bluuebunny/crossref_metadata_2025 huggingface dataset:
High-performance, low-noise enhancement-mode pseudomorphic high-electron-mobility transistor with gate recession by citric acid/hydrogen peroxide selective etching
Authors: Wang Chih-Cheng , Lin Yu-Ju , Huang Hou-Kuei , Wu Chang-Luen , Chang Chian-Sern , Wang Yeong-Her
Published: August 2007

An enhancement-mode pseudomorphic high-electron-mobility transistor (E-PHEMT) using a citric acid/hydrogen peroxide etching solution for gate recession is developed. The etch selectivity between GaAs and Al0.2Ga0.8As is more than 165 at 22°C, while the maximum transconductance for the fabricated device with the gate dimension of 160×0.25μm2 reaches 502mS∕mm at Vds=2.7V. Furthermore, at 12GHz, the 1dB compression output power and gain can reach 13.2dBm and 17.9dB, respectively. Operating at 12GHz, the minimum noise figure at the bias conditions of Vds=2V and Ids=11mA decreases to 0.45dB with the associated gain of 12.95dB, which is comparable to, or even better than that of, reported low-noise depletion- or enhancement-mode PHEMT devices. The standard deviation of the pinch-off voltage for the E-PHEMT across a 4in. diameter wafer could be as low as 50mV.
Ultrathin decoupled plasma nitridation SiON gate dielectrics prepared with various rf powers
Authors: Hu Chan-Yuan , Chen Shih-Chih , Chen Jone F. , Chang Shoou-Jinn , Wang Min-Hong , Yeh Vita , Chen Jung-Che
Published: August 2007

Decoupled plasma nitridation (DPN) SiON films for short channel complementary metal-oxide-semiconductor (CMOS) devices were prepared with various rf powers. As we increased the SiON deposition power from 200to400W, it was found that gate leakage current increased while gate breakdown voltage decreased. It was also found that the authors can achieve more uniform thickness and nitrogen distributions across the wafers, smaller off-state current, and better Vt rolloff characteristics from the PMOS field-effect transistors (MOSFETs) with 400W DPN SiON films. Furthermore, it was found that they can achieve larger hot carrier injection (HCI) lifetime and better HCI resistance from the NMOSFETs with 400W SiON films. However, it was also found that negative bias temperature instability lifetime of the PMOSFETs with 200W SiON films was longer.
Extreme high vacuum field emission microscope for study on the inherent fluctuation of field emission
Authors: Cho B. , Itagaki T. , Ishikawa T. , Rokuta E. , Oshima C.
Published: August 2007

An extreme high vacuum field emission microscope (XHV FEM) was constructed for the study of inherent fluctuations of field emission (FE) current. The damping and fluctuation behaviors of FE current from clean W(111) tips at 90K were observed using the XHV FEM. Exposure to XHV of 7.5×10−10Pa continued to damp FE current for more than 2000min. The fluctuation (∼0.01%) of FE current (1nA) for the clean W(111) tips was comparable to the corresponding shot noise fluctuation (0.005%), demonstrating the suitability of the XHV FEM for the inherent fluctuation study of the FE process. After saturating the tip surface with hydrogen, the FE current showed fluctuations of <0.1%
1.3
μ
m
Ga0.11In0.89As0.24P0.76∕Ga0.27In0.73As0.67P0.33 compressive-strain multiple quantum well with n-type modulation-doped GaInP intermediate-barrier laser diodes
Authors: Lei Po-Hsun
Published: August 2007

The authors report on fabricating and characterizing a 1.3-μm Ga0.11In0.89As0.24P0.76∕Ga0.27In0.73As0.67P0.33 compressive-strain multiple quantum well with n-type modulation-doped GaInP intermediate-barrier laser diodes (LDs). The barrier region contains a 20Å Si-doped modulation-doped GaInP intermediate layer and two 40Å undoped Ga0.11In0.89As0.24P0.76 surrounding layers. The modulation-doped GaInP intermediate barrier can increase the optical gain by the modulated-electron Fermi-Dirac distribution function, and stop the electron overflow by the inherent barrier height. The surrounding layer serves as a buffer layer to prevent the overflow of Si dopant into the well; but it also alleviates the tensile strain of the modulation-doped GaInP intermediate barrier to maintain a compressive-strain quantum well. We measured the threshold current density, differential quantum efficiency, internal quantum efficiency, and internal optical loss as a function of cavity length and doping concentration of the modulation-doped GaInP intermediate barrier. The theoretical and experimental results show that the optimal doping concentration of the modulation-doped GaInP intermediate barrier is 3×1018cm−3. With this optimal condition, the 4μm ridge-stripe and 600μm cavity LDs without facet coating will exhibit the best performance, including a lower threshold current of 29mA, a higher slope efficiency of 0.215W∕A, a higher characteristic temperature of 87.6K, and a wavelength swing of 0.28nm∕K for the LDs operated in a temperature range of 10–80°C under continuous-wave operation.
Evaluation of new materials for plasmonic imaging lithography at 476nm using near field scanning optical microscopy
Authors: Backer Scott A. , Suez Itai , Fresco Zachary M. , Fréchet Jean M. J. , Conway Josh A. , Vedantam Shantha , Lee Hyojune , Yablonovitch Eli
Published: August 2007

A new resist formulation was successfully patterned using near field optical microscopy in order to simulate the conditions prevailing in silver based plasmonic imaging tools. Radiation at 476nm was transmitted through a near field scanning optical microscopy fiber probe tip to cross-link a film of poly(4-methacrylmethyl styrene) via polymerization of pendant methacryloyl groups using camphorquinone and dimethyl aniline as an initiating system. Patterns were generated by scanning at several speeds in order to moderate the dose while maintaining a constant probe height of about 5nm above the sample through shear force feedback. After development, lines corresponding to the exposed regions were observed. At a scanning speed of 4μm∕s, the observed pattern has a full width at half maximum of 275nm and a height of ∼25nm.
Investigation of heteroepitaxial growth of magnetite thin films
Authors: Sterbinsky G. E. , Cheng J. , Chiu P. T. , Wessels B. W. , Keavney D. J.
Published: August 2007

Epitaxial magnetite (Fe3O4) thin films were deposited by molecular beam epitaxy using molecular oxygen as the oxidant. Films deposited on (001) SrTiO3, (001) MgO, and (001) BaTiO3 surfaces are epitaxial with the film (001) parallel to the substrate (001) and the film ⟨100⟩ parallel to the substrate ⟨100⟩. X-ray magnetic circular dichroism was used to determine the relative Fe2+∕Fe3+ stoichiometry of the magnetite films, which was nearly independent of oxygen partial pressure over the range studied. All films show no in-plane magnetic anisotropy. Coercive fields ranged from 0.019to0.039T and depended on film roughness.
Real-time in situ flux monitoring in molecular beam epitaxy by wavelength-modulated atomic absorption spectroscopy
Authors: Vignaud D.
Published: August 2007

Wavelength-modulated atomic absorption spectroscopy has been experimentally studied for real-time flux monitoring in molecular beam epitaxy. Gallium was chosen for the purpose of demonstration. Extension of this technique to other group III element (Al and In) flux measurements should be straightforward, as well as to any other element for which an adequate tunable laser source is available. From the measured absorbance spectra, the scanning parameters (central wavelength and scanning width) were calculated and experimentally optimized. The photon flux dependency of the signal saturates at large flux, which is explained by stimulated transitions within a simple three-level scheme. The variation of the flux-monitoring signal was also studied as a function of the detected even harmonics (from 2f to 6f). For Ga, measurements at 4f or 6f show the best characteristics with respect to signal-to-noise ratio.
Identification of Isopentenol Biosynthetic Genes from
Bacillus subtilis
by a Screening Method Based on Isoprenoid Precursor Toxicity
Authors: Withers Sydnor T. , Gottlieb Shayin S. , Lieu Bonny , Newman Jack D. , Keasling Jay D.
Published: August 2007

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered
Escherichia coli
for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium
Bacillus subtilis
strain 6051 in
E. coli
engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (
yhfR
and
nudF
) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of
nudF
in
E. coli
engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.
Biosynthesis of
Staphylococcus aureus
Autoinducing Peptides by Using the
Synechocystis
DnaB Mini-Intein
Authors: Malone Cheryl L. , Boles Blaise R. , Horswill Alexander R.
Published: August 2007

The Agr quorum-sensing system of
Staphylococcus aureus
modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from
Synechocystis
sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against
S. aureus
. The AIP activity was confirmed through Agr interference and activation profiling with developed
S. aureus
reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of
S. aureus
AIPs for many biological applications.
Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered
Saccharomyces cerevisiae
Strain
Authors: Lu Chenfeng , Jeffries Thomas
Published: August 2007

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for
GND2
and
HXK2
with the genes for transaldolase (
TAL1
), transketolase (
TKL1
), and pyruvate kinase (
PYK1
) in the
Saccharomyces cerevisiae
strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for
TAL1
,
TKL1
, and
PYK1
were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be
GND2
-
TAL1
-
HXK2
-
TKL1
-
HXK2
-
PYK1
. The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.
Multiplex PCR for Simultaneous Detection of Bacteria of the Genus
Listeria
,
Listeria monocytogenes
, and Major Serotypes and Epidemic Clones of
L. monocytogenes
Authors: Chen Yi , Knabel Stephen J.
Published: August 2007

A multiplex PCR assay which combines detection of bacteria of the genus
Listeria
,
Listeria monocytogenes
serotypes 1/2a and 4b, and epidemic clones I, II, and III of
L. monocytogenes
was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.
Effect of Insect Larval Midgut Proteases on the Activity of
Bacillus thuringiensis
Cry Toxins
Authors: Fortier Mélanie , Vachon Vincent , Frutos Roger , Schwartz Jean-Louis , Laprade Raynald
Published: August 2007

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of
Bacillus thuringiensis
insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated
Manduca sexta
midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.
Changes in Bacterial Populations and in Biphenyl Dioxygenase Gene Diversity in a Polychlorinated Biphenyl-Polluted Soil after Introduction of Willow Trees for Rhizoremediation
Authors: de Cárcer Daniel Aguirre , Martín Marta , Karlson Ulrich , Rivilla Rafael
Published: August 2007

The aim of this study was to analyze the structural and functional changes occurring in a polychlorinated-biphenyl (PCB)-contaminated soil ecosystem after the introduction of a suitable host plant for rhizoremediation (
Salix viminalis
). We have studied the populations and phylogenetic distribution of key bacterial groups (
Alpha
- and
Betaproteobacteria
,
Acidobacteria
, and
Actinobacteria
) and the genes encoding iron-sulfur protein α (ISPα) subunits of the toluene/biphenyl dioxygenases in soil and rhizosphere by screening gene libraries using temperature gradient gel electrophoresis. The results, based on the analysis of 415 clones grouped into 133 operational taxonomic units that were sequence analyzed (>128 kbp), show that the rhizospheric bacterial community which evolved from the native soil community during the development of the root system was distinct from the soil community for all groups studied except for the
Actinobacteria. Proteobacteria
were enriched in the rhizosphere and dominated both in rhizosphere and soil. There was a higher than expected abundance of
Betaproteobacteria
in the native and in the planted PCB-polluted soil. The ISPα sequences retrieved indicate a high degree of catabolic and phylogenetic diversity. Many sequences clustered with biphenyl dioxygenase sequences from gram-negative bacteria. A distinct cluster that was composed of sequences from this study, some previously described environmental sequences, and a putative ISPα from
Sphingomonas wittichii
RW1 seems to contain greater diversity than the presently recognized toluene/biphenyl dioxygenase subfamily. Moreover, the rhizosphere selected for two ISPα sequences that accounted for almost 60% of the gene library and were very similar to sequences harbored by
Pseudomonas
species.
Identification of Indole Derivatives as Self-Growth Inhibitors of
Symbiobacterium thermophilum
, a Unique Bacterium Whose Growth Depends on Coculture with a
Bacillus
sp
Authors: Watsuji Tomo-o , Yamada Shinya , Yamabe Tomoya , Watanabe Yuka , Kato Taira , Saito Takao , Ueda Kenji , Beppu Teruhiko
Published: August 2007

Symbiobacterium thermophilum
is a syntrophic bacterium whose growth depends on coculture with a
Bacillus
sp. Recently, we discovered that CO
2
generated by
Bacillus
is the major inducer for the growth of
S. thermophilum
; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by
S. thermophilum
. We succeeded in purifying two substances from an ether extract of the culture supernatant of
S. thermophilum
by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3′-indolyl)indoxyl (BII) and 1,1-bis(3′-indolyl)ethane (BIE). The pure growth of
S. thermophilum
was inhibited by authentic BII and BIE with MICs of 12 and 7 μg/ml, respectively; however, its growth in coculture with
Bacillus
was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 μg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of
S. thermophilum
were far lower than the MICs (<0.1 μg/ml) while a marked amount of BIE (6 to 7 μg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that
Bacillus
benefits
S. thermophilum
by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.
WdStuAp, an APSES Transcription Factor, Is a Regulator of Yeast-Hyphal Transitions in
Wangiella
(
Exophiala
)
dermatitidis
Authors: Wang Qin , Szaniszlo Paul J.
Published: August 2007

APSES transcription factors are well-known regulators of fungal cellular development and differentiation. To study the function of an APSES protein in the fungus
Wangiella dermatitidis
, a conidiogenous and polymorphic agent of human phaeohyphomycosis with yeast predominance, the APSES transcription factor gene Wd
STUA
was cloned, sequenced, disrupted, and overexpressed. Analysis showed that its derived protein was most similar to the APSES proteins of other conidiogenous molds and had its APSES DNA-binding domain located in the amino-terminal half. Deletion of Wd
STUA
in
W. dermatitidis
induced convoluted instead of normal smooth colony surface growth on the rich yeast maintenance agar medium yeast extract-peptone-dextrose agar (YPDA) at 37°C. Additionally, deletion of Wd
STUA
repressed aerial hyphal growth, conidiation, and invasive hyphal growth on the nitrogen-poor, hypha-inducing agar medium potato dextrose agar (PDA) at 25°C. Ectopic overexpression of Wd
STUA
repressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. These new results provide additional insights into the diverse roles played by APSES factors in fungi. They also suggest that the transcription factor encoded by Wd
STUA
is both a positive and negative morphotype regulator in
W. dermatitidis
and possibly other of the numerous human pathogenic, conidiogenous fungi capable of yeast growth.
Genotypic Evolution of Azole Resistance Mechanisms in Sequential
Candida albicans
Isolates
Authors: Coste Alix , Selmecki Anna , Forche Anja , Diogo Dorothée , Bougnoux Marie-Elisabeth , d'Enfert Christophe , Berman Judith , Sanglard Dominique
Published: August 2007

TAC1
(for transcriptional activator of
CDR
genes) is critical for the upregulation of the ABC transporters
CDR1
and
CDR2
, which mediate azole resistance in
Candida albicans
. While a wild-type
TAC1
allele drives high expression of
CDR1/2
in response to inducers, we showed previously that
TAC1
can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of
CDR1/2
. High azole resistance levels are achieved when
C. albicans
carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the
TAC1
locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (
MTL
) locus. Both are located on the Chr 5 left arm along with
ERG11
(target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the
TAC1
and
ERG11
alleles and for Chr 5 alterations. While recovered
ERG11
alleles contained known mutations, 17 new
TAC1
alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired
TAC1
hyperactive alleles and, in most cases,
ERG11
mutant alleles by LOH events not systematically including the
MTL
locus.
TAC1
LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the
TAC1
locus, or the loss and reduplication of the entire Chr 5. In one case, two independent
TAC1
hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them
TAC1
and
ERG11
. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least
ERG11
and
TAC1
) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in
C. albicans
is therefore a powerful instrument for generating genetic diversity.
Differential Regulation and Substrate Preferences in Two Peptide Transporters of
Saccharomyces cerevisiae
Authors: Cai Houjian , Hauser Melinda , Naider Fred , Becker Jeffrey M.
Published: August 2007

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in
Saccharomyces cerevisiae
. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [
14
C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides.
S. cerevisiae
W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [
14
C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of
PTR2, DAL5
expression was influenced by the addition of Leu and by the
CUP9
gene. However,
DAL5
expression was downregulated in the presence of leucine and the absence of
CUP9
, whereas
PTR2
was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.
Host Protein Binding and Adhesive Properties of H6 and H7 Flagella of Attaching and Effacing
Escherichia coli
Authors: Erdem Ayşen L. , Avelino Fabiola , Xicohtencatl-Cortes Juan , Girón Jorge A.
Published: August 2007

It had been suggested that the flagella of enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC
fliC
deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.
Structure of the
Haemophilus influenzae
HMW1B Translocator Protein: Evidence for a Twin Pore
Authors: Li Huilin , Grass Susan , Wang Tao , Liu Tianbo , St. Geme Joseph W.
Published: August 2007

Secretion of the
Haemophilus influenzae
HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded β-barrel domain, the approximate size of the β-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 Å resolution. The native HMW1B structure is a dimer of β-barrels, with each β-barrel measuring 40 Å by 50 Å in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.
Cwp84, a Surface-Associated Protein of
Clostridium difficile
, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins
Authors: Janoir Claire , Péchiné Séverine , Grosdidier Charlotte , Collignon Anne
Published: August 2007

Clostridium difficile
pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of
C. difficile
, which is highly immunogenic in patients with
C. difficile
-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of
C. difficile
. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.
Characterization and Biological Role of the O-Polysaccharide Gene Cluster ofYersinia enterocoliticaSerotype O:9
Authors: Skurnik Mikael , Biedzka-Sarek Marta , Lübeck Peter S. , Blom Tea , Bengoechea José Antonio , Pérez-Gutiérrez Camino , Ahrens Peter , Hoorfar Jeffrey
Published: August 2007

Yersinia enterocoliticaserotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) inY. enterocoliticaO:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer ofN-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of thegndgene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster,galFandgalU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.
Transcriptional Regulation of Stress Response and Motility Functions inHelicobacter pyloriIs Mediated by HspR and HrcA
Authors: Roncarati Davide , Danielli Alberto , Spohn Gunther , Delany Isabel , Scarlato Vincenzo
Published: August 2007

ThehrcAandhspRgenes ofHelicobacter pyloriencode two transcriptional repressor proteins that negatively regulate expression of thegroES-groELandhrcA-grpE-dnaKoperons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain andhspRandhrcAsingly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.
Nanoscale Visualization of a Fibrillar Array in the Cell Wall of Filamentous Cyanobacteria and Its Implications for Gliding Motility
Authors: Read Nicholas , Connell Simon , Adams David G.
Published: August 2007

Many filamentous cyanobacteria are motile by gliding, which requires attachment to a surface. There are two main theories to explain the mechanism of gliding. According to the first, the filament is pushed forward by small waves that pass along the cell surface. In the second, gliding is powered by the extrusion of slime through pores surrounding each cell septum. We have previously shown that the cell walls of several motile cyanobacteria possess an array of parallel fibrils between the peptidoglycan and the outer membrane and have speculated that the function of this array may be to generate surface waves to power gliding. Here, we report on a study of the cell surface topography of two morphologically different filamentous cyanobacteria, using field emission gun scanning electron microscopy (FEGSEM) and atomic force microscopy (AFM). FEGSEM and AFM images ofOscillatoriasp. strain A2 confirmed the presence of an array of fibrils, visible as parallel corrugations on the cell surface. These corrugations were also visualized by AFM scanning of fully hydrated filaments under liquid; this has not been achieved before for filamentous bacteria. FEGSEM images ofNostoc punctiformerevealed a highly convoluted, not parallel, fibrillar array. We conclude that an array of parallel fibrils, beneath the outer membrane ofOscillatoria, may function in the generation of thrust in gliding motility. The array of convoluted fibrils inN. punctiformemay have an alternative function, perhaps connected with the increase in outer membrane surface area resulting from the presence of the fibrils.
Contribution of the FtsQ Transmembrane Segment to Localization to the Cell Division Site
Authors: Scheffers Dirk-Jan , Robichon Carine , Haan Gert Jan , den Blaauwen Tanneke , Koningstein Gregory , van Bloois Edwin , Beckwith Jon , Luirink Joen
Published: August 2007

The
Escherichia coli
cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an
ftsQ
mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.
Binary Toxin Production in
Clostridium difficile
Is Regulated by CdtR, a LytTR Family Response Regulator
Authors: Carter Glen P. , Lyras Dena , Allen David L. , Mackin Kate E. , Howarth Pauline M. , O'Connor Jennifer R. , Rood Julian I.
Published: August 2007

Clostridium difficile
binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various
C. difficile
isolates, including the “hypervirulent” NAP1/027 epidemic strains. In contrast to the two major toxins from
C. difficile
, toxin A and toxin B, little is known about the role of CDT in virulence or how
C. difficile
regulates its production. In this study we have shown that in addition to the
cdtA
and
cdtB
toxin structural genes, a functional
cdt
locus contains a third gene, here designated
cdtR
, which is predicted to encode a response regulator. By introducing functional binary toxin genes into
cdtR
+
and
cdtR
-negative strains of
C. difficile
, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional
cdtR
gene; an internal deletion within
cdtR
resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact
cdtAB
genes or
cdtAB
pseudogenes also did not have
cdtR
, with the entire
cdt
locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from
Staphylococcus aureus
.
Transducing Particles of
Staphylococcus aureus
Pathogenicity Island SaPI1 Are Comprised of Helper Phage-Encoded Proteins
Authors: Tallent Sandra M. , Langston Timothy B. , Moran Richard G. , Christie Gail E.
Published: August 2007

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80α was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80α and SaPI1 virions, and all were encoded by 80α. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.
Defining the Stressome ofMycobacterium aviumsubsp.paratuberculosisIn Vitro and in Naturally Infected Cows
Authors: Wu Chia-wei , Schmoller Shelly K. , Shin Sung Jae , Talaat Adel M.
Published: August 2007

Mycobacterium aviumsubsp.paratuberculosiscauses an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile ofM. aviumsubsp.paratuberculosisexposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis ofM. aviumsubsp.paratuberculosis. In this report, the stress response ofM. aviumsubsp.paratuberculosison a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups ofM. aviumsubsp.paratuberculosisthat were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n= 597), suggesting the high sensitivity ofM. aviumsubsp.paratuberculosisto acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar inM. aviumsubsp.paratuberculosisandMycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g.,sigHandsigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of sixM. aviumsubsp.paratuberculosismutants with inactivation of differentially regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g.,lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could be applied to analyze other intracellular pathogens.
The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in
Salmonella enterica
Authors: Otte Michele M. , Woodson Jesse D. , Escalante-Semerena Jorge C.
Published: August 2007

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a
Salmonella enterica
strain lacking a functional
cobU
gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP:adenosylcobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the
ycfN
gene in a Δ
cobU
strain abrogated Cbi salvaging. Introduction of a plasmid carrying the
ycfN
+
allele into a Δ
cobU
Δ
ycfN
strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.
Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Authors: Smith Darren L. , James Chloë E. , Sergeant Martin J. , Yaxian Yan , Saunders Jon R. , McCarthy Alan J. , Allison Heather E.
Published: August 2007

Infection of
Escherichia coli
by Shiga toxin-encoding bacteriophages (Stx phages) was the pivotal event in the evolution of the deadly Shiga toxin-encoding
E. coli
(STEC), of which serotype O157:H7 is the most notorious. The number of different bacterial species and strains reported to produce Shiga toxin is now more than 500, since the first reported STEC infection outbreak in 1982. Clearly, Stx phages are spreading rapidly, but the underlying mechanism for this dissemination has not been explained. Here we show that an essential and highly conserved gene product, YaeT, which has an essential role in the insertion of proteins in the gram-negative bacterial outer membrane, is the surface molecule recognized by the majority (ca. 70%) of Stx phages via conserved tail spike proteins associated with a short-tailed morphology. The
yaeT
gene was initially identified through complementation, and its role was confirmed in phage binding assays with and without anti-YaeT antiserum. Heterologous cloning of
E. coli yaeT
to enable Stx phage adsorption to
Erwinia carotovora
and the phage adsorption patterns of bacterial species possessing natural
yaeT
variants further supported this conclusion. The use of an essential and highly conserved protein by the majority of Stx phages is a strategy that has enabled and promoted the rapid spread of shigatoxigenic potential throughout multiple
E. coli
serogroups and related bacterial species. Infection of commensal bacteria in the mammalian gut has been shown to amplify Shiga toxin production in vivo, and the data from this study provide a platform for the development of a therapeutic strategy to limit this YaeT-mediated infection of the commensal flora.
Maintaining the Transcription Factor SpoIIID Level Late during Sporulation Causes Spore Defects in
Bacillus subtilis
Authors: Wang Lijuan , Perpich John , Driks Adam , Kroos Lee
Published: August 2007

During sporulation of
Bacillus subtilis
, four regulatory proteins act in the order σ
E
, SpoIIID, σ
K
, and GerE to temporally control gene expression in the mother cell. σ
E
and σ
K
work sequentially with core RNA polymerase to transcribe different sets of genes. SpoIIID and GerE are small, sequence-specific DNA-binding proteins that activate or repress transcription of many genes. Previous studies showed that transcriptionally active σ
K
RNA polymerase inhibits early mother cell gene expression, reducing accumulation of SpoIIID late in sporulation. Here, the effects of perturbing the mother cell gene regulatory network by maintaining the SpoIIID level late during sporulation are reported. Persistent expression was obtained by fusing
spoIIID
to the σ
K
-controlled
gerE
promoter on a multicopy plasmid. Fewer heat- and lysozyme-resistant spores were produced by the strain with persistent
spoIIID
expression, but the number of spores resistant to organic solvents was unchanged, as was their germination ability. Transmission electron microscopy showed structural defects in the spore coat. Reporter fusions to σ
K
-dependent promoters showed lower expression of
gerE
and
cotC
and higher expression of
cotD
. Altered expression of
cot
genes, which encode spore coat proteins, may account for the spore structural defects. These results suggest that one role of negative feedback by σ
K
RNA polymerase on early mother cell gene expression is to lower the level of SpoIIID late during sporulation in order to allow normal expression of genes in the σ
K
regulon.
The T4 RI Antiholin Has an N-Terminal Signal Anchor Release Domain That Targets It for Degradation by DegP
Authors: Tran Tram Anh T. , Struck Douglas K. , Young Ry
Published: August 2007

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a
degP
host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.
Proteins P24 and P41 Function in the Regulation of Terminal-Organelle Development and Gliding Motility in
Mycoplasma pneumoniae
Authors: Hasselbring Benjamin M. , Krause Duncan C.
Published: August 2007

Mycoplasma pneumoniae
is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of
M. pneumoniae
open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28
−
mutant established that the MPN311 mutant phenotype was not a function of loss of P28.
Organization of the Aerotaxis Receptor Aer in the Membrane of
Escherichia coli
Authors: Amin Divya N. , Taylor Barry L. , Johnson Mark S.
Published: August 2007

The Aer receptor guides
Escherichia coli
to specific oxygen and energy-generating niches. The input sensor in Aer is a flavin adenine dinucleotide-binding PAS domain, which is separated from a HAMP/signaling output domain by two membrane-spanning segments that flank a short (four-amino-acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intradimeric collisions from interdimeric collisions. Collisions between proximal residues in the membrane anchor were exclusively intra- or interdimeric but, with one exception, not both. Cross-linking profiles were consistent, with a rigid rather than flexible periplasmic loop and a tilted TM2 helix that crossed TM2′ at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that (i) included a maximum of three Aer dimers, (ii) did not swap neighbors over time, and (iii) appeared to be constrained by interactions in the cytosolic signaling domain.