screening the colour phenotype of a collection of Tn 5-lac insertion mutants, we have isolated a new mutant devoid of carotenoid synthesis. We map the transposon insertion, which co-segregates with the mutant phenotype, to a previously unknown gene designated here as carF . An in frame deletion within carF causes the same phenotype as the Tn 5 - lac insertion. The carF deletion prevents the activation of the normally light-inducible genes, without affecting the expression of any of the regulatory genes known to be expressed in a light-independent manner. Until now, the switch that sets off the regulatory cascade had been identified with light-driven inactivation of protein CarR, an antisigma factor. The exact mechanism of this inactivation has remained elusive. We show by epistatic analysis that the carF gene product participates in the light-dependent inactivation of CarR. The predicted CarF amino acid sequence reveals no known prokaryotic homologues. On the other hand, CarF is remarkably similar to Kua, a family of proteins of unknown function that is widely distributed among eukaryotes.

Notes: The role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron-rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron-mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron-dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.

Notes: It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor. SeqA specifically activates the bacteriophage λpR promoter while revealing no significant effect on the activity of another λ promoter, pL. Here, we demonstrate that lysogenization by bacteriophage λ is impaired in E. coli seqA mutants. Genetic analysis demonstrated that CII-mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function. This was confirmed in in vitro transcription assays. Interestingly, SeqA stimulated CII-dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template. This SeqA-mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates. Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII. Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies. In contrast to pI and paQ, the activity of the third CII-dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro. We conclude that SeqA stimulates transcription from pI and paQ promoters in co-operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region.

Notes: Several members of the two-component signal transduction family have been implicated in the control of  polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD∼P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C. crescentus are discussed.

Notes: Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a Kd of 70 ± 11 nM and 112 ± 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by ɛACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C-terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen-binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C-terminal lysine residues and three (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface-bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.

Notes: To identify proteins expressed in Escherichia coli K-12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native-state high-performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first-dimension fractions to proteins visualized by two-dimensional gel electrophoresis. Co-fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high-throughput gel-independent identification of proteins. It can also be used to assign identities to spots visualized by two-dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.

Notes: In the filamentous fungus Aspergillus nidulans, the multisubunit motor complex cytoplasmic dynein plays essential roles in nuclear migration and septum positioning. The 8 kDa light chain, LC8, the smallest subunit, is conserved among eukaryotic organisms. Besides being a component in the dynein complex, LC8 also interacts with a wide spectrum of mammalian and viral proteins. To date, the function of this small polypeptide is not well understood. To address this issue, we have created a deletion mutation (ΔnudG) at the nudG locus encoding LC8 in A. nidulans. At 42°C, the ΔnudG mutant forms minute colonies lacking asexual reproduction: this phenotype resembles the phenotype of the dynein heavy chain null mutant. The mutant nuclei largely clustered in the spore body after conidial germination, and the septum was often assembled distally toward the hyphal apex, whereas a control germling has its nuclei distributed along the hypha and the septum formed near the spore body. When the mutant was grown at 23°C, however, its colony resembled a control one, and so did the patterns of nuclear distribution and septum positioning. Elevation of the growth temperature gradually reduced colony size and abolished asexual sporulation. After a period of growth at 23°C that allowed the nuclei to move out of the spore end, a temperature shift to 42°C prevented newly divided nuclei from migrating apart, suggesting that LC8/NUDG was required for both initiating and maintaining dynein motor functions at elevated temperatures. A functional GFP-NUDA fusion was used to test whether LC8/NUDG is required for DHC (dynein heavy chain)/NUDA localization. We found that at 23°C GFP-NUDA localized to the hyphal apex and the septation site in ΔnudG cells as in control cells. Such localizations were absent at 42°C in mutant cells, but not in control cells. We conclude that LC8 plays a role in DHC localization/function, and the requirement for such a role in A. nidulans cells is temperature dependent.

Notes: External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

Notes: Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.

Notes: Schizosaccharomyces pombe Rho1p regulates (1,3)β- d -glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different S. pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named rga5 + is a GAP specific for Rho1p. rga5 + overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation. rga5 + deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37°C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p. Overexpression of rho1 + in rga5Δ cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V. In addition (1,3)β- d -glucan synthase activity, regulated by Rho1p, is increased in rga5Δ cells and decreased in rga5 -overexpressing cells. Moreover, the increase in (1,3)β- d -glucan synthase activity caused by rho1 + overexpression is considerably higher in rga5Δ than in wild-type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p.

Notes: Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054–7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mu-tant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

Notes: MinD is a key component of an oscillatory system that spatially regulates cell division in Escherichia coli. It is a peripheral membrane ATPase that recruits MinC and oscillates between the two halves of the cell in a MinE dependent manner. In vitro MinD binds to phospholipid vesicles in an ATP-dependent manner and is released through MinE-stimulated ATP hydrolysis. In this study we examined the function of the conserved C-terminus of MinD. Short truncations of three and ten amino acids dramatically decreased the ability of MinD to localize to the membrane and spatially regulate division. These truncations bound MinC but were deficient in targeting MinC to the septum. In vitro they dimerized, but were deficient in binding to phospholipid vesicles and undergoing MinE stimulation. We suggest a model in which the ATP-dependent dimerization of MinD affects the conformation of the C-terminal region, a potential amphipathic helix, triggering membrane binding.

Notes: Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3′ of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5′ of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5′ end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.

Notes: Lithium and valproate, two structurally different anti-bipolar drugs, cause decreased intracellular inositol in the yeast Saccharomyces cerevisiae and an in-crease in expression of a structural (INO1) and a regulatory (INO2) gene for phospholipid synthesis that responds to inositol depletion (Vaden, D., Ding, D., Peterson, B., and Greenberg, M.L., 2001, J Biol Chem 276: 15466–15471). We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition. In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis. Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3. The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/phosphatidylcholine ratio by both drugs. Because maintenance of the appropriate phosphatidylinositol/phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far-reaching implications for understanding the therapeutic mechanisms of action of these drugs.

Notes: Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Notes: The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA–DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.

Notes: Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N-butyldeoxynojirimycin (NB-DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P-fimbriated Escherichia coli. NB-DNJ blocks the ceramide-specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose-dependent way, and depletion markedly inhibited P-fimbriated bacterial attachment in vitro. NB-DNJ-fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P-fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae-mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.

Notes: In this study, micro-Raman spectroscopy has been used to investigate the vibrational properties of laterally epitaxial overgrown (LEO) GaN. The LEO GaN films were grown by metal organic chemical vapor deposition on a 2 in. sapphire substrate with SiN mask. Photoluminescence and polarized Raman scattering measurements have been performed in the two regions of GaN growth (wing and window regions). Raman scattering results are consistent with the lateral growth of GaN in the overgrown region. We have observed second-order Raman scattering in the wing and window regions of GaN. The observations of longitudinal optical phonon plasmon modes in the overgrown region demonstrate that LEO GaN is doped. We have carried out micro-Raman mapping of the local strain and free carrier concentration in the LEO GaN. Anharmonicity due to temperature in LEO GaN has also been investigated. The anharmonicity was found to increase with increasing temperature, and such temperature-induced anharmonicity introduces changes in the linewidth and line center position of the Raman active phonons. The phonon lifetimes in GaN are estimated in the LEO region as well as in the coherently grown region (window region). © 2002 American Institute of Physics.

Notes: Microstructural observations of gallium arsenide single crystals irradiated with a few tens of MeV C60 incident clusters (fullerenes) were performed. Normal and grazing incidences were investigated. Similar to in the case of silicon and germanium, cylindrical amorphous tracks whose diameters vary as a function of the projectile energy were found. However, for a given energy of the clusters, the track diameters are slightly different from one material to another. Also depending on the fullerene, energy is the length of the amorphous cylinder that formed along the projectile's path. The recrystallization process under an electron beam during transmission electron microscopy observation was analyzed and a higher growth rate for gallium arsenide compared to that of germanium was seen. © 2002 American Institute of Physics.

Notes: This article presents an analysis of the response of silicon carbide to high velocity impact. This includes a wide range of loading conditions that produce large strains, high strain rates, and high pressures. Experimental data from the literature are used to determine constants for the Johnson–Holmquist constitutive model for brittle materials (JH-1). It is possible to directly determine the strength and pressure response of the intact material from test data in the literature. After the ceramic has failed, however, there are not adequate experimental data to directly determine the response of the failed material. Instead, the response is inferred from a comparison of computational results to ballistic penetration test results. After the constants have been obtained for the JH-1 model, a wide range of computational results are compared to experimental data in the literature. Generally, the computational results are in good agreement with the experimental results. Included are computational results that model interface defeat, which occurs when a high velocity projectile impacts a ceramic target and then dwells on the surface of the ceramic with no significant penetration. © 2002 American Institute of Physics.

Notes: The influence of ion-beam-produced lattice defects as well as H, B, C, N, O, and Si, introduced by ion implantation, on the luminescence properties of wurtzite GaN is studied by cathodoluminescence spectroscopy. Results indicate that intrinsic lattice defects produced by ion bombardment mainly act as nonradiative recombination centers and do not give rise to the yellow luminescence (YL) of GaN. Experimental data unequivocally shows that C is involved in the defect-impurity complex responsible for YL. In addition, C-related complexes appear to act as efficient nonradiative recombination centers. Implantation of H produces a broad luminescent peak which is slightly blueshifted with respect to the C-related YL band in the case of high excitation densities. The position of this H-related YL peak exhibits a blueshift with increasing excitation density. Based on this experimental data and results reported previously, the chemical origin of the YL band is discussed. © 2002 American Institute of Physics.

Notes: InGaAs strained quantum well (SQW) samples with lattice matched InGaAsP quaternary barriers are grown on GaAs substrates by metalorganic vapor phase epitaxy. These SQW samples are characterized using photoluminescence, photomodulated reflectance, surface photovoltage spectroscopy and high-resolution x-ray diffraction techniques. The results based on numerical calculations are used to identify the various transitions seen in the spectra. The effect of growth temperature on the indium content of the InGaAs SQW with lattice matched InGaAsP quaternary barriers is studied. Contrary to the reported higher value of indium incorporation in InGaAs SQW with GaAs (In-free) barriers when the growth is performed at low temperatures, we find that the indium content of the InGaAs SQW with InGaAsP (In-based) quaternary barriers decreases if the SQW is grown at lower growth temperatures. A possible explanation for this behavior is provided. © 2002 American Institute of Physics.

Notes: B1–NaCl-structure CrN(001) layers were grown on MgO(001) at 600 °C by ultrahigh vacuum reactive magnetron sputter deposition in pure N2 discharges. X-ray diffraction analyses establish the epitaxial relationship as cube-on-cube, (001)CrN(parallel)(001)MgO with [100]CrN(parallel)[100]MgO, while temperature-dependent measurements show that the previously reported phase transition to the orthorhombic Pnma structure is, due to epitaxial constraints, absent in our layers. The resistivity increases with decreasing temperature, from 0.028 Ω cm at 400 K to 271 Ω cm at 20 K, indicating semiconducting behavior with hopping conduction. Optical absorption is low (α〈2×104 cm−1) for photon energies below 0.7 eV and increases steeply at higher energies. In situ ultraviolet photoelectron spectra indicate that the density of states vanishes at the Fermi level. The overall results provide evidence for CrN exhibiting a Mott–Hubbard type band gap. © 2002 American Institute of Physics.

Notes: We present a simple model for quantum well photodetectors for simultaneous middle infrared and near infrared or visible dual-band detection. We derive analytical formulas for the responsivity and detectivity as functions of the material and structural parameters. It is shown that the characteristics of near infrared and visible radiation detection can be sensitive to parameters of the hole transport, capture into quantum wells, and reflection from the electron emitting contact. We demonstrate that a properly designed photodetector can exhibit comparable performance for both middle infrared and near infrared (or visible) detection. The obtained results can be used to optimize the photodetector design and characteristics in both spectral ranges. © 2002 American Institute of Physics.

Notes: We investigated through ab initio total energy calculations the interaction of arsenic impurities with the core of a 30° partial dislocation in silicon. It was found that when an arsenic atom sits in a crystalline position near the dislocation core, there is charge transfer from the arsenic towards the dislocation core. As a result, the arsenic becomes positively charged and the core negatively charged. The results indicate that the structural changes around the impurity are very small in both environments, namely, the crystal and the dislocation core. In this scenario, the interaction between arsenic and the core is essentially electrostatic, which eventually leads to arsenic segregation. The segregation energy was found to be as large as 0.5 eV/atom. Additionally, it was found that arsenic pairing inside the core is not energetically favorable. © 2002 American Institute of Physics.

Notes: We report on inelastic electron tunneling spectroscopy of a tunneling metal–oxide–semiconductor (MOS) device over an extended energy range compared to previous results. We have clearly observed the vibrations of the hydrogen-passivated (111)Si Pb center in this extended energy range. The assignment of this mode has been confirmed by a comparison with infrared experiments. Capabilities and limitations of the technique to detect and observe molecular vibrations in tunneling MOS devices are discussed. © 2002 American Institute of Physics.

Notes: We report on the effect of rapid thermal annealing (RTA) on the photoluminescence (PL) properties of GaNxAs1−x/GaAs structures. In particular, a blueshift of the PL peak energy is observed when annealing the samples. The results are examined as a consequence of a RTA-induced nitrogen diffusion inside the GaNxAs1−x material rather than diffusion out of the alloy, which homogenizes initial nitrogen composition fluctuations. We propose a simple model that describes the RTA-induced blueshift of the low temperature PL peak energy. This model is in good agreement with experimental results and is consistent with recent studies in which lateral composition fluctuations in the GaNAs alloy were reported. © 2002 American Institute of Physics.

Notes: A quadratic dependence of the band gap energy on the alloy composition x was quantified for CuAl(SxSe1−x)2 films grown by low-pressure metalorganic vapor phase epitaxy, by means of photoreflectance and photoluminescence excitation spectroscopies. The bowing parameter for the A-exciton energy was estimated to be 0.20 eV. Several high-quality films grown on GaAs(001) substrates exhibited excitonic photoluminescence peaks in the blue to ultraviolet spectral ranges. The flow rate of the Al precursor was found to affect the incorporation ratio of S/Se, indicating that the Al–S compound plays a key role in controlling x. All films grown on GaAs(001) showed c(001) orientation. Conversely, the epitaxitial orientation of the films on GaP(001) changed from a(100) to c(001) with an increase in x. The critical value of x was around 0.5. The preferred orientations were explained by the natural selection rule under which the lattice strain in the epilayer is minimized. The residual strain in the 0.5-μm-thick epilayers on GaAs(001) was nearly constant for all x, although the lattice mismatch between the epilayer and the GaAs substrate varied from 0.62% to 5.39% with an increase in x. Consequently the strain was attributed to thermal stress. © 2002 American Institute of Physics.

Notes: The design and manufacture of diode lasers for gas analysis or multilayer thin-film optical devices used at low-temperature require the refractive index and the temperature coefficient of IV–VI compound over a significant temperature range. In this article, the refractive index and the absorption coefficient of Pb0.94Ge0.06Te thin film have been determined from transmission spectra measured at temperature between 80 and 300 K in the spectral range of 2.5–8.5 μm by fitting based on a Lorentz-oscillator model. It is found that the maximum refractive index occurs at 150 K, which corresponds to the structural phase transition from rocksalt to rhombohedrally distorted structure and reflects an increase of lattice polarizability. The value of the index of refraction is 5.350–6.000 in the spectral range of 4.0–8.5 μm for all measured temperatures, which reveals that Pb1−xGexTe is a highly refractive infrared material. The temperature coefficient of refractive index, dn/dT, is found to be −0.006–0.002 K−1 in the spectral range of 3.0–8.5 μm for all measured temperature. An empirical formula that fits the temperature coefficient in the spectral range of 4.0-8.5 μm is presented. The dependence of the transmission and absorption spectra on decreasing temperature can be explained by the modification of the energy-band structure due to rhombohedral distortions. The conclusion can be drawn that anomalies corresponding to the ferroelectric phase transition occur in both refractive index and absorption coefficient of Pb1−xGexTe alloy. © 2002 American Institute of Physics.

Notes: This article uses molecular dynamics simulation to investigate the role of Ar ions in the ion physical vapor deposition (IPVD) process for different Ar+-to-Cu+ ratios, and to analyze the influence of different Ar+-to-Cu+ ratios on the trench filling morphology. Also compared are the trench filling morphology observed for the IPVD process with that found in the conventional collimated magnetron deposition process. The molecular dynamics simulation includes a trench model and a deposition model, and uses the many-body, tight-binding potential method to represent the interatomic force acting among neutral atoms. The interatomic force acting between the ions and the neutral atoms is modeled by the pairwise Moliere potential method. The simulation indicates that the incident Ar ions influence the trench filling mechanisms in two significant ways; peeling of the cluster atoms, which promotes migration of the cluster atoms along the sidewall, and breaking of the bridge which forms when two clusters of atoms join. Both phenomena are beneficial since they promote a more complete filling of the trench. © 2002 American Institute of Physics.

Notes: FeCoV/TiNx and FeCoV/Ti multilayers having tFeCoV=30–700 Å prepared by dc magnetron sputtering are investigated by x-ray diffraction, stress, and magnetization measurements. The x-ray diffraction data of the FeCoV/TiNx system show the presence of interstitial N atoms in the FeCoV layers due to reactive sputtering of Ti with nitrogen. The interstitial N causes an expansion of the FeCoV lattice in FeCoV/TiNx for small tFeCoV. However, for the samples with large tFeCoV, no lattice expansion is observed. In addition to the lattice expansion caused by the intake of N atoms, a change in the crystalline texture of FeCoV layers is also observed as indicated by the enhancement of the FeCoV(200) peaks. The magnetic hysteresis measurements on the samples show that the easy direction of magnetization lies in the plane of the layers. They further show that there are easy and hard axes of magnetization within the plane of the FeCoV layers. The stress anisotropy present in the plane of the samples induces a magnetic anisotropy through magnetostrictive effects leading to the formation of the in- plane easy axis. The hysteresis and stress measurements carried out on these samples clearly show the influence of N on the in-plane magnetic anisotropy. The magnetoelastic energy in the case of the FeCoV/TiNx system, calculated from the stress data and from the magnetization measurements as a function of tFeCoV is found to agree over a large range of thickness, whereas the curves deviate significantly for small layer thickness. This deviation may be due to the role of the FeCoVNx phase. Hysteresis measurements also show that the remanence is about 95% for all the samples of the FeCoV/TiNx system. In contrast, the coercivity increases linearly with increasing tFeCoV in this system. The coercivity of the FeCoV/Ti system is larger and increases more rapidly with tFeCoV, as compared with the FeCoV/TiNx system. This behavior is attributed to a smaller grain size in the FeCoV/TiNx system due to the reactive sputtering of the Ti layers. However, there is no significant influence of N on the saturation magnetization of both systems. © 2002 American Institute of Physics.

Notes: The amorphous alloys Nd40Fe40Co5Al8B7, Nd57Fe20Co5Al10B8, and Nd57Fe20Cu5Al10B8 were prepared by copper mold casting, melt spinning, and mechanical alloying. Despite their similar x-ray diffraction patterns, samples display different magnetic and thermal behavior correlated with the method of preparation. The fully amorphous melt-spun ribbons exhibit relatively soft magnetic properties with coercivities (approximate)40 kA/m at room temperature and a Curie temperature (TC)(approximate)474 K. Apparently only the mold-cast cylinders of 3 mm diameter show hard magnetic behavior with a coercivity in the range of 258–270 kA/m (depending on composition) and have approximately the same TC as that of the melt-spun ribbons. An additional magnetic transition at 585 K due to the presence of Nd2Fe14B phase in the case of Nd40Fe40Co5Al8B7 cast rod has been observed. Heat treatment above crystallization temperature in as-cast Nd57Fe20Co5Al10B8 and Nd57Fe20Cu5Al10B8 samples destroys the hard magnetic properties. In contrast, mechanically alloyed amorphous samples are soft magnetic with maximum coercivity up to 11 kA/m but show an entirely different TC(approximate)680–740 K, which is rather characteristic of an Fe solid solution. The magnetic properties are discussed in terms of different local atomic environment and cluster sizes in amorphous samples prepared by different methods. © 2002 American Institute of Physics.

Notes: The phase evolution, microstructure, and magnetic properties of Nd8Fe86B6−xCx (x=0, 2, 4, 5, 6) melt-spun ribbons were systematically studied as a function of C content. It was found that the addition of C decreases the glass-forming tendency of the as-spun ribbons significantly. A uniform nanoscale exchange coupled Nd2Fe14(BC)/α-Fe microstructure with an average grain size of 20–25 nm can be developed in the directly quenched ribbons with C contents up to 4 at. %. Further increase of C content to x=5 leads to, in the optimally quenched ribbons, the presence of an undesirable Nd2Fe17Cx phase in addition to the 2:14:1 and α-Fe phases, whereas the alloy ribbon containing 6 at. % C consists almost entirely of the soft magnetic Nd2Fe17Cx and α-Fe phases. Subsequent annealing induces a transformation of the 2:17:Cx phase to the 2:14:1 phase +α-Fe in the ribbons with x=5 and 6, resulting in the formation of a composite 2:14:1/α-Fe structure having relatively large crystallite sizes. Magnetic measurements revealed that, for the optimally processed samples, replacement of up to 4 at. % of B by C significantly increases the coercivity iHc, with only slight reduction in remanence Jr; an optimum coercivity of 542 kA/m was obtained in the Nd8Fe86B2C4 ribbon compared with 430 kA/m for the Nd8Fe86B6 ribbon. Excessive substitution of C (x〉4) causes a drastic deterioration of both iHc and Jr due to the microstructural coarsening. Moreover, the Curie temperature of the 2:14:1 phase in the samples decreases progressively with increasing C content from 312 °C for x=0 to 270 °C for x=6. © 2002 American Institute of Physics.

Notes: Domain structures of unpoled as well as poled (along [001]- and [110]-direction) Pb(Zn1/3Nb2/3)O3 (PZN)-8% PbTiO3 (PT) and Pb(Mg1/3Nb2/3)O3 (PMN)-29% PT single crystals have been investigated by scanning force microscopy (SFM) in the piezoresponse mode, at room temperature. Antiparallel domain structures have been detected mostly in unpoled crystals of both materials, with a fingerprint pattern in (001)-oriented PZN-8% PT crystal. The ferroelastic domain wall has been identified in poled (110)-oriented PZN-8% PT crystal. "Writing" of ferroelectric domains has been performed by applying a dc voltage to the SFM tip. Local re-poling has been observed for all unpoled as well as for poled (001)-oriented crystals at the voltage ±60 V. Local electrical switching was successful in poled (110)-oriented PMN-29% PT at higher voltage (±120 V) but was not successful in poled (110)-oriented PZN-8% PT crystal. Domain-engineered crystals poled in [110]-direction seem to exhibit more stable (in the sense of local re-poling properties) domain arrangement. Hysteretic d(E) dependencies were observed by local application of an ac voltage. © 2002 American Institute of Physics.

Notes: In the study of elastic and piezoelectric fields in semiconductors due to buried quantum dots (QDs), the semicoupled piezoelectric model is commonly adopted. However, its accuracy and suitability have never been studied. In this article, by developing a fully coupled piezoelectric model and deriving the analytical elastic and piezoelectric fields based on this and the semicoupled models, we are able to verify that when the piezoelectric coupling is weak, like GaAs with the electromechanical coupling factor g=0.04, the semicoupled model predicts very accurate results as compared to those based on the fully coupled model. However, if the piezoelectric coupling is relatively strong, like AlN with g=0.32, we have shown that the semicoupled model gives very serious errors or even totally wrong results. Applying these two models to a uniformly strained AlN layer grown along the polar axis has also confirmed our observation. Therefore, for semiconductors like AlN, the fully coupled model presented in this article must be employed in order to give a reliable and accurate prediction for the elastic and piezoelectric fields. Also presented in this article is the distribution of the piezoelectric field on the surface of a half-space GaAs due to a buried QD located at 2 nm below the surface with a volume 4π/3 (nm)3. It is observed that the horizontal electric field on the traction-free and insulating surface shows some special features and its maximum magnitude can be as high as 3.5×107 V/m when the uniform mismatch eigenstrain is 0.07. Furthermore, the piezoelectric field on the traction-free and conducting surface exhibits different characters as compared to the traction-free and insulating case. © 2002 American Institute of Physics.

Notes: Optically thin palladium metal films evaporated on different silicon based substrates are investigated following exposure to different concentrations of hydrogen gas in air. Laser modulated reflectance off the palladium surface of silicon oxide and silicon nitride substrates is used to recover information regarding the reflectivity inversion and α/β-phases of the palladium complex after both first and multiple gas cycling. Atomic force microscopy confirms the formation of metal nanostructures following exposure to hydrogen of the optically thin palladium films. © 2002 American Institute of Physics.

Notes: A detailed systematic study on the growth morphology of carbon nanotubes (CNTs) on Si in atmospheric pressure thermal chemical vapor deposition was undertaken. The role of NH3 for vertical alignment of CNTs was investigated. The direct cause for the alignment was a dense distribution of the catalytic metal particles, but that the particles are maintained catalytically active under amorphous carbon deposits was established by NH3. It allows a dense nucleation of the CNTs, and consequently, assists vertical alignment through entanglement and mechanical leaning among the tubes. The CNTs grew in a base growth mode. Since Ni is consumed both by silicide reaction and by capture into the growing tube, the growth stops when Ni is totally depleted. It occurs earlier for smaller particles, and thus a long time of growth results in a thin bottom with poor adhesion. © 2002 American Institute of Physics.

Notes: Pt/Ti/WSi/Ni ohmic contacts to n-SiC, initially annealed at 950 and 1000 °C for 30 s, were evaluated for thermal stability via pulsed/cyclic thermal fatigue and aging experiments at 650 °C. Modifications of material properties in response to cyclic thermal fatigue and aging tests were quantitatively assessed via current–voltage measurements, field emission scanning microscopy, atomic force microscopy, and Rutherford backscattering spectrometry. Negligible changes in the electrical properties, microstructure, and surface morphology/roughness were observed for both annealed ohmic contacts in response to 100 cycles of acute cyclic thermal fatigue. Aging of the 950 °C annealed contact for 75 h at 650 °C resulted in electrical failure and chemical interdiffusion/reaction between the contact and SiC substrate. The 1000 °C annealed contact retained ohmicity after 100 h of aging and was found to be chemically and microstructurally stable. These findings indicate that the 1000 °C annealed Pt/Ti/WSi/Ni ohmic contact to n-SiC is thermally stable and merits strong potential for utilization in high temperature and pulsed power devices. © 2002 American Institute of Physics.

Notes: Transmission electron microscopy and x-ray diffraction measurements reveal the presence of stacking faults (SFs) in undoped cubic GaN thin layers. We demonstrate the importance of the defects in the interfacial region of the films by showing that the SFs act as nucleation sites for precipitates of residual impurities such as C and Si present in the GaN layers grown on SiC(001) substrates. We used the imaging secondary ion mass spectroscopy technique to locate these impurities. The systematic decrease of the SF density as a function of the layer thickness is explained by an annihilation mechanism. Finally, the effects of usual dopants on the structural properties of GaN layers are discussed. It is shown that Mg has a tendency to incorporate out of the Ga site by forming Mg precipitates for a concentration higher than 1019 cm−3 in contrast with the results found for heavily Si doped layers. © 2002 American Institute of Physics.

Notes: Results of thermally stimulated photoluminescence (TSL) measurements in poly(2,5-dioctoxy p-phenylene vinylene) (DOO–PPV) are reported. The obtained results are analyzed in terms of the hopping model of TSL in disordered organic materials. It is shown that the experimentally obtained TSL curve can be fitted on the basis of a double-peak Gaussian density-of-states distribution. The upper peak is associated with intrinsic localized states while the lower one can be ascribed to aggregates. The latter assignment is also supported by measurements of steady-state and time-resolved photoluminescence in DOO–PPV films and solutions. Possible mechanisms of charge carrier photogeneration in DOO–PPV are discussed. © 2002 American Institute of Physics.

Notes: A theoretical analysis based on a perturbation method is used to elucidate the results of attenuated total reflection (ATR) measurements performed on silicon oxide layers of different thicknesses on silicon substrates. This analysis shows that the absorbance ATR spectrum in p polarization is the image of the layer energy loss function, under specific conditions. It is pointed out that the enhanced sensitivity of ATR is controlled by the air gap thickness, the optical properties of the media involved, and the probing light polarization. An exact ATR spectrum simulation using a matrix formalism showed that straightforward interpretation in terms of the layer dielectric function is limited to a very narrow layer thickness range. The ATR spectrum fitting process is considered for layers out of this range and evaluated for the interpretation of experimental silicon oxide layer spectra. © 2002 American Institute of Physics.

Notes: The effect of Nb codoping on the optical properties of the PbWO4:Mo scintillator is investigated by radio- and thermoluminescence, scintillation decay, and light yield measurements. Steady-state radioluminescence efficiency of PbWO4:Mo,Nb with optimized doping concentrations (2750 and 350 molar ppm, respectively) becomes up to 20 times higher with respect to that of undoped PbWO4 and is comparable to that of Bi3Ge4O12. However, slow components down to several tens of microseconds appear in the time decay. Their existence may be related to the presence of traps monitored by thermoluminescence. © 2002 American Institute of Physics.

Notes: The thermal expansion coefficients of a single crystal and ceramic of Nb2O5 are measured in the temperature range of −200–500 °C by the dilatometer technique. Both single crystals and ceramics of Nb2O5 and Nb2O5(1−x):xTiO2 show negative thermal expansion in this temperature range. Some contribution to the result could be due to the presence of the Magneli phases. The main phase transition temperature, which also matches with the dielectric anomaly, occurs at (approximate)150 °C. © 2002 American Institute of Physics.

Notes: Germanium-doped ZnSe epilayers have been grown on (001)-oriented semi-insulating GaAs substrates by molecular beam epitaxy. The photoluminescence (PL) spectra exhibit strong near-band edge emission similar to those from undoped, chlorine-doped and gallium-doped samples, though some differences exist. The prominent PL peak at 2.795 eV (10 K) is attributed to the germanium-bound exciton recombination and is accompanied by free exciton (2.802 eV), Ia-type exciton (2.785 eV) and Iv-type exciton (2.775 eV) emission peaks. Following an increase in temperature, the intensity of all the IGe, Ia, and Iv emission peaks decreases gradually, indicating the presence of nonradiative recombination mechanisms with thermal activation energies of 40, 70, and 50 meV respectively. However, for the Iv peak, there is one additional nonradiative recombination mechanism in accordance with the thermally activated transfer of excitons from the Iv-type centers to Ia-type centers. This nonradiative recombination mechanism with activation energy of 9 meV is responsible for the decrease of the Iv peak intensity when the sample temperature is changed from 15 to 100 K. Following an increase in temperature, the Iv peak, Ia peak, and germanium-related peak disappear gradually and successively. Finally, the PL spectrum is dominated by free exciton emission at temperatures exceeding 210 K. © 2002 American Institute of Physics.

Notes: Electronic property variations of a two-dimensional electron gas (2DEG) in modulation-doped step quantum wells due to an embedded potential barrier were studied by performing Shubnikov–de Haas (SdH), Van der Pauw–Hall-effect, and cyclotron resonance measurements on two kinds of InxGa1−xAs/InyAl1−yAs step quantum wells which were one without and the other with an embedded barrier. The fast Fourier transformation results for the SdH data at 1.5 K indicated the electron occupation of two subbands in both step quantum wells. The total electron carrier density and the mobility of the 2DEG in the step quantum well with an embedded barrier were smaller than those in the quantum well without an embedded barrier. The electron effective masses were determined from the slopes of the main peak absorption energies as functions of the magnetic field, and satisfied qualitatively the nonparabolicity effects in both quantum wells. The electronic subband energies, the wave functions, and the Fermi energies were calculated by using a self-consistent method taking into account exchange-correlation effects together with strain and nonparabolicity effects. These present results indicate that the electronic parameters in modulation-doped InxGa1−xAs/InyAl1−yAs step quantum wells are significantly affected by an embedded barrier. © 2002 American Institute of Physics.

Notes: The direct and assisted-by-trap elastic tunnel current in metal–oxide–semiconductor capacitors with ultrathin gate oxide (1.5–3.6 nm) has been studied. Bardeen's method has been adapted to obtain the assisted tunnel current, in addition to the direct tunnel current. The dependence of the assisted current on the trap distribution in energy has also been analyzed. This allows us to obtain the trap distribution in energy from experimental current curves. Finally, we have analyzed the role of the image force, the inclusion of which can avoid a barrier height dependence on the oxide thickness. © 2002 American Institute of Physics.

Notes: X-ray photoemission and x-ray photoabsorption were used to study the composition and the electronic structure of ytterbium-doped strontium fluoroapatite (Yb:S-FAP). High resolution photoemission measurements on the valence band electronic structure and Sr 3d, P 2p and 2s, Yb 4d and 4p, F 1s, and O 1s core lines were used to evaluate the surface and near surface chemistry of this fluoroapatite. Element specific density of unoccupied electronic states in Yb:S-FAP were probed by x-ray absorption spectroscopy at the Yb 4d (N4,5 edge), Sr 3d (M4,5 edge), P 2p (L2,3 edge), F 1s, and O 1s (K edges) absorption edges. These results provide measurements of the electronic structure and surface chemistry of this material. © 2002 American Institute of Physics.

Notes: Extremely thin titanium nitride (TiN) barrier layers for Cu based interconnects were deposited using metal organic chemical vapor deposition. The effect of the subsequently performed nitrogen/hydrogen plasma treatment on the microstructure, composition, and electrical properties of these films is studied using conventional and high resolution transmission electron microscopy, Auger electron spectroscopy, and four point probe resistivity measurements. In the studied system the crystallization of the TiN film starts from an amorphous matrix and a polycrystalline morphology is developed upon the H2/N2 plasma treatment. After a short plasma treatment, most of the film is already crystalline and consists of grains of a few nanometers in diameter. Continued plasma treatment leads to grain growth and a significant reduction of contaminants such as oxygen and carbon. The resistivity of the films drops with plasma treatment time, and a correlation between resistivity and oxygen content is found, which suggests that oxygen in the grain boundaries plays a decisive role for the resistivity of the films. It is shown that the oxygen in the grain boundaries leads to an electron reflectance of 0.9. Thus, the oxygen accumulation in the grain boundaries is the limiting factor for the reduction of film resistivity by plasma treatment. © 2002 American Institute of Physics.

Notes: The effect of periods on the accumulation and release of stress in GaAs/AlInAs superlattices structure is reported here. It is observed that in GaAs/AlInAs superlattices, when the Indium (In) content is greater than 10%, stress accumulates monotonically as the number of period increases. In GaAs/AlInAs superlattices with an In content of 5%, the accumulated stress is larger when the number of periods is less than 10. However when the number of periods exceeds 10, it was observed that suddenly there is a significant increase in defects and stress release. However, with any further increase in period number, there is once again an accumulation of stress. © 2002 American Institute of Physics.

Notes: Five electron traps were detected successfully in heavily Si-doped GaAs and AlxGa1−xAs of low Al content with a Si concentration of above 1×1019 cm−3 using deep level transient spectroscopy. The junctions were grown by liquid phase epitaxy and were strongly compensated. The traps were investigated for functions of the Si concentration and the AlAs mole fraction. The traps are discussed in terms using their spectra and concentration as opposed to the previous results which used point defects in the GaAs and AlGaAs. The traps show distinctive features, which can be attributed to strongly Si-compensated crystals. Three traps among them were confirmed to be DX centers. © 2002 American Institute of Physics.

Notes: Using a theoretical model of a bilayer organic light emitting diode, we calculate numerically the evolution of carrier densities and electric fields inside the device. The obtained results allow us to detail injection and accumulation of carriers during transient excitation. Charge densities as a function of applied voltage present two distinct thresholds which determine three operating ranges: no injection, unipolar injection, and bipolar injection. Dynamically these thresholds depend on the rise time of the applied voltage and have a clear signature in the current densities. We show that the electroluminescence threshold has static and dynamic values which may be different. Calculated external current in response to a voltage ramp presents two steps related to the evolution of the capacity of the device. This capacitive behavior is observed experimentally. © 2002 American Institute of Physics.

Notes: This paper analyzes a simplified rate equation model of localized exciton emission in GaInN. Expressions for temperature dependent photoluminescence (PL) efficiency and decay time are derived and compared with time integrated (TIPL) and time resolved photoluminescence (TRPL) data for a series of multiquantum well light emitting diodes with varying In composition in the active region. Time resolved photoluminescence is measured up to relatively high temperature (540 K) and a decreasing efficiency coupled with a peak energy decay time that is weakly dependent on temperature is observed. The decay time at peak emission energy begins to decrease at a temperature that depends on the In content in the quantum wells. The analysis developed here demonstrates that application of the expressions τr=τpl/η and τnr=τpl/(1−η) is not sufficient to determine radiative and nonradiative lifetimes from TRPL and TIPL data in the GaInN system. (Here τr is the radiative decay time, τnr is the nonradiative decay time, τpl is the measured PL decay time, and η is the measured TIPL intensity normalized to the low temperature intensity.) GaInN with even small amounts of In exhibits highly efficient luminescence due to recombination through localizing centers. As relaxation occurs into both defects and localizing states after initial generation with the above GaInN band gap excitation, the number of carriers arriving at localization centers can change with temperature. This temperature dependent change should be considered when calculating relevant decay times from TRPL and TIPL data. This mechanism is distinct from an increase in the intrinsic radiative decay time obtained by applying conventional analysis to extract radiative and nonradiative lifetimes from TRPL and TIPL data. © 2002 American Institute of Physics.

Notes: Avalanche breakdown voltages were determined for a series of In0.53Ga0.47As p-i-n diodes with i-region thicknesses ranging from 0.4 to 4.79 μm using measurements of reverse dark current and phase-sensitive photomultiplication. Despite its narrower bandgap In0.53Ga0.47As is found to have a very similar breakdown voltage to GaAs. © 2002 American Institute of Physics.

Notes: Multi-quantum-well GaN/InGaN heterojunction diodes prepared by metalorganic chemical vapor deposition on sapphire showed effects of strong tunneling in their I–V characteristics. The space charge region was shown to be located in the GaN/InGaN superlattice (SL). The injection of moderately high forward currents through the structure for several hours enhanced the overall tunneling through the structure and facilitated faster tunneling between the layers in the GaN/InGaN SL. These results may have relevance to the aging characteristics of light-emitting diodes under bias. © 2002 American Institute of Physics.

Notes: Current and voltage noise measurements have been carried out on nanoparticle WO3 films. The fluctuation dissipation theorem holds, which indicates that the observed noise is an equilibrium phenomenon. Results on the thinnest films show that noise measurements can be used for quality assessment of nanocrystalline insulating films. © 2002 American Institute of Physics.

Notes: Fe–Co–Nd–Dy–B glassy alloys are a current type of ferromagnetic material. To increase their glass-forming ability against the growth of a crystallization phase, the effects of the addition of transition metals TM (TM=V, Nb, Ta, Cr, Mo, and W) on the glass-formation ability and magnetic properties of Fe62Co9.5Nd3Dy0.5B25 glassy alloy have been investigated. The substitution of 2 at. % elements TM (=Nb, Ta, Mo, and W) for Fe and Co increases crystallization temperature Tx and decreases the onset temperature of solidification Tm, leading to a significant increase in the thermal stability against crystallization for Fe60.3Co9.2TM2Nd3Dy0.5B25. The difference (ΔTx=Tx−Tg) between Tx and the glass transition temperature Tg increases from 55 K at 0 at. % TM to 87 K at 2 at. % TM. The bulk glassy alloy with a diameter up to 1.2 mm was produced by copper mold casting. Also no distinct changes in Tg, Tx and ΔTx are seen for the addition of Cr and V. The results can be explained by the difference of atomic size in the additional elements. The saturation magnetization decreases slightly by the addition of 2 at. % TM elements. The saturation magnetization and coercive force of glassy Fe60.3Co9.2TM2Nd3Dy0.5B25 alloys are 1.13 to 1.19 T and 3.85–4.98 A/m, respectively. © 2002 American Institute of Physics.

Notes: Ti/Co/NiFe/Al-oxide/NiFe (F1) and Co/NiFe/Al-oxide/NiFe (F2) junction films were characterized using high-resolution electron microscopy (HREM), Lorentz transmission electron microscopy (LTEM), and alternating gradient force magnetometry (AGFM). HREM images showed that the Ti seed layer induced a strong 〈111〉 texture in the bottom Co/NiFe bilayer. The ferromagnet/Al-oxide interfaces in F1 showed correlated waviness, while the interface waviness in F2 appeared uncorrelated. Thus, "orange-peel" coupling effect was more significant in F1 than in F2, which was confirmed by the steep slope of the magnetization curve in the "antiparallel" magnetization configuration for F1. The LTEM in situ magnetizing experiment results and the AGFM measurement of magnetization curves showed that both junction films possessed a two-stage magnetization reversal characteristic—magnetization of the top NiFe layer reversed first followed by the reversal of the bottom Co/NiFe bilayer. LTEM observation revealed that the magnetization reversal of the top NiFe layer was via domain wall motion, while the reversal of the bottom Co/NiFe bilayers was mainly by wall motion together with a small degree of moment rotation. Domain wall mobility in the Co/NiFe bilayer of F1 was higher due to the strong crystallographic texture and large grain size appeared in the bilayer. © 2002 American Institute of Physics.

Notes: We present here a magnetic tunnel junction device using perpendicular magnetization films designed for magnetic random access memory (MRAM). In order to achieve high-density MRAM, magnetic tunnel junction devices with a small area of low aspect ratio (length/width) is required. However, all MRAMs reported so far consist of in-plane magnetization films, which require an aspect ratio of 2 or more in order to reduce magnetization curling at the edge. Meanwhile, a perpendicular magnetic tunnel junction (pMTJ) can achieve an aspect ratio=1 because the low saturation magnetization does not cause magnetization curling. Magnetic-force microscope shows that stable and uniform magnetization states were observed in 0.3 μm×0.3 μm perpendicular magnetization film fabricated by focused-ion beam. In contrast, in-plane magnetization films clearly show the presence of magnetization vortices at 0.5 μm×0.5 μm, which show the impossibility of information storage. The PMTJ shows a magnetoresistive (MR) ratio larger than 50% with a squareness ratio of 1 and no degradation of MR ratio at 103 Ω μm2 ordered junction resistance. © 2002 American Institute of Physics.

Notes: For an infinitely long bar with rectangular cross section 2a×2b and constant susceptibility χ in a uniform transverse applied field Ha along dimension a, the fluxmetric and magnetometric demagnetizing factors Nf,m as functions of a/b and χ are accurately computed using a finite-elements method. By comparison of the computed results with exact analytical results for χ=−1,0,∞ and with a set of conjugate relations derived in this work, the elements distributions have been optimized to give a minimum discretization error. This error is further greatly reduced in the final results of Nf,m by using an error correction approach previously proposed in a similar work for cylinders. © 2002 American Institute of Physics.

Notes: The effects of thermal stress on the electrical characteristics of metal–oxide–semiconductor diodes with oxides in an ultrathin regime were studied. By centering a quartz ring as a heat sink beneath the silicon wafer, the introduced temperature gradient results in a corresponding hat-like shape thickness distribution for an oxide grown on the wafer with a rapid thermal processing system. The enhanced exterior tensile and compressive thermal stresses due to introduced temperature gradient make the oxides exhibit less and more substrate injection saturation current Jsat, respectively, in comparison to control oxides. Their flatband voltage VFB data also clearly show the dependency of effective charge number density Neff on exterior thermal stress. A stress distribution model is proposed to explain the observation. Co-60 irradiation was also performed on the stressed samples to observe this stress extent by examining the variation of electrical characteristics. It was found that an oxide grown on a wafer in exterior compression exhibited better radiation hardness than one in tension. © 2002 American Institute of Physics.

Notes: The layer-by-layer hydrogen plasma treatment method, alternatively repeating the process of nanometer thickness film deposition and hydrogen plasma chemical annealing, was applied to fabricate amorphous carbon (a-C) films. It was shown that hydrogen plasma treatment reduced the size of sp2 clusters and resulted in the increase of the optical band gap. Consequently, a stable vacuum electron emission with a low threshold field was achieved from layer-by-layer hydrogen plasma annealed a-C films compared with that from conventionally deposited samples. The threshold electric field was as low as 2 V/μm. The improvement of field emission characteristics could be attributed to the large field enhancement effect due to the inhomogeneous distribution of nanometer scale sp2 clusters. © 2002 American Institute of Physics.

Notes: A Fokker–Planck equation for carrier transport in semiconductors is derived from the Boltzmann transport equation by expanding in Legendre polynomials and assuming the phonon energy exchanged at momentum randomizing collisions is small compared with the mean carrier energy. The method is used to compute impact ionization probability distributions in space and time and the results agree well with those generated by an equivalent Monte Carlo model over a wide range of electric fields from 300 kV/cm to 1 MV/cm. © 2002 American Institute of Physics.

Notes: The dynamic high-strain-rate behavior of two-phase TiB2+Al2O3 ceramics with biased microstructures was investigated in this study. The microstructural bias includes differences in phase (grain) size and phase distribution such that in one case a continuous (interconnected) TiB2 network surrounds the Al2O3 phase (qualitatively termed "T@A") and in another case the TiB2 and Al2O3 phases are interdispersed and uniformly intertwined with each other (qualitatively termed "TinA"). Quantitative microscopy was used to characterize the phase size and the integral curvature which is taken as a measure of TiB2 phase connectivity around Al2O3. Dynamic compression and tension (spall) properties were measured using plate impact experiments. The measurements used piezoelectric polyvinyldine fluoride stress gauges to obtain the loading profile and to determine the Hugoniot elastic limit. In addition, velocity interferometry system for any reflector interferometry was used to obtain the spall signal and the tensile dynamic strengths of the materials. Experimental results reveal that while the σHEL and the compressive strengths of TiB2+Al2O3 ceramics are dependent on the average grain (phase) size, the tensile (spall) strength scales with the TiB2-phase connectivity. This result suggests that the interconnected TiB2/Al2O3 microstructural morphology provides a stronger impediment to failure in tension compared with the morphology with simply interdispersed phases. © 2002 American Institute of Physics.

Notes: Two spatial dimension front tracking simulations have been performed to study the growth of polycrystalline, faceted films from randomly oriented nuclei by varying the deposition angle of the incident flux during physical vapor deposition. The orientation of grain columns, the porosity, the crystallographic texture, and grain size are sensitive to the deposition angle. The origin of this effect is widely believed to be associated with shadowing. In order to isolate the effects of shadowing from other physical effects (such as surface diffusion, deposition species size, flux divergence, etc.), we have constructed a simulation where all of these effects are completely removed. These simulations demonstrate that while many of the observed structural properties of obliquely deposited films are controlled by shadowing, a few key properties cannot be attributed solely to shadowing. © 2002 American Institute of Physics.

Notes: © 2002 American Institute of Physics.

Notes: © 2002 American Institute of Physics.

Notes: © 2002 American Institute of Physics.

Notes: © 2002 American Institute of Physics.

Notes: Optical waveguide amplifiers based on polymer materials offer a low-cost alternative for inorganic waveguide amplifiers. Due to the fact that their refractive index is similar to that of standard optical fibers, they can be easily coupled to existing fibers with low coupling losses. Doping the polymer with rare-earth ions that yield optical gain is not straightforward, as the rare-earth salts are poorly soluble in the polymer matrix. This review article focuses on two different approaches to dope a polymer waveguide with rare-earth ions. The first approach is based on organic cage-like complexes that encapsulate the rare-earth ion and are designed to provide coordination sites to bind the rare-earth ion and to shield it from the surrounding matrix. These complexes also offer the possibility of attaching a highly absorbing antenna group, which increases the pump efficiency significantly. The second approach to fabricate rare-earth doped polymer waveguides is obtained by combining the excellent properties of SiO2 as a host for rare-earth ions with the easy processing of polymers. This is done by doping polymers with Er-doped silica colloidal spheres. © 2002 American Institute of Physics.

Notes: We present a method for calculating band structures for one-dimensional Kerr nonlinear photonic crystals, which exhibit an optical switching function. The band structure shows the allowed modes for the nonlinear photonic crystal as a function of the magnitude of the nonlinearity. The dielectric band is found to be most suited for the control beam as it is least effected by the nonlinearity. The third band is more sensitive to the nonlinearity and thus suited for the probe beam. Also the wavelength dependence of the switching function can be estimated using our method. The applied Fourier method is found to be robust in describing the effect of the nonlinearity. © 2002 American Institute of Physics.