bluuebunny/crossref_metadata_2025 huggingface dataset:
Self-powered near field electron lithography
Authors: Lu Yuerui , Yoshimizu Norimasa , Lal Amit
Published: December 2009

Electron beam exposure is the tool of choice for highest resolution lithography but suffers from the low throughput during serial beam writing [T. Ito and S. Okazaki, Nature (London) 406, 1027 (2000); R. F. Pease and S. Y. Chou, Proc. IEEE 96, 248 (2008)]. The authors designed and developed a low-cost self-powered near-field electron lithography (SPEL) technique, which utilizes the spontaneously emitted energetic electrons from beta-emitting radioisotope thin films. This approach enables massively parallel e-beam lithography, with potentially arbitrarily large concurrently exposed surface area, controlled by the size of the radioactive source. This method potentially eliminates the need for vacuum systems and the electron focusing column as needed in the existing electron beam lithography systems. This will greatly simplify the overall lithographic system and reduce the cost of deep-subnanometer lithography. In SPEL system, emitted electrons are spatially blocked using a nanostenciled micromachined mask that is placed in proximity to an electron sensitive resist on the silicon substrate (Fig. 1). The electrons that are not blocked, impact and enter the e-beam resist, along with secondary electrons generated by primary electrons impacting the sidewalls of the stencil layer. Using three-dimensional 3D Monte Carlo (MC) simulations of electron paths, the authors show that the critical dimension (CD) in the system could be down to 20nm with 14.9keV electrons emitted from Ni63. The 3D MC simulation considered both elastic scattering and inelastic scattering for the high energetic primary electrons as well as the cascade secondary electrons generated. The 20nm limit is imposed by the secondary emission scattering. In order to prove the concept, experiments were conducted using the safe and low-activity (1mCi∕cm2) beta particle emitting Ni63 thin film source with electrons emitted at an average energy of 14.9keV. They exposed negative tone resist NEB31A, and a minimum gap between ebeam resist posts or CD of 100nm was achieved. The secondary electrons generated by the primary electron impact onto mask are also useful for exposure. Compared to traditional electron beam lithography, with serial raster scanning taking days to expose a wafer, the lithography system will enable parallel exposure of large patterns on arbitrarily large wafers in several minutes. SPEL may enable massively parallel top-down approach to realizing nanostructures in bulk quantities.
Limiting factors in sub-10nm scanning-electron-beam lithography
Authors: Cord Bryan , Yang Joel , Duan Huigao , Joy David C. , Klingfus Joseph , Berggren Karl K.
Published: December 2009

Achieving the highest possible resolution using scanning-electron-beam lithography (SEBL) has become an increasingly urgent problem in recent years, as advances in various nanotechnology applications [F. S. Bates and G. H. Fredrickson, Annu. Rev. Phys. Chem. 41, 525 (1990); Black et al., IBM J. Res. Dev. 51, 605 (2007); Yang et al., J. Chem. Phys. 116, 5892 (2002)] have driven demand for feature sizes well into the sub-10nm domain, close to the resolution limit of the current generation of SEBL processes. In this work, the authors have used a combination of calculation, modeling, and experiment to investigate the relative effects of resist contrast, beam scattering, secondary electron generation, system spot size, and metrology limitations on SEBL process resolution. In the process of investigating all of these effects, they have also successfully yielded dense structures with a pitch of 12nm at voltages as low as 10keV.
Off-axis emission properties for the extended Schottky electron source
Authors: Liu K. , Schwind G. A. , Swanson L. W.
Published: December 2009

The total energy distribution (TED) and reduced brightness from the ZrO∕W(100) Schottky electron source are extended to positions 4.4° off of the optical axis of the emitter. The faceted nature of the stable end form precludes a monotonic variation in emission properties with the beam angle. Both the full width at half maximum of the TED and the reduced brightness of the source increase by 37% and 18%, respectively, at an off-axis beam angle, consistent with the facet edge of the emitting (100) plane and at a constant current angular density of 0.5mA∕sr and 1800K source temperature. Similarly, the angular magnification and work function increase by 27% and 6%, respectively, as the beam angle increases from 0° to the facet edge at 4.4°.
Application of neural network to controlling three-dimensional electron-beam exposure distribution in resist
Authors: Guo C. , Lee S.-Y. , Lee S. H. , Kim B.-G. , Cho H.-K.
Published: December 2009

Electron-beam (e-beam) lithography is often employed for the fabrication of binary patterns with nanoscale features and grayscale structures. In such applications, exposure distribution along the depth dimension of the resist layer can significantly affect the geometry of the written patterns or structures. Most of the previous e-beam dose control schemes adopted a two-dimensional exposure model, ignoring the depth-dependent exposure variation. In this paper, a method which utilizes a neural network for explicitly controlling three-dimensional (3D) (vertical as well as lateral) distribution of exposure is described. A neural network allows one to achieve more balanced exposure distributions compared to a conventional region-by-region recursive approach. Through an extensive computer simulation, performance of the proposed approach to controlling (3D) exposure distribution has been analyzed in detail.
Amino-propyl-triethoxy-silane on aluminum fiducial grids for spatial-phase-locked electron-beam lithography
Authors: Samantaray C. B. , Hastings J. T.
Published: December 2009

A new fiducial grid suitable for low-energy (1–5keV) spatial-phase locked electron-beam lithography (SPLEBL) has been developed based on an aluminum layer with an organosilane grid pattern. The grids (400nm period) were made of two to three layers of 3-amino-propyl-triethoxy-silane (APTES) that was microcontact printed onto Al coated polymethyl methacrylate (PMMA). Signal-to-noise ratios (SNRs) were measured at beam energies ranging from 1to5keV at a dose of 20μC∕cm2 and compared to the SNRs of octadecanethiol (ODT) based fiducial grids on Au, Ag, and Cu. Although fiducial grids made of ODT on Au provided excellent SNR, Au is not suitable because of poor penetration and strong scattering of the primary electrons. ODT based grids on lower atomic number metal such as Ag or Cu yielded inadequate SNRs. In contrast, APTES-based grids on Al provide SNRs approaching ODT on Au with greatly reduced electron scattering. The authors successfully developed a grid removal process that allows patterns to be resolved in the underlying PMMA. These results represent a key step in the development of low-energy SPLEBL.
Method for improving the aspect ratio of ultrahigh-resolution structures in negative electron-beam resist
Authors: Sidorkin V. A. , Alkemade P. F. A. , Salemink H. W. M. , Schmits R. , van der Drift E.
Published: December 2009

A method for improving the aspect ratio of ultrahigh-resolution structures in negative electron-beam resist is provided for enhanced pattern-transfer capabilities. The essence of the proposed method is to form a protective “cap” on top of the resist structure by means of electron-beam-induced deposition (EBID) in a self-aligned approach. This is implemented by a combination of electron-beam lithography and EBID during exposure of the resist material in the presence of a precursor gas. The results of the proposed method using hydrogen silsesquioxane resist material are presented and discussed, including various attempts to further optimize this method.
RIAM Regulates the Cytoskeletal Distribution and Activation of PLC-γ1 in T Cells
Authors: Patsoukis Nikolaos , Lafuente Esther M. , Meraner Paul , Kim Jin sub , Dombkowski David , Li Lequn , Boussiotis Vassiliki A.
Published: December 2009

RIAM, an adaptor protein that regulates integrin signaling, is found to also function in T cell receptor–proximal signaling.
Memories of Receptors Endocytosed
Authors: Ray L. Bryan
Published: December 2009

Altered phosphorylation of a neurotransmitter receptor leads to specific loss of spatial memory.
Sealed with a Kinase
Authors: Foley John F.
Published: December 2009

G protein βγ subunits stimulate a Fyn-dependent pathway that reverses thrombin-induced permeability of endothelial cells.
Notch and Self-Renewal
Authors: Adler Elizabeth M.
Published: December 2009

PEDF promotes Notch signaling in adult neural stem cells by stimulating the p65-dependent nuclear export of the transcriptional corepressor N-CoR.
Simply Mycoplasma
Authors: Ray L. Bryan
Published: December 2009

A set of comprehensive analyses of the proteome, metabalome, and transcriptome of a relatively simple bacterium reveals insights into signaling, gene regulation, metabolism, and beyond.
Living by the Numbers
Authors: Yaffe Michael B.
Published: December 2009

Articles should be judged on their own merit, not the Impact Factor of the journal in which they are published.
Quantitative Analysis of Protein-Lipid Interactions Using Tryptophan Fluorescence
Authors: Kraft Catherine A. , Garrido Jose Luis , Leiva-Vega Luis , Romero Guillermo
Published: December 2009

The intrinsic fluorescence of tryptophan can be used to quantify the interaction between proteins and lipids.
bla
CMY-2
-Positive IncA/C Plasmids from
Escherichia coli
and
Salmonella enterica
Are a Distinct Component of a Larger Lineage of Plasmids
Authors: Call Douglas R. , Singer Randall S. , Meng Da , Broschat Shira L. , Orfe Lisa H. , Anderson Janet M. , Herndon David R. , Kappmeyer Lowell S. , Daniels Joshua B. , Besser Thomas E.
Published: December 2009

Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from
Escherichia coli
(cattle) and
Salmonella enterica
serovar Newport (human) and that carry the cephamycinase gene
bla
CMY-2
. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of
bla
CMY-2
, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in
floR
. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the
bla
CMY-2
plasmids from
E. coli
and
S. enterica
are genetically distinct from those originating from
Yersinia pestis
and
Photobacterium damselae
and distal to one originating from
Yersinia ruckeri
. While the overall similarity of these plasmids supports the likelihood of recent movements among
E. coli
and
S. enterica
hosts, their greater divergence from
Y. pestis
or
Y. ruckeri
suggests less recent plasmid transfer among these pathogen groups.
Effects of Ceftobiprole and Oxacillin on
mecA
Expression in Methicillin-Resistant
Staphylococcus aureus
Clinical Isolates
Authors: Shang Wenchi , Davies Todd A. , Flamm Robert K. , Bush Karen
Published: December 2009

Induction of
mecA
by ceftobiprole and oxacillin in 18 methicillin-resistant
Staphylococcus aureus
clinical isolates with various SCC
mec
cassettes was examined using reverse transcriptase PCR. The magnitude of
mecA
induction, 3- to 65-fold for ceftobiprole and 2- to 69-fold for oxacillin, did not correlate with ceftobiprole MICs (≤4 μg/ml), although the 11 isolates with at least 13-fold induction by oxacillin all had oxacillin MICs of ≥256 μg/ml. No correlation between magnitude of induction and SCC
mec
type was found.
Transferable Quinolone Resistance in
Vibrio cholerae
Authors: Kim Hong Bin , Wang Minghua , Ahmed Sabeena , Park Chi Hye , LaRocque Regina C. , Faruque Abu S. G. , Salam Mohammed A. , Khan Wasif A. , Qadri Firdausi , Calderwood Stephen B. , Jacoby George A. , Hooper David C.
Published: December 2009

Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of
Vibrio cholerae
over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in
gyrA
, which encodes a subunit of DNA gyrase, followed by mutation in
parC
, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing
qnrVC3
, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that
qnrVC3
confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of
V. cholerae
and on the chromosomes of
Escherichia coli
transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in
V. cholerae
are linked to cumulative mutations in quinolone targets and most recently to a
qnr
gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.
Chloroquine Modulates HIV-1-Induced Plasmacytoid Dendritic Cell Alpha Interferon: Implication for T-Cell Activation
Authors: Martinson Jeffrey A. , Montoya Carlos J. , Usuga Xiomara , Ronquillo Rollie , Landay Alan L. , Desai Seema N.
Published: December 2009

Plasmacytoid dendritic cells (pDC) contribute to antiviral immunity mainly through recognition of microbial products and viruses via intracellular Toll-like receptor 7 (TLR7) or TLR9, resulting in the production of type I interferons (IFNs). Although interferons reduce the viral burden in the acute phase of infection, their role in the chronic phase is unclear. The presence of elevated plasma IFN-α levels in advanced HIV disease and its association with microbial translocation in chronic HIV infection lead us to hypothesize that IFN-α could contribute to immune activation. Blocking of IFN-α production using chloroquine, an endosomal inhibitor, was tested in a novel
in vitro
model system with the aim of characterizing the effects of chloroquine on HIV-1-mediated TLR signaling, IFN-α production, and T-cell activation. Our results indicate that chloroquine blocks TLR-mediated activation of pDC and MyD88 signaling, as shown by decreases in the levels of the downstream signaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-α synthesis. Chloroquine decreased CD8 T-cell activation induced by aldrithiol-2-treated HIV-1 in peripheral blood mononuclear cell cultures. In addition to blocking pDC activation, chloroquine also blocked negative modulators of the T-cell response, such as indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1). Our results indicate that TLR stimulation and production of IFN-α by pDC contribute to immune activation and that blocking of these pathways using chloroquine may interfere with events contributing to HIV pathogenesis. Our results suggests that a safe, well-tolerated drug such as chloroquine can be proposed as an adjuvant therapeutic candidate along with highly active antiretroviral therapy to control immune activation in HIV-1 infection.
Blockade of X4-Tropic HIV-1 Cellular Entry by GSK812397, a Potent Noncompetitive CXCR4 Receptor Antagonist
Authors: Jenkinson Stephen , Thomson Michael , McCoy David , Edelstein Mark , Danehower Susan , Lawrence Wendell , Wheelan Pat , Spaltenstein Andrew , Gudmundsson Kristjan
Published: December 2009

GSK812397 is a potent entry inhibitor of X4-tropic strains of HIV-1, as demonstrated in multiple
in vitro
cellular assays (e.g., in peripheral blood mononuclear cells [PBMCs] and a viral human osteosarcoma [HOS] assay, mean 50% inhibitory concentrations [IC
50
s] ± standard errors of the means were 4.60 ± 1.23 nM and 1.50 ± 0.21 nM, respectively). The primary
in vitro
potency of GSK812397 was not significantly altered by the addition of serum proteins (2.55 [±0.12]-fold shift in the presence of human serum albumin and α-acid glycoprotein in the PBMC assay). Pharmacological characterization of GSK812397 in cell-based functional assays revealed it to be a noncompetitive antagonist of the CXCR4 receptor, with GSK812397 producing a concentration-dependent decrease in both an SDF-1-mediated chemotaxis and intracellular calcium release (IC
50
s were 0.34 ± 0.01 nM and 2.41 ± 0.50 nM, respectively). With respect to the antiviral activity of GSK812397, it was effective against a broad range of X4- and X4R5-utilizing clinical isolates. The potency and efficacy of GSK812397 were dependent on the individual isolate, with complete inhibition of infection observed with 24 of 30 isolates. GSK812397 did not show any detectable
in vitro
cytotoxicity and was highly selective for CXCR4, as determined using a wide range of receptors, enzymes, and transporters. Moreover, GSK812397 demonstrated acceptable pharmacokinetic properties and bioavailability across species. The data demonstrate that GSK812397 has antiviral activity against a broad range of X4-utilizing strains of HIV-1 via a noncompetitive antagonism of the CXCR4 receptor.
Effect of Biostimulation and Bioaugmentation on Degradation of Polyurethane Buried in Soil
Authors: Cosgrove L. , McGeechan P. L. , Handley P. S. , Robson G. D.
Published: December 2009

This work investigated biostimulation and bioaugmentation as strategies for removing polyurethane (PU) waste in soil. Soil microcosms were biostimulated with the PU dispersion agent “Impranil” and/or yeast extract or were bioaugmented with PU-degrading fungi, and the degradation of subsequently buried PU was determined. Fungal communities in the soil and colonizing buried PU were enumerated on solid media and were analyzed using denaturing gradient gel electrophoresis (DGGE). Biostimulation with yeast extract alone or in conjunction with Impranil increased PU degradation 62% compared to the degradation in untreated control soil and was associated with a 45% increase in putative PU degraders colonizing PU. Specific fungi were enriched in soil following biostimulation; however, few of these fungi colonized the surface of buried PU. Fungi used for soil bioaugmentation were cultivated on the surface of sterile wheat to form a mycelium-rich inoculum. Wheat, when added alone to soil, increased PU degradation by 28%, suggesting that wheat biomass had a biostimulating effect. Addition of wheat colonized with
Nectria haematococca
,
Penicillium viridicatum
,
Penicillium ochrochloron
, or an unidentified
Mucormycotina
sp. increased PU degradation a further 30 to 70%, suggesting that biostimulation and bioaugmentation were operating in concert to enhance PU degradation. Interestingly, few of the inoculated fungi could be detected by DGGE in the soil or on the surface of the PU 4 weeks after inoculation. Bioaugmentation did, however, increase the numbers of indigenous PU-degrading fungi and caused an inoculum-dependent change in the composition of the native fungal populations, which may explain the increased degradation observed. These results demonstrate that both biostimulation and bioaugmentation may be viable tools for the remediation of environments contaminated with polyurethane waste.
Quantitative Detection of Human Adenoviruses in Wastewater and Combined Sewer Overflows Influencing a Michigan River
Authors: Fong Theng-Theng , Phanikumar Mantha S. , Xagoraraki Irene , Rose Joan B.
Published: December 2009

Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (
n
= 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 × 10
6
viruses/liter and 5.35 × 10
5
viruses/liter, respectively. Adenovirus removal was <2 log
10
(99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (
n
= 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 × 10
3
viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.
Synergistic Effects of the
Lactobacillus acidophilus
Surface Layer and Nisin on Bacterial Growth
Authors: Prado-Acosta Mariano , Ruzal Sandra M. , Allievi Mariana C. , Palomino María Mercedes , Sanchez Rivas Carmen
Published: December 2009

We have previously described a murein hydrolase activity for the surface layer (S-layer) of
Lactobacillus acidophilus
ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative
Salmonella enterica
and potential pathogenic Gram-positive bacteria,
Staphylococcus aureus
and
Bacillus cereus.
In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.
Determination of the Diversity of
Rhodopirellula
Isolates from European Seas by Multilocus Sequence Analysis
Authors: Winkelmann Nadine , Jaekel Ulrike , Meyer Carolin , Serrano Wilbert , Rachel Reinhard , Rosselló-Mora Ramon , Harder Jens
Published: December 2009

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of
Rhodopirellula
sp., an attached-living planctomycete. The development of an MLSA for
Rhodopirellula baltica
enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes
acsA
,
guaA
,
trpE
,
purH
,
glpF
,
fumC
,
icd
,
glyA
, and
mdh
indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable
Rhodopirellula
species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living
Rhodopirellula
species.
Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization
Authors: Penny Christian , Nadalig Thierry , Alioua Malek , Gruffaz Christelle , Vuilleumier Stéphane , Bringel Françoise
Published: December 2009

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.
Rapid Evaluation of the Mycobactericidal Efficacy of Disinfectants in the Quantitative Carrier Test EN 14563 by Using Fluorescent
Mycobacterium terrae
Authors: Steinhauer Katrin , Eschenbacher Iris , Radischat Nadine , Detsch Christian , Niederweis Michael , Goroncy-Bermes Peter
Published: December 2009

To prevent transmission of mycobacterial pathogens, medical devices must be disinfected by germicides with proven mycobactericidal activity. The quantitative carrier test EN 14563 provides an international standard for evaluation of the mycobactericidal activity of disinfectants under practical conditions. However, tests according to the EN 14563 standard are based on cultivation, and results are available only after 21 days. The aim of this study was to accelerate assessment of dosage and contact times of mycobactericidal preparations based on the EN 14563 standard. To this end, a
gfp
gene was constructed with a codon usage adapted for
Mycobacterium tuberculosis
. Expression of the
gfp
m
2+
gene in
Mycobacterium terrae
improved the detection sensitivity by 10-fold over that with a previously used reporter strain. Peracetic acid and a cation-active formulation were tested as commercially available disinfectants for medical devices.
M. terrae
expressing
gfp
m
2+
was used to determine dosage and contact times for the two test germicides. Fluorescence measurements correlated well with growth of the reporter strain, demonstrating that the fluorescence reliably indicated the number of viable cells. The fluorescence enabled us to determine the mycobactericidal efficacy of the test disinfectants according to the quantitative carrier test EN 14563 standard within at least 15 days. In conclusion, this study establishes
gfp
m
2+
-expressing
M. terrae
as a new reporter strain for reliable evaluation of mycobactericidal activities of disinfectants with a superior sensitivity and in a significantly shorter time than previously possible.
Molecular Detection of Viable Bacterial Pathogens in Water by Ratiometric Pre-rRNA Analysis
Authors: Cangelosi Gerard A. , Weigel Kris M. , Lefthand-Begay Clarita , Meschke John S.
Published: December 2009

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples.
Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical
Burkholderia
Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions
Authors: Ferreira Ana S. , Leitão Jorge H. , Silva Inês N. , Pinheiro Pedro F. , Sousa Sílvia A. , Ramos Christian G. , Moreira Leonilde M.
Published: December 2009

The genus
Burkholderia
includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative
Burkholderia cepacia
complex (
Bc
c) and non-
Bc
c strains for the presence of the
bce-I
gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the
bce-II
cluster were named
bceM
to
bceU
and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the
B. cepacia
IST408
bceQ
and
bceR
genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the
bceS
gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the
bce-I
and
bce-II
clusters in a
B
urkholderia multivorans
EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10
Burkholderia
isolates tested was cepacian. The ability of
Burkholderia
strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.
Conjugative Transfer of Insecticidal Plasmid pHT73 from
Bacillus thuringiensis
to
B. anthracis
and Compatibility of This Plasmid with pXO1 and pXO2
Authors: Yuan Yongming , Zheng Dasheng , Hu Xiaomin , Cai Quanxin , Yuan Zhiming
Published: December 2009

Bacillus anthracis
, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with
B. cereus
and
B. thuringiensis
. In view of the human pathogenicity and the long persistence in the environment of
B. anthracis
, there is growing concern about the effects of genetic exchange with
B. anthracis
on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from
B. thuringiensis
strain KT0 could be efficiently transferred into two attenuated
B. anthracis
strains, Ba63002R (pXO1
+
pXO2
−
) and Ba63605R (pXO1
−
pXO2
+
), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 × 10
−4
and 1.6 × 10
−4
CFU/donor, respectively. The
B. anthracis
transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to
Helicoverpa armigera
larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of
B. thuringiensis
and closely related strains in food and in the environment.
Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7
Escherichia coli
Strain
Authors: Rump Lydia V. , Feng Peter C. H. , Fischer Markus , Monday Steven R.
Published: December 2009

The O-antigen (
rfb
) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic
Escherichia coli
strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS
629
, was observed within its
gne
gene.
trans
complementation with a functional
gne
gene from O157:H7 restored O157 antigen expression in MA6.
Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster, Representing a Core Group of the Betaproteobacterial Genus
Limnohabitans
Authors: Šimek Karel , Kasalický Vojtěch , Jezbera Jan , Jezberová Jitka , Hejzlar Josef , Hahn Martin W.
Published: December 2009

The distribution of the phylogenetically narrow R-BT065 cluster (
Betaproteobacteria
) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.
Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox
Authors: Vlaeminck Siegfried E. , Terada Akihiko , Smets Barth F. , De Clippeleir Haydée , Schaubroeck Thomas , Bolca Selin , Demeestere Lien , Mast Jan , Boon Nico , Carballa Marta , Verstraete Willy
Published: December 2009

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.
Archaeal and Bacterial Communities Respond Differently to Environmental Gradients in Anoxic Sediments of a California Hypersaline Lake, the Salton Sea
Authors: Swan Brandon K. , Ehrhardt Christopher J. , Reifel Kristen M. , Moreno Lilliana I. , Valentine David L.
Published: December 2009

Sulfidic, anoxic sediments of the moderately hypersaline Salton Sea contain gradients in salinity and carbon that potentially structure the sedimentary microbial community. We investigated the abundance, community structure, and diversity of
Bacteria
and
Archaea
along these gradients to further distinguish the ecologies of these domains outside their established physiological range. Quantitative PCR was used to enumerate 16S rRNA gene abundances of
Bacteria
,
Archaea
, and
Crenarchaeota
. Community structure and diversity were evaluated by terminal restriction fragment length polymorphism (T-RFLP), quantitative analysis of gene (16S rRNA) frequencies of dominant microorganisms, and cloning and sequencing of 16S rRNA.
Archaea
were numerically dominant at all depths and exhibited a lesser response to environmental gradients than that of
Bacteria
. The relative abundance of
Crenarchaeota
was low (0.4 to 22%) at all depths but increased with decreased carbon content and increased salinity. Salinity structured the bacterial community but exerted no significant control on archaeal community structure, which was weakly correlated with total carbon. Partial sequencing of archaeal 16S rRNA genes retrieved from three sediment depths revealed diverse communities of
Euryarchaeota
and
Crenarchaeota
, many of which were affiliated with groups previously described from marine sediments. The abundance of these groups across all depths suggests that many putative marine archaeal groups can tolerate elevated salinity (5.0 to 11.8% [wt/vol]) and persist under the anaerobic conditions present in Salton Sea sediments. The differential response of archaeal and bacterial communities to salinity and carbon patterns is consistent with the hypothesis that adaptations to energy stress and availability distinguish the ecologies of these domains.
Alanine Mutagenesis of the Primary Antigenic Escape Residue Cluster, C1, of Apical Membrane Antigen 1
Authors: Dutta Sheetij , Dlugosz Lisa S. , Clayton Joshua W. , Pool Christopher D. , Haynes J. David , Gasser Robert A. , Batchelor Adrian H.
Published: December 2009

Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion ofPlasmodiummerozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strainPlasmodium falciparumAMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.
Role of RpoS in Virulence of Pathogens
Authors: Dong Tao , Schellhorn Herb E.
Published: December 2009

Understanding mechanisms of bacterial pathogenesis is critical for infectious disease control and treatment. Infection is a sophisticated process that requires the participation of global regulators to coordinate expression of not only genes coding for virulence factors but also those involved in other physiological processes, such as stress response and metabolic flux, to adapt to host environments. RpoS is a key response regulator to stress conditions in
E
scherichia coli
and many other proteobacteria. In contrast to its conserved well-understood role in stress response, effects of RpoS on pathogenesis are highly variable and dependent on species. RpoS contributes to virulence through either enhancing survival against host defense systems or directly regulating expression of virulence factors in some pathogens, while RpoS is dispensable, or even inhibitory, to virulence in others. In this review, we focus on the distinct and niche-dependent role of RpoS in virulence by surveying recent findings in many pathogens.
Global Regulation of Gene Expression and Cell Differentiation inCaulobacter crescentusin Response to Nutrient Availability
Authors: England Jennifer C. , Perchuk Barrett S. , Laub Michael T. , Gober James W.
Published: December 2009

In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments,Caulobacter crescentuscells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest thatCaulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period.
Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “
Candidatus
Liberibacter asiaticus”
Authors: Vahling Cheryl M. , Duan Yongping , Lin Hong
Published: December 2009

ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “
Candidatus
Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “
Ca.
Liberibacter asiaticus” gene (
nttA
) encoding the translocase was subsequently expressed in
Escherichia coli
and shown to enable
E.
coli
to import ATP directly into the cell. Competition assays with the heterologous
E. coli
system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “
Ca.
Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “
Ca.
Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment.
In
Salmonella enterica
, 2-Methylcitrate Blocks Gluconeogenesis
Authors: Rocco Christopher J. , Escalante-Semerena Jorge C.
Published: December 2009

Strains of
Salmonella enterica
serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report
in vivo
and
in vitro
data which show that a target of the 2-MC isomer produced by GltA (2-MC
GltA
) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MC
GltA
was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an
fbp
allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MC
GltA
and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MC
PrpC
) are not equally toxic to the cell, with 2-MC
GltA
being significantly more toxic than 2-MC
PrpC
. This difference in 2-MC toxicity is likely due to the fact that as a
si
-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.
Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in
Methanosarcina mazei
Authors: Welte Cornelia , Kallnik Verena , Grapp Marcel , Bender Gunes , Ragsdale Steve , Deppenmeier Uwe
Published: December 2009

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a
Methanosarcina mazei
Δ
ech
mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C
1
compounds.
Genome Sequence of the Cellulosome-Producing Mesophilic Organism
Clostridium cellulovorans
743B
Authors: Tamaru Yutaka , Miyake Hideo , Kuroda Kouichi , Nakanishi Akihito , Kawade Yujiro , Yamamoto Kousuke , Uemura Masaaki , Fujita Yasuhiro , Doi Roy H. , Ueda Mitsuyoshi
Published: December 2009

Clostridium cellulovorans
743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of
C. cellulovorans
743B.