bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of interfacial defects in high-k gate stack films by spectroscopic ellipsometry
Authors: Price J. , Bersuker G. , Lysaght P. S.
Published: February 2009

The authors present a spectroscopic study of defects in HfO2, Hf0.8Si0.2O2, Al2O3, and SiO2 dielectric gate stacks. The results indicate that all optically observable dielectric-related defects are associated with the interfacial SiO2 layer rather than the bulk high-k film. The identified defects, located at 2.9, 3.5, 3.9, and 4.75eV within the dielectric film’s band gap, are found to be strongly affected by subsequent postdeposition anneal treatments and trend consistently with recent electron spin resonance results and theoretical calculations of optical transitions associated with negatively charged vacancies in SiO2 media. The close connection between our results and both atomistic calculations and experimental findings motivates the use of spectroscopic ellipsometry as a potential in-line characterization method for identifying process-induced defects during complementary metal oxide semiconductor device fabrication.
Al
2
O
3
–
Si
O
2
stack with enhanced reliability
Authors: Lisiansky M. , Fenigstein A. , Heiman A. , Raskin Y. , Roizin Y. , Bartholomew L. , Owyang J. , Gladkikh A. , Brener R. , Geppert I. , Lyakin E. , Meyler B. , Shnieder Y. , Yofis S. , Eizenberg M.
Published: February 2009

The authors developed a new Al2O3–SiO2 (A-O) stack for application as a high voltage complementary metal oxide semiconductor (CMOS) dielectric and/or top oxide in electrically erasable programmable read only memory floating gate and polysilicon-oxide-nitride-oxide-silicon embedded memories in advanced technology nodes. An amorphous atomic layer deposited alumina is doped with nitrogen and transformed into the crystalline phase by further rapid thermal process annealing. The 65Å effective oxide thickness (EOT) stack allows operating voltages twice exceeding the values for thermal SiO2 of the same EOT, has extremely low leakage currents, has negligible charge trapping, and is immune to degradation. Moreover, after alumina removal, the remaining strongly nitrided bottom oxide layer can be used as the gate dielectric of CMOS devices.
Spatial distribution of electrically active defects in dual-layer (SiO2∕HfO2) gate dielectric n-type metal oxide semiconductor field effect transistors
Authors: Nguyen T. , Savio A. , Militaru L. , Plossu C.
Published: February 2009

Spatial distributions of electrically active defects in dual-layer (SiO2∕HfO2) gate dielectric n-type metal oxide semiconductor transistors have been determined by charge pumping and 1∕f low-frequency noise measurements. Oxide trap concentration levels extracted from both techniques are in good agreement and appear one decade greater for HfO2 oxides compared to the reference SiO2 one. Moreover an increase in the trap concentration at the SiO2∕HfO2 is observed that could explain threshold voltage instabilities currently observed in HfO2 based transistors.
On the impact of silicon nitride technology on charge trap NAND memories
Authors: Sebastiani A. , Scozzari C. , Mauri A. , Modelli A. , Albini G. , Piagge R. , Bacciaglia P. , Del Vitto A. , Alessandri M. , Grossi A. , Tessariol P. , Ghidini G.
Published: February 2009

The aim of this work is to study the impact of silicon nitride deposition/treatment technologies on charge trap (CT) nonvolatile memory performances. The authors have found that the technology modifies the charge trapping behavior with a one to one correlation between write/erase and charge retention characteristics. In particular, they used rapid thermal chemical vapor deposition techniques to obtain films with different compositions, but they were not able to improve CT performances with respect to standard low pressure chemical vapor deposition (LPCVD). Besides, an in situ steam generated treatment applied to standard LPCVD silicon nitride modifies the film properties inducing a lower programming efficiency, but improving charge retention characteristics.
Investigation of voltage dependent relaxation, charge trapping, and stress induced leakage current effects in HfO2∕Dy2O3 gate stacks grown on Ge (100) substrates
Authors: Rahman M. S. , Evangelou E. K. , Androulidakis I. I. , Dimoulas A. , Mavrou G. , Tsipas P.
Published: February 2009

Germanium is a very promising material and has an extra advantage due to its higher mobility than silicon. At the same time, high-κ gate dielectrics such as HfO2 are already used for the replacement of SiO2 in advanced complementary metal-oxide-semiconductor (MOS) devices. A buffer interfacial layer is required to have better interfacial quality between HfO2 and the semiconductor substrate. In the present work the authors investigate the voltage dependent relaxation effects and charge trapping characteristics of Pt∕HfO2∕Dy2O3∕p-Ge MOS devices. The devices have been subjected to constant voltage stress and show relaxation effects in the whole range of applied stress voltages (−1to−5V). Charge trapping is negligible at low stress field while at higher fields (>4MV∕cm) it is significant. Also interesting is the fact that the trapped charge is negative at low stress fields but changes to positive at higher fields.
Lift-off and hybrid applications with ma-n 1405 negative-tone resist
Authors: Aassime A. , Mathet V.
Published: February 2009

The authors evaluated the performance of the negative-tone resist ma-n 1405 for lift-off and hybrid processes by combining electron-beam and/or laser lithography techniques with metal deposition. Electron lithography and gold deposition allow the fabrication of useful structures such as circles, lines, and coaxes with a resolution of nearly 70nm. The ability of this resist to withstand a hybrid process was also tested. A constricted line was transferred to the silicon substrate by lift-off, where the large (1μm linewidth) and the small (200nm linewidth) features were, respectively, patterned by laser and electron-beam direct writing on a single resist level.
Implanted and irradiated SiO2∕Si structure electrical properties at the nanoscale
Authors: Porti M. , Nafria N. , Gerardin S. , Aymerich X. , Cester A. , Paccagnella A. , Ghidini G.
Published: February 2009

In this work, a conductive atomic force microscope (C-AFM), a scanning capacitance microscope (SCM), and a kelvin probe force microscope (KPFM) have been used to qualitatively study at the nanoscale the electrical properties of irradiated and implanted gate oxides of metal-oxide-semiconductor structures. These techniques have allowed to investigate the electrical conduction (C-AFM) and the presence of charge (SCM and KPFM) in the oxide of the analyzed structures. The impact of the energy of the impinging ions has also been qualitatively evaluated.
Trap related dielectric absorption of HfSiO films in metal-insulator-semiconductor structures
Authors: Kerber M. , Fachmann C. , Heitmann J. , Kudelka S. , Schröder U. , Reisinger H.
Published: February 2009

Dielectric absorption in HfSiO has been investigated with transient floating potential measurements. This has been achieved by peripheral component interconnect-based recording after the application of conditioning pulses. Metal-insulator-semiconductor (TiN∕HfSiO∕n+Si) capacitors show an increase in dielectric absorption with increasing annealing temperature when the film is covered with TiN. This effect is attributed to electrically active defects introduced by the presence of titanium during annealing.
Self-assembly solder process to form three-dimensional structures on silicon
Authors: Rao M. , Lusth J. C. , Burkett S. L.
Published: February 2009

The self-assembly of microscale polyhedra driven by surface-tension constraints has been previously described in the literature. Lithographic techniques were used to fabricate two-dimensional structures that, when freed from the underlying substrate, folded into polyhedral shapes. In this article, a modified technique is described in which one face of each polyhedron remains attached to the silicon substrate. The advantage of this new approach is that precise arrays of polyhedra can be formed. Five different polyhedral shapes were fabricated and their corresponding yields are given. The yield values show that the success of the autofolding process depends on the shape of the three-dimensional structures. Truncated shapes appear to be the most readily assembled.
Stress-induced leakage current and random telegraph signal
Authors: Teramoto Akinobu , Kumagai Yuki , Abe Kenichi , Fujisawa Takafumi , Watabe Shunichi , Suwa Tomoyuki , Miyamoto Naoto , Sugawa Shigetoshi , Ohmi Tadahiro
Published: February 2009

Stress-induced leakage current (SILC) and random telegraph signal (RTS) in n-type metal-oxide-semiconductor field-effect-transistor (n-MOSFETs) caused by the Fowler-Nordheim tunneling stress are studied by using the author’s newly developed test pattern. MOSFETs having large RTS increase can be induced by electrical stress in parallel with the inducing of SILC. Generation and recovery characteristics of SILC and RTS against the stress time and measurement temperature are very similar. However, the MOSFETs having large RTS are not related to ones having large anomalous SILC in this study. We consider that the traps that cause the RTS and anomalous SILC are the same, but their locations in SiO2 are different.
Radiation damage on dielectrics: Single event effects
Authors: Paccagnella Alessandro , Gerardin Simone , Cellere Giorgio
Published: February 2009

Single event effects are due to ionizing particles impinging on sensitive circuit regions, directly or as a by-product of nuclear reactions. Atmospheric neutrons originating from the interaction of cosmic rays with the outer layers of the atmosphere, alpha particles from radioactive contaminants at sea level, and heavy ions in space continuously hit electronic devices, causing a plethora of effects, some of which are governed by the properties and geometry of the dielectric layers used in the struck devices. This contribution will review these effects, focusing first on the thin gate dielectrics used in complementary metal-oxide semiconductor circuits and then on tunnel oxide of floating-gate memories.
Impact of a γ-Al2O3(001) barrier on LaAlO3 metal-oxide-semiconductor capacitor electrical properties
Authors: Becerra L. , Merckling C. , El-Kazzi M. , Baboux N. , Vilquin B. , Saint-Girons G. , Plossu C. , Hollinger G.
Published: February 2009

Amorphous LaAlO3 high-κ oxides were grown in a molecular beam epitaxy reactor on p-Si(001) employing a thin γ-Al2O3 epitaxial layer as buffer. Interfaces are free of SiO2 and silicates and remain abrupt even after high temperature annealing as demonstrated by x-ray photoelectron spectroscopy. Electrical measurements performed on as-deposited samples reveal dielectric constant values close to the bulk ones, small equivalent oxide thickness, and low interface state densities. Some negative charges are present leading to a flatband voltage shift. Postdeposition annealing can correct this effect.
High-k materials and their response to gamma ray radiation
Authors: Zhao C. Z. , Taylor S. , Werner M. , Chalker P. R. , Potter R. J. , Gaskell J. M. , Jones A. C.
Published: February 2009

The radiation response of four different high-k materials has been investigated by irradiating them using a 979 MBq Cs137 γ-ray source and a dose absorption rate of 0.71rad(Si)∕s. Acceptorlike electron traps and donorlike traps were observed in HfO2 and ZrO2 metal-oxide-semiconductor capacitors originating from radiation-induced defects. A lower density of donor-like traps were created in LaAlO3 and NdAlO3 capacitors, but both electron and hole trapping play a role in shifting the flat band voltage. The radiation hardness of the LaAlO3 and NdAlO3 thin films is similar to thermal SiO2 but better than the HfO2 and ZrO2.
New Electropositive Filter for Concentrating Enteroviruses and Noroviruses from Large Volumes of Water
Authors: Karim Mohammad R. , Rhodes Eric R. , Brinkman Nichole , Wymer Larry , Fout G. Shay
Published: February 2009

The U.S. Environmental Protection Agency's information collection rule requires the use of 1MDS electropositive filters for concentrating enteric viruses from water, but unfortunately, these filters are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the NanoCeram filter, was evaluated for its ability to concentrate enteroviruses and noroviruses from large volumes of water. Seeded viruses were concentrated using the adsorption-elution procedure. The mean percent retention of seeded polioviruses by NanoCeram filters was 84%. To optimize the elution procedure, six protocols, each comprising two successive elutions with various lengths of filter immersion, were evaluated. The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 minute during the first elution and for 15 min during the second elution. The recovery efficiencies of poliovirus, coxsackievirus B5, and echovirus 7 from 100-liter samples of seeded tap water were 54%, 27%, and 32%, respectively. There was no significant difference in virus recovery from tap water with a pH range of 6 to 9.5 and a water flow rate range of 5.5 liters/min to 20 liters/min. Finally, poliovirus and Norwalk virus recoveries by NanoCeram filters were compared to those by 1MDS filters, using tap water and Ohio River water. Poliovirus and Norwalk virus recoveries by NanoCeram filters from tap and river water were similar to or higher than those by the 1MDS filters. These data suggest that NanoCeram filters can be used as an inexpensive alternative to 1MDS filters for routine viral monitoring of water.
Development of Bacteriocinogenic Strains of
Saccharomyces cerevisiae
Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from
Enterococcus faecium
L50
Authors: Basanta Antonio , Herranz Carmen , Gutiérrez Jorge , Criado Raquel , Hernández Pablo E. , Cintas Luis M.
Published: February 2009

A segregationally stable expression and secretion vector for
Saccharomyces cerevisiae
, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (
MF
α
1

s

) into the
S. cerevisiae
high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from
Enterococcus faecium
L50 were cloned, separately (
entL50A
or
entL50B
) and together (
entL50AB
), into pYABD01 under the control of the galactose-inducible promoter P
GAL1
. The generation of recombinant
S. cerevisiae
strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the
MF
α
1

s

-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the
S. cerevisiae
Sec system.
Time-Resolved Metabolic Footprinting for Nonlinear Modeling of Bacterial Substrate Utilization
Authors: Behrends Volker , Ebbels Tim M. D. , Williams Huw D. , Bundy Jacob G.
Published: February 2009

Untargeted profiling of small-molecule metabolites from microbial culture supernatants (metabolic footprinting) has great potential as a phenotyping tool. We used time-resolved metabolic footprinting to compare oneEscherichia coliand threePseudomonas aeruginosastrains growing on complex media and show that considering metabolite changes over the whole course of growth provides much more information than analyses based on data from a single time point. Most strikingly, there was pronounced selectivity in metabolite uptake, even when the bacteria were growing apparently exponentially, with certain groups of metabolites not taken up until others had been entirely depleted from the medium. In addition, metabolite excretion showed some complex patterns. Fitting nonlinear equations (four-parameter sigmoids) to individual metabolite data allowed us to model these changes for metabolite uptake and visualize them by back-projecting the curve-fit parameters onto the original growth curves. These “uptake window” plots clearly demonstrated strain differences, with the uptake of some compounds being reversed in order between different strains. Comparison of an undefined rich medium with a defined complex medium designed to mimic cystic fibrosis sputum showed many differences, both qualitative and quantitative, with a greater proportion of excreted to utilized metabolites in the defined medium. Extending the strain comparison to a more closely related set of isolates showed that it was possible to discriminate two species of theBurkholderia cepaciacomplex based on uptake dynamics alone. We believe time-resolved metabolic footprinting could be a valuable tool for many questions in bacteriology, including isolate comparisons, phenotyping deletion mutants, and as a functional complement to taxonomic classifications.
Characterization of Regulatory Pathways in
Xylella fastidiosa
: Genes and Phenotypes Controlled by
gacA
Authors: Shi Xiang Yang , Dumenyo C. Korsi , Hernandez-Martinez Rufina , Azad Hamid , Cooksey Donald A.
Published: February 2009

The xylem-limited, insect-transmitted bacterium
Xylella fastidiosa
causes Pierce's disease in grapes through cell aggregation and vascular clogging. GacA controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in
Pseudomonas syringae
pv. tomato DC3000. Cloned
gacA
of
X. fastidiosa
was found to restore the hypersensitive response and pathogenicity in
gacA
mutants of
P. syringae
pv. tomato DC3000 and
Erwinia amylovora
. A
gacA
mutant of
X. fastidiosa
(DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines, compared with the parent (A05). cDNA microarray analysis identified 7 genes that were positively regulated by GacA, including
xadA
and
hsf
, predicted to encode outer membrane adhesion proteins, and 20 negatively regulated genes, including
gumC
and an antibacterial polypeptide toxin gene,
cvaC
. These results suggest that GacA of
X. fastidiosa
regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of
X. fastidiosa
to environmental stresses and the competition within the host xylem.
Oenococcus oeni
Genome Plasticity Is Associated with Fitness
Authors: Bon Elisabeth , Delaherche Arnaud , Bilhère Eric , De Daruvar Antoine , Lonvaud-Funel Aline , Le Marrec Claire
Published: February 2009

Oenococcus oeni
strains are well-known for their considerable phenotypic variations in terms of tolerance to harsh wine conditions and malolactic activity. Genomic subtractive hybridization (SH) between two isolates with differing enological potentials was used to elucidate the genetic bases of this intraspecies diversity and identify novel genes involved in adaptation to wine. SH revealed 182 tester-specific fragments corresponding to 126 open reading frames (ORFs). A large proportion of the chromosome-related ORFs resembled genes involved in carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication, recombination, and repair. Six regions of genomic plasticity were identified, and their analysis suggested that both limited recombination and insertion/deletion events contributed to the vast genomic diversity observed in
O. oeni
. The association of selected sequences with adaptation to wine was further assessed by screening a large collection of strains using PCR. No sequences were found to be specific to highly performing (HP) strains alone. However, there was a statistically significant positive association between HP strains and the presence of eight gene sequences located on regions 2, 4, and 5. Gene expression patterns were significantly modified in HP strains, following exposure to one or more of the common stresses in wines. Regions 2 and 5 showed no traces of mobile elements and had normal GC content. In contrast, region 4 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhances the fitness of
O. oeni
strains.
Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum
Chloroflexi
Authors: Speirs Lachlan , Nittami Tadashi , McIlroy Simon , Schroeder Sarah , Seviour Robert J.
Published: February 2009

Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum
Chloroflexi
. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-β-hydroxyalkanoates or polyphosphate.
Functional Expression of a Bacterial Xylose Isomerase in
Saccharomyces cerevisiae
Authors: Brat Dawid , Boles Eckhard , Wiedemann Beate
Published: February 2009

In industrial fermentation processes, the yeast
Saccharomyces cerevisiae
is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like
d
-xylose and
l
-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in
S. cerevisiae
have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium
Clostridium phytofermentans
in
S. cerevisiae
. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize
d
-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from
Piromyces
sp. strain E2 and
Thermus thermophilus
, which were the only two XIs previously functionally expressed in
S. cerevisiae
. The activity and kinetic parameters of the new enzyme are comparable to those of the
Piromyces
XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of
S. cerevisiae
. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.
Elimination of
Escherichia coli
O157:H7 from Alfalfa Seeds through a Combination of High Hydrostatic Pressure and Mild Heat
Authors: Neetoo Hudaa , Pizzolato Thompson , Chen Haiqiang
Published: February 2009

Escherichia coli
O157:H7 has been associated with contaminated seed sprout outbreaks. The majority of these outbreaks have been traced to sprout seeds contaminated with low levels of pathogens. Sanitizing sprout seeds presents a unique challenge in the arena of produce safety in that even a low residual pathogen population remaining on contaminated seed after treatments appears capable of growing to very high levels during sprouting. In this study, the effectiveness of high-pressure treatment in combination with low and elevated temperatures was assessed for its ability to eliminate
E. coli
O157:H7 on artificially contaminated alfalfa seeds. Inoculated seed samples were treated at 600 MPa for 2 min at 4, 20, 25, 30, 35, 40, 45, and 50°C. The pressure sensitivity of the pathogenic bacteria was strongly dependent on the treatment temperature. At 40°C, the process was adequate in eliminating a 5-log-unit population on the seeds with no adverse effect on seed viability. Three treatments carried out at reduced pressure levels and/or extended treatment time, 550 MPa for 2 min at 40°C, 300 MPa for 2 min at 50°C, and 400 MPa for 5 min at 45°C, were equally lethal to the pathogen. When all three treatments were compared in terms of their impact on seed viability, the process of 550 MPa for 2 min at 40°C was the most desirable, achieving final germination percentages and sprout sizes statistically similar to those of control untreated seeds (
P
> 0.05).
Hierarchical Oligonucleotide Primer Extension as a Time- and Cost-Effective Approach for Quantitative Determination of
Bifidobacterium
spp. in Infant Feces
Authors: Hong Pei-Ying , Yap Gaik Chin , Lee Bee Wah , Chua Kaw Yan , Liu Wen-Tso
Published: February 2009

The
Bifidobacterium
spp. present in 10 infant fecal samples (4 from infants with eczema and 6 from healthy infants) were quantified with both hierarchical oligonucleotide primer extension (HOPE) and fluorescence in situ hybridization-flow cytometry. The relative abundances of
Bifidobacterium longum
and
B
.
catenulatum
with respect to the total bifidobacteria had a poor correlation (ρ, <0.600;
P
value, >0.208), presumably due to differences in primer specificity and the level of hybridization stringency of both methods. In contrast, the relative abundances of organisms of the genus
Bifidobacterium
against the total amplified 16S rRNA genes and those of
B
.
adolescentis
,
B
.
bifidum
, and
B
.
breve
against the genus
Bifidobacterium
exhibited a good statistical correlation (ρ, >0.783;
P
value, <0.066). This good comparability supports HOPE as a method to achieve high-throughput quantitative determination of bacterial targets in a time- and cost-effective manner.
Ecophysiology of “
Halarsenatibacter silvermanii
” Strain SLAS-1
T
, gen. nov., sp. nov., a Facultative Chemoautotrophic Arsenate Respirer from Salt-Saturated Searles Lake, California
Authors: Blum Jodi Switzer , Han Sukkyun , Lanoil Brian , Saltikov Chad , Witte Brian , Tabita F. Robert , Langley Sean , Beveridge Terry J. , Jahnke Linda , Oremland Ronald S.
Published: February 2009

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1
T
was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced
arrABD
gene cluster, cell envelope lipids, and its phylogenetic alignment within the order
Halanaerobacteriales
, assigning it the name “
Halarsenatibacter silvermanii
” strain SLAS-1
T
. We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order
Desulfovibriales
, the archaeon
Halorhabdus utahensis
, as well as a close homolog of strain SLAS-1
T
.
Rhizobia from Lanzarote, the Canary Islands, That Nodulate
Phaseolus vulgaris
Have Characteristics in Common with
Sinorhizobium meliloti
Isolates from Mainland Spain
Authors: Zurdo-Piñeiro José Luis , García-Fraile Paula , Rivas Raúl , Peix Alvaro , León-Barrios Milagros , Willems Anne , Mateos Pedro Francisco , Martínez-Molina Eustoquio , Velázquez Encarna , van Berkum Peter
Published: February 2009

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of
Phaseolus vulgaris
grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within
Sinorhizobium meliloti
(assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The
P. vulgaris
isolates and the group II LMW RNA
S. meliloti
isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of
S. meliloti
. The isolates from
P. vulgaris
nodulated bean but not
Medicago sativa
, while those recovered from
Medicago
,
Melilotus
, and
Trigonella
spp. nodulated both host legumes. The bean isolates also were distinguished from those of
Medicago
,
Melilotus
, and
Trigonella
spp. by
nodC
sequence analysis. The
nodC
sequences of the bean isolates were most similar to those reported for
S. meliloti
bv. mediterranense and
Sinorhizobium fredii
bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus
Rhizobium
, which perhaps is inconsistent with seed-borne transmission of
Rhizobium etli
from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.
p21-Activated Kinases Cla4 and Ste20 Regulate Vacuole Inheritance in
Saccharomyces cerevisiae
Authors: Bartholomew Clinton R. , Hardy Christopher F. J.
Published: February 2009

Each time
Saccharomyces cerevisiae
cells divide they ensure that both the mother and daughter cell inherit a vacuole by actively transporting a portion of the vacuole into the bud. As the mother cell begins budding, a tubular and vesicular segregation structure forms that is transported into the bud by the myosin V motor Myo2, which is bound to the vacuole-specific myosin receptor, Vac17 (41, 59, 70, 79). Upon arriving in the bud the segregation structure is resolved to found the daughter vacuole. The mechanism that regulates segregation structure resolution in a spatially dependent manner is unknown. In addition to resolving the segregation structure, Vac17 is degraded specifically in the bud to provide directionality to vacuole inheritance. It has been proposed that bud-specific degradation of Vac17 is promoted by proteins localized to or activated solely in the bud (77). The p21-activated kinases (PAKs) Cla4 and Ste20 are localized to and activated in the bud. Here we report that Cla4 is localized to the segregation structure just prior to segregation structure resolution, and cells lacking PAK function fail to resolve the segregation structure. Overexpression of either Cla4 or Ste20 inhibited vacuole inheritance and this inhibition was suppressed by the expression of nondegradable
VAC17
. Finally, PAK activity was required for Vac17 degradation in late M phase and
CLA4
overexpression promoted Vac17 degradation. We propose that Cla4 and Ste20 are bud-specific proteins that play roles in both segregation structure resolution and the degradation of Vac17.
Degradation of
Saccharomyces cerevisiae
Transcription Factor Gcn4 Requires a C-Terminal Nuclear Localization Signal in the Cyclin Pcl5
Authors: Streckfuss-Bömeke Katrin , Schulze Florian , Herzog Britta , Scholz Eva , Braus Gerhard H.
Published: February 2009

Pcl5 is a
Saccharomyces cerevisiae
cyclin that directs the phosphorylation of the general amino acid control transcriptional activator Gcn4 by the cyclin-dependent kinase (CDK) Pho85. Phosphorylation of Gcn4 by Pho85/Pcl5 initiates its degradation via the ubiquitin/proteasome system and is regulated by the availability of amino acids. In this study, we show that Pcl5 is a nuclear protein and that artificial dislocation of Pcl5 into the cytoplasm prevents the degradation of Gcn4. Nuclear localization of Pcl5 depends on the β-importin Kap95 and does not require Pho85, Gcn4, or the CDK inhibitor Pho81. Pcl5 nuclear import is independent on the availability of amino acids and is mediated by sequences in its C-terminal domain. The nuclear localization signal is distinct from other functional domains of Pcl5. This is corroborated by a C-terminally truncated Pcl5 variant, which carries the N-terminal nuclear domain of Pho80. This hybrid is still able to fulfill Pcl5 function, whereas Pho80, which is another Pho85 interacting cyclin, does not mediate Gcn4 degradation.
Efficient Gene Replacements in
Toxoplasma gondii
Strains Deficient for Nonhomologous End Joining
Authors: Fox Barbara A. , Ristuccia Jessica G. , Gigley Jason P. , Bzik David J.
Published: February 2009

A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite
Toxoplasma gondii
. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted
KU80
gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in
KU80
knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in
KU80
knockouts compared to wild-type strains. Target DNA flanks of only ∼500 bp were found to be sufficient for efficient gene replacements in
KU80
knockouts.
KU80
knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or γ-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in
Toxoplasma gondii
. Integration essentially occurs only at the homologous targeted sites in the
KU80
knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology.
Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80
Authors: Huynh My-Hang , Carruthers Vern B.
Published: February 2009

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, onToxoplasma gondiiare now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novelT. gondiigenes.
Subspecies IIIa and IIIb Salmonellae Are Defective for Colonization of Murine Models of Salmonellosis Compared to
Salmonella enterica
subsp. I Serovar Typhimurium
Authors: Katribe Erin , Bogomolnaya Lydia M. , Wingert Heather , Andrews-Polymenis Helene
Published: February 2009

Non-subspecies I salmonellae are commensals of cold-blooded vertebrates and cause sporadic disease in mammals. The reasons why non-subspecies I salmonellae do not circulate in populations of warm-blooded vertebrates, but instead only cause occasional disease in this niche, are unknown. We examined the ability of
Salmonella enterica
subsp. IIIa (subsp.
arizonae
) and subsp. IIIb (subsp.
diarizonae
) isolates to grow competitively with subspecies I (serovar Typhimurium) ATCC 14028 in vitro, to colonize
Salmonella
-sensitive BALB/c mice, and to persist in the intestine of
Salmonella
-resistant CBA/J mice in competitive infections. Subspecies IIIa had severely reduced intestinal colonization, intestinal persistence, and systemic spread in mice. Subspecies IIIa is nonmotile on swarming agar and thus may also have reduced motility under viscous conditions in vivo. Surprisingly, subspecies IIIb colonizes the intestinal tract of BALB/c mice normally yet does not spread systemically. Subspecies IIIb colonization of the intestine of CBA/J mice is reduced late in infection. In order to understand why these isolates do not colonize systemic sites, we determined that subspecies IIIa and IIIb are not internalized well and do not replicate in J774-A.1 murine macrophages, despite normal adherence to these cells. We further show that selected effectors of both type III secretion systems 1 and 2 are secreted by subspecies IIIa and IIIb in vitro but that each of these isolates secretes a different combination of effectors. We outline the phenotypic differences between these subspecies and subspecies I and provide a possible explanation for the inability of these strains to spread systemically in murine models.
An
Aeromonas caviae
Genomic Island Is Required for both O-Antigen Lipopolysaccharide Biosynthesis and Flagellin Glycosylation
Authors: Tabei S. Mohammed B. , Hitchen Paul G. , Day-Williams Michaela J. , Merino Susana , Vart Richard , Pang Poh-Choo , Horsburgh Gavin J. , Viches Silvia , Wilhelms Markus , Tomás Juan M. , Dell Anne , Shaw Jonathan G.
Published: February 2009

Aeromonas caviae
Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G+C content than the average aeromonad G+C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both
Campylobacter jejuni
and
Helicobacter pylori
. Two of the genes,
lst
and
lsg
, are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS; this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by
flmA
,
flmB
,
neuA
,
flmD
, and
neuB
are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the
A. caviae
wild-type strain, and to be absent from the
A. caviae flmD
mutant strain.
Characterization of a
Mesorhizobium loti
α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation
Authors: Kalloniati Chrysanthi , Tsikou Daniela , Lampiri Vasiliki , Fotelli Mariangela N. , Rennenberg Heinz , Chatzipavlidis Iordanis , Fasseas Costas , Katinakis Panagiotis , Flemetakis Emmanouil
Published: February 2009

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO
2
to bicarbonate, a reaction that participates in many biochemical and physiological processes.
Mesorhizobium loti
, the microsymbiont of the model legume
Lotus japonicus
, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the
CAA1
open reading frame from
M. loti
strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the
CAA1
gene in free-living and symbiotic rhizobia were examined by using an
M. loti
R7A disruption mutant strain. Our analysis revealed that
CAA1
is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH.
L. japonicus
plants inoculated with the
CAA1
mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ
13
C abundance. Based on these results, we propose that although
CAA1
is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH
3
protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ
13
C abundance suggest the recycling of at least part of the HCO
3
−
produced by CAA1.
Regulation ofigaAand the Rcs System by the MviA Response Regulator inSalmonella enterica
Authors: García-Calderón Clara B. , Casadesús Josep , Ramos-Morales Francisco
Published: February 2009

IgaA is a membrane protein that prevents overactivation of the Rcs regulatory system in enteric bacteria. Here we provide evidence thatigaAis the first gene in a σ70-dependent operon ofSalmonella entericaserovar Typhimurium that also includesyrfG,yrfH, andyrfI. We also show that the Lon protease and the MviA response regulator participate in regulation of theigaAoperon. Our results indicate that MviA regulatesigaAtranscription in an RpoS-dependent manner, but the results also suggest that MviA may regulate RcsB activation in an RpoS- and IgaA-independent manner.
Nanoscale Structural and Mechanical Properties of Nontypeable
Haemophilus influenzae
Biofilms
Authors: Arce Fernando Terán , Carlson Ross , Monds James , Veeh Richard , Hu Fen Z. , Stewart Philip S. , Lal Ratnesh , Ehrlich Garth D. , Avci Recep
Published: February 2009

Nontypeable
Haemophilus influenzae
(NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their importance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.
X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from
Burkholderia
sp. Strain FA1
Authors: Jitsumori Keiji , Omi Rie , Kurihara Tatsuo , Kurata Atsushi , Mihara Hisaaki , Miyahara Ikuko , Hirotsu Ken , Esaki Nobuyoshi
Published: February 2009

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from
Burkholderia
sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the α/β hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N
ε1
atom of Trp150 and the N
ε2
atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.
Functional Genomics ofEnterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome
Authors: Ballering Katie S. , Kristich Christopher J. , Grindle Suzanne M. , Oromendia Ana , Beattie David T. , Dunny Gary M.
Published: February 2009

The ability ofEnterococcus faecalisto form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistantE. faecalisinfections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome ofE. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified byrecombinaseinvivoexpressiontechnology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809ebrA(enterococcalbiofilmregulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery.