bluuebunny/crossref_metadata_2025 huggingface dataset:
Special Relativity and Magnetism in an Introductory Physics Course
Authors: Piccioni R. G.
Published: February 2007

Too often, students in introductory courses are left with the impression that Einstein's special theory of relativity comes into play only when the relative speed of two objects is an appreciable fraction of the speed of light (c). In fact, relativistic length contraction, along with Coulomb's law, accounts quantitatively for the force on a charged particle as it moves relative to a current-carrying wire. That force, which in the reference frame of the wire we call “magnetic,” is measurable and important even at relative speeds on the order of 10−12c. This paper offers a straightforward way of introducing students to the connection between magnetism and special relativity and provides references to more in-depth treatments, especially those of E.R. Huggins1 and of E.M. Purcell as simplified by D.V. Schroeder.2
An Elementary Model of the Earth's Magnetic Field
Authors: Watt Jeanie I. , Roth Bradley J.
Published: February 2007

This article is intended as a guide for teaching geomagnetism in a high school or university introductory physics class. Many students find this subject fascinating. Instructors, however, often have a difficult time introducing this topic at an elementary level. Suggestions will be made on how to accomplish this using three electricity and magnetism problems commonly encountered in an introductory class.
Mentor-Mentee Relationships: A Two-Way Street
Authors: Hughes Shannon , Riendeau Diane
Published: February 2007

The mentor-mentee relationship should be a source of joy and growth for both participants. Too often, the mentee feels intimidated and the mentor overburdened. Mentor Diane Riendeau and mentee Shannon Hughes share their thoughts and experiences in the hopes that new teachers and experienced teachers alike will gain insights into the role each play in encouraging and promoting the teaching profession. These are the elements they describe as key to building a successful relationship with a fellow teacher and setting the climate for a valuable experience for both parties.
Interpretation of Genotype and Pharmacokinetics for Resistance to Fosamprenavir-Ritonavir-Based Regimens in Antiretroviral-Experienced Patients
Authors: Pellegrin Isabelle , Breilh Dominique , Coureau Gaelle , Boucher Sébastien , Neau Didier , Merel Patrick , Lacoste Denis , Fleury Hervé , Saux Marie-Claude , Pellegrin Jean-Luc , Lazaro Estibaliz , Dabis François , Thiébaut Rodolphe
Published: February 2007

In this study, named the Zephir study (Telzir-pharmacokinetics), 121 antiretroviral-experienced human immunodeficiency virus (HIV) patients failing on highly active antiretroviral therapy (HAART) were included in a prospective cohort and received a fosamprenavir-ritonavir (700 mg/100 mg twice a day)-based regimen. The impact of baseline HIV type 1 (HIV-1) mutations, pharmacokinetic (PK) parameters, and genotype inhibitory quotient (GIQ) on the virological response at week 12 (W12) was assessed. HIV reverse transcriptase and protease were sequenced at W0. The response at W12 was defined as <2.3 log10HIV-1 RNA copies/ml or a virus load decrease of ≥1 log10copies/ml. W4 amprenavir PK were determined by high-performance liquid chromatography. Patients had a median of nine previous treatments over 8 years. Median W0 values were as follows: 295 CD4+/μl, 4.4 log10HIV-1 RNA copies/ml, and 6 protease- and 5 nucleotide reverse transcription inhibitor-related mutations. Respective values for minimum concentration of drug in serum (Cmin) and area under the concentration-time curve (AUC) from 0 to 24 h were 1,400 ng/ml and 35 mg·h/ml. At W12, 52% of the patients were successes, with a median decrease of −0.7 log10HIV-1 RNA copies/ml. The Zephir mutation score included 12 IAS protease mutations associated with poorer virological response: L10I/F/R/V, L33F, M36I, M46I/L, I54L/M/T/V, I62V, L63P, A71I/L/V/T, G73A/C/F/T, V82A/F/S/T, I84V, L90M, and polymorphism mutations I13V, L19I, K55R, and L89M. Comparing <4 versus ≥4 mutations, HIV-1 RNA decreases were −2.3 log10copies/ml versus −0.1 log10copies/ml (P< 10−4) with 93% versus 19% successes (P< 10−4), respectively. This score predicted W12 failure with 94% sensitivity, versus 31% for the ANRS 2005 algorithm.Cmin(<1,600 ng/ml), AUC (<40 mg·h/ml), and GIQ (<300) values were associated with failure (allPvalues were <10−4). The need to test genotype-based algorithms using different patient databases before their implementation in clinical practice is highlighted. Specific mutations, PK and GIQ, provide relevant information for monitoring fosamprenavir-ritonavir-based HAART.
Determination of Antibiotic Efficacy against
Bacillus anthracis
in a Mouse Aerosol Challenge Model
Authors: Heine Henry S. , Bassett Jennifer , Miller Lynda , Hartings Justin M. , Ivins Bruce E. , Pitt M. Louise , Fritz David , Norris Sarah L. , Byrne W. Russell
Published: February 2007

An anthrax spore aerosol infection mouse model was developed as a first test of in vivo efficacy of antibiotics identified as active against
Bacillus anthracis
. Whole-body, 50% lethal dose (LD
50
) aerosol challenge doses in a range of 1.9 × 10
3
to 3.4 × 10
4
CFU with spores of the fully virulent Ames strain were established for three inbred and one outbred mouse strain (A/J, BALB/c, C57BL, and Swiss Webster). The BALB/c strain was further developed as a model for antibiotic efficacy. Time course microbiological examinations of tissue burdens in mice after challenge showed that spores could remain dormant in the lungs while vegetative cells disseminated to the mediastinal lymph nodes and then to the spleen, accompanied by bacteremia. For antibiotic efficacy studies, BALB/c mice were challenged with 50 to 100 LD
50
of spores followed by intraperitoneal injection of either ciprofloxacin at 30 mg/kg of body weight (every 12 h [q12h]) or doxycycline at 40 mg/kg (q6h). A control group was treated with phosphate-buffered saline (PBS) q6h. Treatment was begun 24 h after challenge with groups of 10 mice for 14 or 21 days. The PBS-treated control mice all succumbed (10/10) to inhalation anthrax infection within 72 h. Sixty-day survival rates for ciprofloxacin and doxycycline-treated groups were 8/10 and 9/10, respectively, for 14-day treatment and 10/10 and 7/10 for 21-day treatment. Delayed treatment with ciprofloxacin initiated 36 and 48 h postexposure resulted in 80% survival and was statistically no different than early (24 h) postexposure treatment. Results using this mouse model correlate closely with clinical observations of inhalational anthrax in humans and with earlier antibiotic studies in the nonhuman primate inhalational anthrax model.
Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years
Authors: Katayama Taiki , Tanaka Michiko , Moriizumi Jun , Nakamura Toshio , Brouchkov Anatoli , Douglas Thomas A. , Fukuda Masami , Tomita Fusao , Asano Kozo
Published: February 2007

Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.
Carbohydrate Starvation Causes a Metabolically Active but Nonculturable State in
Lactococcus lactis
Authors: Ganesan Balasubramanian , Stuart Mark R. , Weimer Bart C.
Published: February 2007

This study characterized the ability of lactococci to become nonculturable under carbohydrate starvation while maintaining metabolic activity. We determined the changes in physiological parameters and extracellular substrate levels of multiple lactococcal strains under a number of environmental conditions along with whole-genome expression profiles. Three distinct phases were observed, logarithmic growth, sugar exhaustion, and nonculturability. Shortly after carbohydrate starvation, each lactococcal strain lost the ability to form colonies on solid media but maintained an intact cell membrane and metabolic activity for over 3.5 years. ML3, a strain that metabolized lactose rapidly, reached nonculturability within 1 week. Strains that metabolized lactose slowly (SK11) or not at all (IL1403) required 1 to 3 months to become nonculturable. In all cases, the cells contained at least 100 pM of intracellular ATP after 6 months of starvation and remained at that level for the remainder of the study. Aminopeptidase and lipase/esterase activities decreased below detection limits during the nonculturable phase. During sugar exhaustion and entry into nonculturability, serine and methionine were produced, while glutamine and arginine were depleted from the medium. The cells retained the ability to transport amino acids via proton motive force and peptides via ATP-driven translocation. The addition of branched-chain amino acids to the culture medium resulted in increased intracellular ATP levels and new metabolic products, indicating that branched-chain amino acid catabolism resulted in energy and metabolic products to support survival during starvation. Gene expression analysis showed that the genes responsible for sugar metabolism were repressed as the cells entered nonculturability. The genes responsible for cell division were repressed, while autolysis and cell wall metabolism genes were induced neither at starvation nor during nonculturability. Taken together, these observations verify that carbohydrate-starved lactococci attain a nonculturable state wherein sugar metabolism, cell division, and autolysis are repressed, allowing the cells to maintain transcription, metabolic activity, and energy production during a state that produces new metabolites not associated with logarithmic growth.
Succession and Diel Transcriptional Response of the Glycolate-Utilizing Component of the Bacterial Community during a Spring Phytoplankton Bloom
Authors: Lau Winnie W. Y. , Keil Richard G. , Armbrust E. Virginia
Published: February 2007

The influence of the phytoplankton-specific organic compound glycolate on bacterial community structure was examined during the 2004 spring phytoplankton bloom (February to April) in Dabob Bay in Washington. The diversity of the bacteria able to utilize glycolate during the phytoplankton bloom was determined using previously developed PCR primers to amplify the gene for the D subunit of glycolate oxidase (
glcD
). Many of the
glcD
sequences obtained represented novel sequences that appeared to be specific to marine environments. Overall, the
glcD
sequence diversity decreased as the phytoplankton bloom progressed. Phylotype-specific
glcD
quantitative PCR primers were designed for the six most commonly detected
glcD
phylotypes that represented distinct phylogenetic groups of heterotrophic bacteria. Three patterns of phylotype abundance were detected: four phylotypes were most abundant during the onset of the bloom; the abundance of one phylotype increased as the bloom progressed; and one phylotype was abundant throughout the bloom. Quantitative reverse transcriptase PCR with the same phylotype-specific primers was used to determine the levels of day and night
glcD
RNA transcription over the course of the bloom.
glcD
transcripts, when detectable, were always more abundant in the day than at night for each phylotype, suggesting that the bacteria responded to the glycolate produced by phytoplankton during the day. The nearly constant low in situ glycolate concentrations suggested that bacteria rapidly utilized the available glycolate. This study provided evidence for direct phytoplankton-bacterium interactions and the resulting succession in a single functional group of marine bacteria.
Gamma Study of pH, Nitrite, and Salt Inhibition of
Aeromonas hydrophila
Authors: Lambert Ronald J. W. , Bidlas Eva
Published: February 2007

The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of
Aeromonas hydrophila
challenged with pH, NaNO
2
, and salt combinations at 30°C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO
2
and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter
−1
), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter
−1
). This change is not due to a “synergy” between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO
2
, and salt act independently on the growth of
A. hydrophila
. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot.
54:
429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.
Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization
Authors: Smolina Irina , Lee Charles , Frank-Kamenetskii Maxim
Published: February 2007

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes.
Isolation and Characterization of Soil Bacteria That Define
Terriglobus
gen. nov., in the Phylum
Acidobacteria
Authors: Eichorst Stephanie A. , Breznak John A. , Schmidt Thomas M.
Published: February 2007

Bacteria in the phylum
Acidobacteria
are widely distributed and abundant in soils, but their ecological roles are poorly understood, owing in part to a paucity of cultured representatives. In a molecular survey of acidobacterial diversity at the Michigan State University Kellogg Biological Station Long-Term Ecological Research site, 27% of acidobacterial 16S rRNA gene clones in a never-tilled, successional plant community belonged to subdivision 1, whose relative abundance varied inversely with soil pH. Strains of subdivision 1 were isolated from these never-tilled soils using low-nutrient medium incubated for 3 to 4 weeks under elevated levels of carbon dioxide, which resulted in a slightly acidified medium that matched the pH optima of the strains (between 5 and 6). Colonies were approximately 1 mm in diameter and either white or pink, the latter due to a carotenoid(s) that was synthesized preferentially under 20% instead of 2% oxygen. Strains were gram-negative, aerobic, chemo-organotrophic, nonmotile rods that produced an extracellular matrix. All strains contained either one or two copies of the 16S rRNA encoding gene, which along with a relatively slow doubling time (10 to 15 h at ca. 23°C) is suggestive of an oligotrophic lifestyle. Six of the strains are sufficiently similar to one another, but distinct from previously named
Acidobacteria
, to warrant creation of a new genus,
Terriglobus
, with
Terriglobus roseus
defined as the type species. The physiological and nutritional characteristics of
Terriglobus
are consistent with its potential widespread distribution in soil.
Cellular Identification of a Novel Uncultured Marine Stramenopile (MAST-12 Clade) Small-Subunit rRNA Gene Sequence from a Norwegian Estuary by Use of Fluorescence In Situ Hybridization-Scanning Electron Microscopy
Authors: Kolodziej Karolina , Stoeck Thorsten
Published: February 2007

Revealing the cellular identity of organisms behind environmental eukaryote rRNA gene sequences is a major objective in microbial diversity research. We sampled an estuarine oxygen-depleted microbial mat in southwestern Norway and retrieved an 18S rRNA gene signature that branches in the MAST-12 clade, an environmental marine stramenopile clade. Detailed phylogenetic analyses revealed that MAST-12 branches among the heterotrophic stramenopiles as a sister of the free-living Bicosoecida and the parasitic genus
Blastocystis
. Specific sequence signatures confirmed a relationship to these two groups while excluding direct assignment. We designed a specific oligonucleotide probe for the target sequence and detected the corresponding organism in incubation samples using fluorescence in situ hybridization (FISH). Using the combined FISH-scanning electron microscopy approach (T. Stoeck, W. H. Fowle, and S. S. Epstein, Appl. Environ. Microbiol.
69:
6856-6863, 2003), we determined the morphotype of the target organism among the very diverse possible morphologies of the heterotrophic stramenopiles. The unpigmented cell is spherical and about 5 μm in diameter and possesses a short flagellum and a long flagellum, both emanating anteriorly. The long flagellum bears mastigonemes in a characteristic arrangement, and its length (30 μm) distinguishes the target organism from other recognized heterotrophic stramenopiles. The short flagellum is naked and often directed posteriorly. The organism possesses neither a lorica nor a stalk. The morphological characteristics that we discovered should help isolate a representative of a novel stramenopile group, possibly at a high taxonomic level, in order to study its ultrastructure, physiological capabilities, and ecological role in the environment.
Campylobacter jejuni
Strains Compete for Colonization in Broiler Chicks
Authors: Konkel Michael E. , Christensen Jeffrey E. , Dhillon A. Singh , Lane Alison B. , Hare-Sanford Rebekah , Schaberg Dennis M. , Larson Charles L.
Published: February 2007

Campylobacter jejuni
isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among
C. jejuni
isolates, we hypothesized that
C. jejuni
strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two
C. jejuni
strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal
r
strain) and one streptomycin-resistant strain (the 02-833L Str
r
strain). In vitro binding assays revealed that the
C. jejuni
F38011 Nal
r
and 02-833L Str
r
strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the
C. jejuni
02-833L Str
r
strain relative to that of the
C. jejuni
F38011 Nal
r
strain competitively inhibited the binding of the
C. jejuni
F38011 Nal
r
strain to LMH cells in a dose-dependent fashion. Similarly, the
C. jejuni
02-833L Str
r
strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the
C. jejuni
F38011 Nal
r
strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that
C. jejuni
strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which
C. jejuni
colonizes the cecum.
Reaerosolization of Fluidized Spores in Ventilation Systems
Authors: Krauter Paula , Biermann Arthur
Published: February 2007

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of
Bacillus atrophaeus
, a nonpathogenic surrogate for
Bacillus anthracis
, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 10
10
to 10
11
viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10
−3
and 10
−5
per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours.
Salt-Inducible Bionylon Polymer from
Bacillus megaterium
Authors: Shimizu Kazuki , Nakamura Hisaaki , Ashiuchi Makoto
Published: February 2007

Poly-γ-glutamate (PGA) is a chiral polyamide material that possesses a nylon-like backbone, a bionylon polymer. We examined the PGA productivity of
Bacillus megaterium
and found NaCl-responsive PGA production in the bacterium. In the system of
B. megaterium
, salt would be significant in controlling the yield, molecular size, and stereochemistry of bionylon.
Potential for Mercury Reduction by Microbes in the High Arctic
Authors: Poulain Alexandre J. , Ní Chadhain Sinéad M. , Ariya Parisa A. , Amyot Marc , Garcia Edenise , Campbell Peter G. C. , Zylstra Gerben J. , Barkay Tamar
Published: February 2007

The contamination of polar regions due to the global distribution of anthropogenic pollutants is of great concern because it leads to the bioaccumulation of toxic substances, methylmercury among them, in Arctic food chains. Here we present the first evidence that microbes in the high Arctic possess and express diverse
merA
genes, which specify the reduction of ionic mercury [Hg(II)] to the volatile elemental form [Hg(0)]. The sampled microbial biomass, collected from microbial mats in a coastal lagoon and from the surface of marine macroalgae, was comprised of bacteria that were most closely related to psychrophiles that had previously been described in polar environments. We used a kinetic redox model, taking into consideration photoredox reactions as well as
mer
-mediated reduction, to assess if the potential for Hg(II) reduction by Arctic microbes can affect the toxicity and environmental mobility of mercury in the high Arctic. Results suggested that
mer
-mediated Hg(II) reduction could account for most of the Hg(0) that is produced in high Arctic waters. At the surface, with only 5% metabolically active cells, up to 68% of the mercury pool was resolved by the model as biogenic Hg(0). At a greater depth, because of incident light attenuation, the significance of photoredox transformations declined and
merA
-mediated activity could account for up to 90% of Hg(0) production. These findings highlight the importance of microbial redox transformations in the biogeochemical cycling, and thus the toxicity and mobility, of mercury in polar regions.
l
-Valine Production with Pyruvate Dehydrogenase Complex-Deficient
Corynebacterium glutamicum
Authors: Blombach Bastian , Schreiner Mark E. , Holátko Jiří , Bartek Tobias , Oldiges Marco , Eikmanns Bernhard J.
Published: February 2007

Corynebacterium glutamicum
was engineered for the production of
l
-valine from glucose by deletion of the
aceE
gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the
ilvBNCE
genes encoding the
l
-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth,
C. glutamicum
Δ
aceE
showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate,
l
-alanine, and
l
-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of
ilvBNCE
in
C. glutamicum
Δ
aceE
resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and
l
-alanine towards
l
-valine. In fed-batch fermentations at high cell densities and an excess of glucose,
C. glutamicum
Δ
aceE
(pJC4ilvBNCE) produced up to 210 mM
l
-valine with a volumetric productivity of 10.0 mM h
−1
(1.17 g l
−1
h
−1
) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.
Extraintestinal PathogenicEscherichia coliSurvives within Neutrophils
Authors: Nazareth Helen , Genagon Stacy A. , Russo Thomas A.
Published: February 2007

Extracellular pathogenicEscherichia coli(ExPEC) strains are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, nosocomial pneumonia, neonatal meningitis, and sepsis. ExPEC strains are extracellular bacterial pathogens; therefore, the innate immune response (e.g., professional phagocytes) plays a crucial role in the host defense against them. Studies using the model ExPEC strain CP9 demonstrated that it is relatively resistant to neutrophil-mediated bactericidal activity. Although this could be due to resistance to phagocytosis, the ability of CP9 to survive the intracellular killing mechanisms of neutrophils is another possibility. Using a variation of the intracellular invasion assay, we studied the survival of CP9 within peripheral blood-derived human neutrophils. Our results indicated that CP9 did survive within human neutrophils, but we were unable to demonstrate that intracellular replication occurred. This finding was not unique to CP9, since when a conservative assessment of survival was used, four of six additional ExPEC strains, but not anE. colilaboratory strain, were also capable of survival within neutrophils. Initial studies in which we began to decipher the mechanisms by which CP9 is able to successfully survive intracellular neutrophil-mediated bactericidal activity demonstrated that CP9 was at least partially susceptible to the neutrophil oxidative burst. Therefore, absolute resistance to the oxidative burst is not a mechanism by which ExPEC survives within neutrophils. In addition, electron microscopy studies showed that CP9 appeared to be present in phagosomes within neutrophils. Therefore, avoidance of phagosomal uptake or subsequent escape from the phagosome does not appear to be a mechanism that contributes to CP9's survival. These findings suggest that survival of ExPEC within neutrophils may be an important virulence mechanism.
Candidate Live, Attenuated
Salmonella enterica
Serotype Typhimurium Vaccines with Reduced Fecal Shedding Are Immunogenic and Effective Oral Vaccines
Authors: Abd El Ghany Moataz , Jansen Angela , Clare Simon , Hall Lindsay , Pickard Derek , Kingsley Robert A. , Dougan Gordon
Published: February 2007

Environmental shedding of genetically manipulated microorganisms is an issue impeding the development of new live vaccines. We have investigated the immunogenicity of a number of novel
Salmonella enterica
serotype Typhimurium oral vaccine candidates that express the fragment C (TetC) component of tetanus toxin and harbor combinations of additional mutations in genes
shdA
,
misL
, and
ratB
that contribute to the persistence of serotype Typhimurium's colonization of the intestine. Serotype Typhimurium
aroA
(TetC) derivatives harboring additional mutations in either
shdA
or
misL
or combinations of these mutations exhibited a marked decrease in shedding of the vaccine strain in the feces of orally vaccinated mice. However, equivalent levels of anti-TetC and anti-
Salmonella
lipopolysaccharide immunoglobulin G (IgG), IgG1, IgG2a, and IgA were detected in sera of the vaccinated but not of the control mice. Cellular immune responses to TetC were detected in all vaccinated mice, regardless of the presence of the additional mutations in
shdA
or
misL
. Further, immunization with serotype Typhimurium
aroA
candidate vaccines harboring
shdA
and
misL
afforded complete protection against challenge with a virulent strain of serotype Typhimurium.
Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and α-Sarcin
Authors: Brigotti Maurizio , Carnicelli Domenica , Ravanelli Elisa , Vara Antonio González , Martinelli Chiara , Alfieri Roberta R. , Petronini Pier Giorgio , Sestili Piero
Published: February 2007

Treatment of human endothelial cells with Shiga toxin 1 and 2 leads to the upregulation of genes encoding proinflammatory molecules involved in the pathogenesis of hemolytic-uremic syndrome. The paradoxical effect of inhibitors of mRNA translation, such as Shiga toxins, that at the same time induce protein expression was investigated by studying the relationship between their enzymatic activity (abstraction of adenine from nucleic acids) and the induction of interleukin-8 and granulocyte-macrophage colony-stimulating factor in human endothelial cells. As a positive control, the fungal toxin α-sarcin, acting on the same rRNA sequence targeted by Shiga toxins with a different mechanism (RNase activity), was used. The three toxins caused ribosomal lesions that, in turn, induced the activation of p38 stress kinase with kinetics that paralleled the inhibition of translation. α-Sarcin was devoid of activity on DNA. Shiga toxin 2 targeted nuclear DNA with more rapid kinetics than did Shiga toxin 1. Since the fungal ribotoxin was fully effective in the induction of proinflammatory proteins, we conclude that damage to ribosomes is indispensable and sufficient to activate protein expression via induction of the stress-kinase cascade. However, gene upregulation events induced by Shiga toxin 2 were much more efficient than those triggered by Shiga toxin 1, although the two toxins impaired translation to the same extent and had overlapping time courses of stress kinase activation. Regulations independent of the ribotoxic stress were assumed to operate in intoxicated cells. We hypothesized that the two bacterial toxins recognize different DNA sequences inducing different regulating effects on gene expression.
Modulation of Naive CD4+T-Cell Responses to an Airway Antigen during Pulmonary Mycobacterial Infection
Authors: Anis Mursalin M. , Fulton Scott A. , Reba Scott M. , Harding Clifford V. , Boom W. Henry
Published: February 2007

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+T cells after airway antigen challenge. BALB/c mice were infected by aerosol withMycobacterium bovisBCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+T cells in the lungs in response to airway antigen challenge.
Invasion and Persistence ofMycobacterium aviumsubsp.paratuberculosisduring Early Stages of Johne's Disease in Calves
Authors: Wu Chia-wei , Livesey Michael , Schmoller Shelly K. , Manning Elizabeth J. B. , Steinberg Howard , Davis William C. , Hamilton Mary Jo , Talaat Adel M.
Published: February 2007

Infection withMycobacterium aviumsubsp.paratuberculosiscauses Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves withM. aviumsubsp.paratuberculosis. To our surprise, strains ofM. aviumsubsp.paratuberculosiswere able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition ofM. aviumsubsp.paratuberculosisdespite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutantM. aviumsubsp.paratuberculosisstrain (with an inactivatedgcpEgene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain ofM. aviumsubsp.paratuberculosisdeficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.
A Defined O-Antigen Polysaccharide Mutant ofFrancisella tularensisLive Vaccine Strain Has Attenuated Virulence while Retaining Its Protective Capacity
Authors: Sebastian Shite , Dillon Simon T. , Lynch Jillian G. , Blalock LeeAnn T. , Balon Emmy , Lee Kristin T. , Comstock Laurie E. , Conlan J. Wayne , Rubin Eric J. , Tzianabos Arthur O. , Kasper Dennis L.
Published: February 2007

Francisella tularensis, the causative agent of tularemia, has been designated a CDC category A select agent because of its low infective dose (<10 CFU), its ready transmission by aerosol, and its ability to produce severe morbidity and high mortality. The identification and characterization of this organism's virulence determinants will facilitate the development of a safe and effective vaccine. We report that inactivation of thewbtA-encoded dehydratase of the O-antigen polysaccharide (O-PS) locus of the still-unlicensed live vaccine strain ofF. tularensis(LVS) results in a mutant (the LVSwbtAmutant) with remarkably attenuated virulence. Western blot analysis and immune electron microscopy studies associate this loss of virulence with a complete lack of surface O-PS expression. A likely mechanism for attenuation is shown to be the transformation from serum resistance in the wild-type strain to serum sensitivity in the mutant. Despite this significant attenuation in virulence, the LVSwbtAmutant remains immunogenic and confers protective immunity on mice against challenge with an otherwise lethal dose of eitherF. tularensisLVS or a fully virulent clinical isolate ofF. tularensistype B. Recognition and characterization of the pivotal role of O-PS in the virulence of this intracellular bacterial pathogen may have broad implications for the creation of a safe and efficacious vaccine.
Mucosal and Systemic Immune Responses in Patients with Diarrhea Due to CS6-Expressing Enterotoxigenic
Escherichia coli
Authors: Qadri Firdausi , Ahmed Tanvir , Ahmed Firoz , Bhuiyan M. Saruar , Mostofa Mohammad Golam , Cassels Frederick J. , Helander Anna , Svennerholm Ann-Mari
Published: February 2007

Colonization factor CS6 expressed by enterotoxigenic
Escherichia coli
(ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (
n
= 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.
Contributions of [4Fe-4S]-FNR and Integration Host Factor to
fnr
Transcriptional Regulation
Authors: Mettert Erin L. , Kiley Patricia J.
Published: February 2007

Maintaining appropriate levels of the global regulator FNR is critical to its function as an O
2
sensor. In this study, we examined the mechanisms that control transcription of
fnr
to increase our understanding of how FNR protein levels are regulated. Under anaerobic conditions, one mechanism that controls
fnr
expression is negative autoregulation by the active [4Fe-4S] form of FNR. Through DNase I footprinting and in vitro transcription experiments, we observed that direct binding of [4Fe-4S]-FNR to the predicted downstream FNR binding site is sufficient for repression of the
fnr
promoter in vitro. In addition, the downstream FNR binding site was required for repression of transcription from
fnr
′-
lacZ
fusions in vivo. No repression of
fnr
was observed in vivo or in vitro with the apoprotein form of FNR, indicating that repression requires the dimeric, Fe-S cluster-containing protein. Furthermore, our in vitro and in vivo data suggest that [4Fe-4S]-FNR does not bind to the predicted upstream FNR binding site within the
fnr
promoter. Rather, we provide evidence that integration host factor binds to this upstream region and increases in vivo expression of P
fnr
under both aerobic and anaerobic conditions.
Assembly of Fimbrial Structures inPseudomonas aeruginosa: Functionality and Specificity of Chaperone-Usher Machineries
Authors: Ruer Ségolène , Stender Silke , Filloux Alain , de Bentzmann Sophie
Published: February 2007

Fimbrial or nonfimbrial adhesins assembled by the bacterial chaperone-usher pathway have been demonstrated to play a key role in pathogenesis. Such an assembly mechanism has been exemplified in uropathogenicEscherichia colistrains with the Pap and the Fim systems. InPseudomonas aeruginosa, three gene clusters (cupA,cupB, andcupC) encodingchaperone-usherpathway components have been identified in the genome sequence of the PAO1 strain. The Cup systems differ from the Pap or Fim systems, since they obviously lack numbers of genes encoding fimbrial subunits. Nevertheless, the CupA system has been demonstrated to be involved in biofilm formation on solid surfaces, whereas the role of the CupB and CupC systems in biofilm formation could not be clearly elucidated. Moreover, these gene clusters were described as poorly expressed under standard laboratory conditions. ThecupBandcupCclusters are directly under the control of a two-component regulatory system designated RocA1/S1/R. In this study, we revealed that Roc1-dependent induction of thecupBandcupCgenes resulted in a high level of biofilm formation, with CupB and CupC acting with synergy in clustering bacteria for microcolony formation. Very importantly, this phenotype was associated with the assembly of cell surface fimbriae visualized by electron microscopy. Finally, we observed that the CupB and CupC systems are specialized in the assembly of their own fimbrial subunits and are not exchangeable.
Characterization of the Variants, Flanking Genes, and Promoter Activity of the
Leifsonia xyli
subsp.
cynodontis
Insertion Sequence IS
1237
Authors: Lin Hui , Li Tai-Yuan , Xie Mao-Hua , Zhang Yi
Published: February 2007

We performed a comprehensive study of the distribution and function of an insertion sequence (IS) element, IS
1237
, in the genome of
Leifsonia xyli
subsp.
cynodontis
, a useful genetic carrier for expressing beneficial foreign genes in plants. Two shorter IS
1237
isoforms, IS
1237
d1 and IS
1237
d2 resulting from precise deletion between two nonperfect repeats, were found in the bacterial genome at a level that was one-fifth the level of wild-type IS
1237
. Both the genome and native plasmid pCXC100 harbor a truncated toxin-antitoxin cassette that is precisely fused with a 5′-truncated IS
1237
sequence at one nonperfect repeat, indicating that it is a hot site for DNA rearrangement. Nevertheless, no transposition activity was detected when the putative transposase of IS
1237
was overexpressed in
Escherichia coli
. Using thermal asymmetric interlaced PCR, we identified 13 upstream and 10 downstream unique flanking sequences, and two pairs of these sequences were from the same loci, suggesting that IS
1237
has up to 65 unique loci in the
L. xyli
subsp.
cynodontis
chromosome. The presence of TAA or TTA direct repeat sequences at most insertion sites indicated that IS
1237
inserts into the loci by active transposition. IS
1237
showed a high propensity for insertion into other IS elements, such as IS
Lxc1
and IS
Lxc2
, which could offer IS
1237
a nonautonomous transposition pathway through the host IS elements. Interestingly, we showed that IS
1237
has a strong promoter at the 3′ end and a weak promoter at the 5′ end, and both promoters promote the transcription of adjacent genes in different gram-positive bacteria. The high-copy-number nature of IS
1237
and its promoter activity may contribute to bacterial fitness.
Characterization of an Exceedingly Active NADH Oxidase from the Anaerobic Hyperthermophilic Bacterium
Thermotoga maritima
Authors: Yang Xianqin , Ma Kesen
Published: February 2007

An NADH oxidase from the anaerobic hyperthermophilic bacterium
Thermotoga maritima
was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The

V
max

was 230 ± 14 μmol/min/mg (
k
cat
/

K
m

= 548,000 min
−1
mM
−1
), and the

K
m

values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of
T. maritima
.
The DeoR-Type Regulator SugR Represses Expression ofptsGinCorynebacterium glutamicum
Authors: Engels Verena , Wendisch Volker F.
Published: February 2007

Corynebacterium glutamicumgrows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression ofptsG, which encodes the glucose-specific PTS enzyme II. Overexpression ofsugRresulted in reducedptsGmRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lackingsugR, expression of theptsG′-′catfusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, includingptsSand the putative NCgl1859-fruK-ptsFoperon. Purified SugR bound to DNA regions upstream ofptsG,ptsS, and NCgl1859, and a 75-bpptsGpromoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to theptsGpromoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR repressesptsG,ptsGexpression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allowsC. glutamicumto adapt glucose uptake to carbon source availability.
A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System inRhodobacter sphaeroides
Authors: Poggio Sebastian , Abreu-Goodger Cei , Fabela Salvador , Osorio Aurora , Dreyfus Georges , Vinuesa Pablo , Camarena Laura
Published: February 2007

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure.Rhodobacter sphaeroideshas two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system ofR. sphaeroidescan be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system ofR. sphaeroidesproduces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related toR. sphaeroideshave also acquired a set of flagellar genes similar to the set found inR. sphaeroides, suggesting that a common ancestor received this gene cluster.