bluuebunny/crossref_metadata_2025 huggingface dataset:
Sources of uncertainty in flood inundation maps
Authors: Bales J.D. , Wagner C.R.
Published: April 2009

Flood inundation maps typically have been used to depict inundated areas for floods having specific exceedance levels. The uncertainty associated with the inundation boundaries is seldom quantified, in part, because all of the sources of uncertainty are not recognized and because data available to quantify uncertainty seldom are available. Sources of uncertainty discussed in this paper include hydrologic data used for hydraulic model development and validation, topographic data, and the hydraulic model. The assumption of steady flow, which typically is made to produce inundation maps, has less of an effect on predicted inundation at lower flows than for higher flows because more time typically is required to inundate areas at high flows than at low flows. Difficulties with establishing reasonable cross sections that do not intersect and that represent water‐surface slopes in tributaries contribute additional uncertainties in the hydraulic modelling. As a result, uncertainty in the flood inundation polygons simulated with a one‐dimensional model increases with distance from the main channel.
H‐Cadherin expression reduces invasion of malignant melanoma
Authors: Kuphal Silke , Martyn Adam C. , Pedley Julie , Crowther Lisa M. , Bonazzi Vanessa F. , Parsons Peter G. , Bosserhoff Anja K. , Hayward Nicholas K. , Boyle Glen M.
Published: April 2009

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.
Collective Information Processing and Pattern Formation in Swarms, Flocks, and Crowds
Authors: Moussaid Mehdi , Garnier Simon , Theraulaz Guy , Helbing Dirk
Published: April 2009

The spontaneous organization of collective activities in animal groups and societies has attracted a considerable amount of attention over the last decade. This kind of coordination often permits group‐living species to achieve collective tasks that are far beyond single individuals' capabilities. In particular, a key benefit lies in the integration of partial knowledge of the environment at the collective level. In this contribution, we discuss various self‐organization phenomena in animal swarms and human crowds from the point of view of information exchange among individuals. In particular, we provide a general description of collective dynamics across species and introduce a classification of these dynamics not only with respect to the way information is transferred among individuals but also with regard to the knowledge processing at the collective level. Finally, we highlight the fact that the individual’s ability to learn from past experiences can have a feedback effect on the collective dynamics, as experienced with the development of behavioral conventions in pedestrian crowds.
What is your diagnosis? Hemorrhagic effusion in a dog
Authors: Barger Anne , Riensche Melissa
Published: April 2009

Abstract: A 2‐year‐old spayed female, German Shepherd dog was presented to the University of Illinois Veterinary Teaching Hospital with a history of intermittent lethargy. On physical examination, lung sounds were increased. The dog had a mild fever (103.7°F) and mild tachycardia (120 bpm). Thoracic ultrasound revealed a sternal mass and pericardial effusion, both of which were aspirated. On cytologic examination, the pericardial fluid contained a large amount of blood and was interpreted as a hemorrhagic effusion. Nucleated cells consisted mainly of macrophages containing phagocytized RBCs and hemosiderin and many clusters of reactive mesothelial cells. The majority of mesothelial cells contained variable amounts of rod‐shaped brown pigment granules that were suspected to be iron. The granules were positive for Prussian blue and carbol–fuschin, indicative of iron potentially mixed with lipofuscin. The granules stained negatively with Melan A, rhodamine, Hall's, and periodic acid‐Schiff. The iron within the mesothelial cells was likely secondary to hemorrhage, based on the erythrophagia and accumulation of hemosiderin in macrophages. Iron deposition and phagocytic activity in mesothelial cells has been reported previously in humans and rats, but not in dogs.
What is your diagnosis? Marked hyperchloremia in a dog
Authors: Piperisova Ida , Neel Jennifer A. , Papich Mark G.
Published: April 2009

Abstract:A 5‐year‐old neutered male Cavalier King Charles Spaniel was evaluated for a 3‐week history of progressive paresis. The dog had been receiving potassium citrate capsules to acidify urine for the past 2 years because of an earlier history of urolithiasis. Results of neurologic examination, spinal cord radiography, and magnetic resonance imaging of the skull and spinal cord revealed no lesions that could have accounted for the neurologic signs. The main abnormalities on a clinical chemistry profile were marked hyperchloremia (179 mmol/L, reference interval 108–122 mmol/L) and an anion gap of −50.4 mmol/L (reference interval 16.3–28.6 mmol/L). Because of the severe hyperchloremia, serum bromide concentration was measured (400 mg/dL; toxic concentration >150 mg/dL; some dogs may tolerate up to 300 mg/dL). Analysis of the potassium citrate capsules, which had been compounded at a local pharmacy, yielded a mean bromide concentration of 239 mg/capsule. Administration of the capsules was discontinued and there was rapid resolution of the dog's neurologic signs. This case of extreme bromide toxicity, which apparently resulted from inadvertent use of bromide instead of citrate at the pharmacy, illustrates the importance of knowing common interferents with analyte methodologies and of pursing logical additional diagnostic tests based on clinical and laboratory evidence, even when a patient's history appears to rule out a potential etiology.
Validation of the Sysmex XT‐2000iV hematology system for dogs, cats, and horses. I. Erythrocytes, platelets, and total leukocyte counts
Authors: Lilliehöök Inger , Tvedten Harold
Published: April 2009

Background:The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories.

Objective:The purpose of this study was to validate the Sysmex XT‐2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses.

Methods:Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV and the CELL‐DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results.

Results:Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL‐DYN (r≥0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33–0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL‐DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts.

Conclusion:The Sysmex XT‐2000iV performed as well as the CELL‐DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.
Angiotensin II‐induced increase in myocardial distensibility and its modulation by the endocardial endothelium in the rabbit heart
Authors: Castro‐Chaves Paulo , Fontes‐Carvalho Ricardo , Pintalhao Mariana , Pimentel‐Nunes Pedro , Leite‐Moreira Adelino F.
Published: April 2009

As recently demonstrated, angiotensin II (Ang II) induces an increase in myocardial distensibility. Although endothelin‐1 and the endocardial endothelium (EE) also modulate myocardial diastolic properties, their interaction with Ang II at this level has not yet been investigated. Increasing concentrations of Ang II (from 10−8to 10−5m) were studied in rabbit right papillary muscles in the following conditions: (1) baseline; (2) after selective removal of EE with Triton X‐100; and (3) with intact EE in presence of a non‐selective endothelin receptor antagonist (PD‐145065), a selective endothelin type A receptor antagonist (BQ‐123), an inhibitor of nitric oxide synthesis (NG‐nitro‐l‐arginine (l‐NA) or an inhibitor of the NAD(P)H oxidase (apocynin). At baseline, Ang II induced a concentration‐dependent positive inotropic effect and an increase in passive muscle length (L) up to 1.020 ± 0.004L/Lmax. After restoring muscle length to maximal physiological length (Lmax), passive tension decreased by 46.1 ± 4.0%. When the EE was removed, the effect on myocardial distensibility was abolished. With intact EE in presence of PD‐145065, BQ‐123 orl‐NA, the effects of Ang II on myocardial distensibility were attenuated, with a maximal increase in passive muscle length of 1.0087 ± 0.0012, 1.0068 ± 0.0022 and 1.0066 ± 0.0020L/Lmaxand a decrease in resting tension of 22.6 ± 3.6, 16.1 ± 6.0 and 20.4 ± 5.6%, respectively. In the presence of apocynin, the effect on myocardial distensibility was abolished. In conclusion, the Ang II‐dependent acute increase in myocardial distensibility is abolished by the selective removal of the EE and attenuated in the presence of endothelin‐1 receptor antagonists, an inhibitor of nitric oxide synthesis or an inhibitor of NAD(P)H oxidase.
Cutin synthesis: A slippery paradigm
Authors: Heredia Antonio , Heredia-Guerrero José A. , Domínguez Eva , Benítez José J.
Published: April 2009

Despite its biological importance, the mechanism of construction of cutin, the polymer matrix of plant cuticles, has not yet been elucidated. Recently, progress on lipid barrier formation of polymers such as cutin and suberin has been recently reviewed by Pollard et al.1 In their review the authors state that the ubiquitous cutin is the least understood of the plant extracellular polymers and that major questions about cutin structure and its macromolecular assembly remain to be resolved. At the time this paper was being published our research group has developed a new hypothesis on plant cutin synthesis.2
Hypoxic Adaptation by Efg1 Regulates Biofilm Formation by
Candida albicans
Authors: Stichternoth Catrin , Ernst Joachim F.
Published: April 2009

Hypoxia is encountered frequently by
Candida albicans
during systemic infection of the human host. We tested if hypoxia allows biofilm formation by
C. albicans
, which is a major cause of perseverance and antifungal resistance in
C. albicans
infections. Using an in vitro biofilm system, we unexpectedly discovered that several positive regulators of biofilm formation during normoxia, including Tec1, Ace2, Czf1, Och1, and Als3, had little or no influence on biofilm development during hypoxia, irrespective of the carbon dioxide level, indicating that
C. albicans
biofilm pathways differ depending on the oxygen level. In contrast, the Efg1 and Flo8 regulators were required for both normoxic and hypoxic biofilm formation. To explore the role of Efg1 during hypoxic and/or biofilm growth, we determined transcriptome kinetics following release of
EFG1
expression by a system under transcriptional control of a doxycycline-inducible promoter. During hypoxia, Efg1 rapidly induced expression of all major classes of genes known to be associated with normoxic biofilm formation, including genes involved in glycolysis, sulfur metabolism, and antioxidative and peroxisome activities, as well as genes for iron uptake. The results suggest that hypoxic adaptation mediated by the Efg1 and Flo8 regulators is required even during normoxic biofilm development, while hypoxic biofilm formation in deep tissues or in organs may generate foci of
C. albicans
infections.
Lactobacillus plantarum
WCFS1 Electron Transport Chains
Authors: Brooijmans R. J. W. , de Vos W. M. , Hugenholtz J.
Published: April 2009

Lactobacillus plantarum
WCFS1 requires both heme and menaquinone to induce respiration-like behavior under aerobic conditions. The addition of these compounds enhanced both biomass production, without progressive acidification, and the oxygen consumption rate. When both heme and menaquinone were present,
L. plantarum
WCFS1 was also able to reduce nitrate. The ability to reduce nitrate was severely inhibited by the glucose levels that are typically found in
L. plantarum
growth media (1 to 2% [vol/vol] glucose). In contrast, comparable mannitol levels did not inhibit the reduction of nitrate.
L. plantarum
reduced nitrate with concomitant formation of nitrite and ammonia. Genes that encode a
bd
-type cytochrome (
cydABCD
) and a nitrate reductase (
narGHJI
) were identified in the genome of
L. plantarum
. The
narGHJI
operon is part of a cluster of genes that includes the molybdopterin cofactor biosynthesis genes and
narK
. Besides a menaquinone source, isogenic mutants revealed that
cydA
and
ndh1
are required for the aerobic-respiration-like response and
narG
for nitrate reduction. The
ndh1
mutant was still able to reduce nitrate. The existence of a nonredundant branched electron transport chain in
L. plantarum
WCFS1 that is capable of using oxygen or nitrate as a terminal electron acceptor is proposed.
Production of Recombinant Proteins in thelon-Deficient BL21(DE3) Strain ofEscherichia coliin the Absence of the DnaK Chaperone
Authors: Ratelade Julien , Miot Marie-Caroline , Johnson Emmett , Betton Jean-Michel , Mazodier Philippe , Benaroudj Nadia
Published: April 2009

To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) ΔdnaKstrain ofEscherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.
Gut-Associated Denitrification and In Vivo Emission of Nitrous Oxide by the Earthworm Families Megascolecidae and Lumbricidae in New Zealand
Authors: Wüst Pia K. , Horn Marcus A. , Henderson Gemma , Janssen Peter H. , Rehm Bernd H. A. , Drake Harold L.
Published: April 2009

Previous studies have documented the capacity of European earthworms belonging to the family Lumbricidae to emit the greenhouse gas nitrous oxide (N2O), an activity attributed primarily to the activation of ingested soil denitrifiers. To extend the information base to earthworms in the Southern Hemisphere, four species of earthworms in New Zealand were examined for gut-associated denitrification.Lumbricus rubellusandAporrectodea rosea(introduced species of Lumbricidae) emitted N2O, whereas emission of N2O byOctolasion cyaneum(an introduced species of Lumbricidae) and emission of N2O byOctochaetus multiporus(a native species of Megascolecidae) were variable and negligible, respectively. Exposing earthworms to nitrite or nitrate and acetylene significantly increased the amount of N2O emitted, implicating denitrification as the primary source of N2O and indicating that earthworms emitted dinitrogen (N2) in addition to N2O. The alimentary canal displayed a high capacity to produce N2O when it was supplemented with nitrite, and alimentary canal contents contained large amounts of carbohydrates and organic acids indicative of fermentation (e.g., succinate, acetate, and formate) that could serve as sources of reductant for denitrification.nosZencodes a portion of the terminal oxidoreductase used in denitrification. ThenosZsequences detected in the alimentary canals ofL. rubellusandO. multiporuswere similar to those retrieved from soil and were distantly related to sequences of uncultured soil bacteria and genera common in soils (i.e.,Bradyrhizobium,Azospirillum,Rhodopseudomonas,Rhodospirillum,Pseudomonas,Oligotropha, andSinorhizobium). These findings (i) suggest that the capacity to emit N2O and N2is a general trait of earthworms and not geographically restricted, (ii) indicate that species belonging to different earthworm families (i.e., Megascolecidae and Lumbricidae) may not have equal capacities to emit N2O, and (iii) also corroborate previous findings that link this capacity to denitrification in the alimentary canal.
Metabolic Engineering of
Saccharomyces cerevisiae
for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids
Authors: Steinle Anna , Bergander Klaus , Steinbüchel Alexander
Published: April 2009

Cyanophycin (multi-
l
-arginyl-poly-
l
-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant
Saccharomyces cerevisiae
strains defective in arginine metabolism and expressing the cyanophycin synthetase of
Synechocystis
sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.
Campylobacter
Colonization of the Turkey Intestine in the Context of Microbial Community Development
Authors: Scupham Alexandra J.
Published: April 2009

Patterns of microbial community dynamics in the turkey intestine were examined. Every week of the 18-week production cycle, cecal bacterial communities and
Campylobacter
loads were examined from five birds for each of two flocks. Molecular fingerprinting via the automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (T-RFLP) of the cecal samples revealed that microbial communities changed in a time-dependent manner, and during both trials they developed via transition through three phases during the production cycle. A core component of the microbiota consisting of 11
Bacteroidetes
types was present throughout both trials. In contrast, constant succession was detected in the
Clostridiales
populations until week 10 or 11, with few shared sequences between the flocks. Changes in
Campylobacter jejuni
and
Campylobacter coli
loads were correlated to, but not dependent on, the two acute transitions delimiting the three developmental phases.
CO
2
Production as an Indicator of Biofilm Metabolism
Authors: Kroukamp Otini , Wolfaardt Gideon M.
Published: April 2009

Biofilms are important in aquatic nutrient cycling and microbial proliferation. In these structures, nutrients like carbon are channeled into the production of extracellular polymeric substances or cell division; both are vital for microbial survival and propagation. The aim of this study was to assess carbon channeling into cellular or noncellular fractions in biofilms. Growing in tubular reactors, biofilms of our model strain
Pseudomonas
sp. strain CT07 produced cells to the planktonic phase from the early stages of biofilm development, reaching pseudo steady state with a consistent yield of ∼10
7
cells·cm
−2
·h
−1
within 72 h. Total direct counts and image analysis showed that most of the converted carbon occurred in the noncellular fraction, with the released and sessile cells accounting for <10% and <2% of inflowing carbon, respectively. A CO
2
evolution measurement system (CEMS) that monitored CO
2
in the gas phase was developed to perform a complete carbon balance across the biofilm. The measurement system was able to determine whole-biofilm CO
2
production rates in real time and showed that gaseous CO
2
production accounted for 25% of inflowing carbon. In addition, the CEMS made it possible to measure biofilm response to changing environmental conditions; changes in temperature or inflowing carbon concentration were followed by a rapid response in biofilm metabolism and the establishment of new steady-state conditions.
Escherichia coli
and
Salmonella enterica
Are Protected against Acetic Acid, but Not Hydrochloric Acid, by Hypertonicity
Authors: Chapman B. , Ross T.
Published: April 2009

Chapman et al. (B. Chapman, N. Jensen, T Ross, and M. B. Cole, Appl. Environ. Microbiol. 72:5165-5172, 2006) demonstrated that an increased NaCl concentration prolongs survival of
Escherichia coli
O157 SERL 2 in a broth model simulating the aqueous phase of a food dressing or sauce containing acetic acid. We examined the responses of five other
E. coli
strains and four
Salmonella enterica
strains to increasing concentrations of NaCl under conditions of lethal acidity and observed that the average “lag” time prior to inactivation decreases in the presence of hydrochloric acid but not in the presence of acetic acid. For
E. coli
in the presence of acetic acid, the lag time increased with increasing NaCl concentrations up to 2 to 4% at pH 4.0, up to 4 to 6% at pH 3.8, and up to 4 to 7% (wt/wt of water) NaCl at pH 3.6.
Salmonella
was inactivated more rapidly by combined acetic acid and NaCl stresses than
E. coli
, but increasing NaCl concentrations still decreased the lag time prior to inactivation in the presence of acetic acid; at pH 4.0 up to 1 to 4% NaCl was protective, and at pH 3.8 up to 1 to 2% NaCl delayed the onset of inactivation. Sublethal injury kinetics suggest that this complex response is a balance between the lethal effects of acetic acid, against which NaCl is apparently protective, and the lethal effects of the NaCl itself. Compared against 3% NaCl, 10% (wt/wt of water) sucrose with 0.5% NaCl (which has similar osmotic potential) was found to be equally protective against adverse acetic acid conditions. We propose that hypertonicity may directly affect the rate of diffusion of acetic acid into cells and hence cell survival.
Molecular Diversification in the Quorum-Sensing System of
Vibrio cholerae
: Role of Natural Selection in the Emergence of Pandemic Strains
Authors: Talyzina Nina M. , Ingvarsson Pär K. , Zhu Jun , Wai Sun N. , Andersson Agneta
Published: April 2009

Two haplotypes of the
Vibrio cholerae
quorum-sensing system regulator
hapR
are described:
hapR1
, common among nonpandemic, non-O1, non-O139 strains, and
hapR2
, associated with pandemic O1 and O139 and epidemic O37
V. cholerae
strains. The
hapR2
has evolved under strong natural selection, implying that its fixation was influenced by conditions that led to cholera pandemics.
Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur
Authors: Raina Jean-Baptiste , Tapiolas Dianne , Willis Bette L. , Bourne David G.
Published: April 2009

Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species,Montipora aequituberculataandAcropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera,Roseobacter, Spongiobacter, Vibrio, andAlteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated withRoseobactersp. constituted 28% of theM. aequituberculatatissue libraries, while 59% of the clones from theA. milleporalibraries were affiliated with sequences related to theSpongiobactergenus.Vibriospp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous todddD, anddddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.
Quantitative Method To Determine Sporicidal Decontamination of Building Surfaces by Gaseous Fumigants, and Issues Related to Laboratory-Scale Studies
Authors: Rastogi Vipin K. , Wallace Lalena , Smith Lisa S. , Ryan Shawn P. , Martin Blair
Published: April 2009

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores ofBacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 μl containing the target number (1 × 106, 1 × 107, or 1 × 108) of avirulent spores ofB. anthracisNNR1Δ1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%.
Simultaneous Cellulose Degradation and Electricity Production by
Enterobacter cloacae
in a Microbial Fuel Cell
Authors: Rezaei Farzaneh , Xing Defeng , Wagner Rachel , Regan John M. , Richard Tom L. , Logan Bruce E.
Published: April 2009

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was
Enterobacter cloacae
. An isolate designated
E. cloacae
FR from the enrichment was found to be 100% identical to
E. cloacae
ATCC 13047
T
based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m
2
, compared to 5.4 ± 0.3 mW/m
2
for strain ATCC 13047
T
. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.
Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis, Lucinidae) and Its Symbiont Population
Authors: Caro Audrey , Got Patrice , Bouvy Marc , Troussellier Marc , Gros Olivier
Published: April 2009

The bivalveCodakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.
Characterization of Rhamnosidases from
Lactobacillus plantarum
and
Lactobacillus acidophilus
Authors: Beekwilder Jules , Marcozzi Daniela , Vecchi Samuele , de Vos Ric , Janssen Patrick , Francke Christof , van Hylckama Vlieg Johan , Hall Robert D.
Published: April 2009

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of
Lactobacillus plantarum
, two putative rhamnosidase genes (
ram1
Lp
and
ram2
Lp
) were identified, while in
Lactobacillus acidophilus
, one rhamnosidase gene was found (
ramA
La
). Gene products from all three genes were produced after introduction into
Escherichia coli
and were then tested for their enzymatic properties. Ram1
Lp
, Ram2
Lp
, and RamA
La
were able to efficiently hydrolyze rutin and other rutinosides, while RamA
La
was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of
Lactobacillus
rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1
Lp
and RamA
La
enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1
Lp
for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium
Lactococcus lactis
. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into
L. lactis
appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.
Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms byPseudomonas aeruginosaandStaphylococcus aureus
Authors: Tran Phat L. , Hammond Adrienne A. , Mosley Thomas , Cortez Janette , Gray Tracy , Colmer-Hamood Jane A. , Shashtri Mayank , Spallholz Julian E. , Hamood Abdul N. , Reid Ted W.
Published: April 2009

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibitingPseudomonas aeruginosaandStaphylococcus aureusbiofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibitedP. aeruginosaandS. aureusbiofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.
Diversity and Morphology of Members of the Phylum “
Synergistetes
” in Periodontal Health and Disease
Authors: Vartoukian S. R. , Palmer R. M. , Wade W. G.
Published: April 2009

Members of the phylum “
Synergistetes
” have frequently been detected in the human oral cavity at sites of dental disease, but they have rarely been detected in studies of oral health. Only two oral “
Synergistetes
” taxa are cultivable. The aims of this study were to investigate the diversity of “
Synergistetes
” in the oral cavity, to establish whether “
Synergistetes
” taxa are more strongly associated with periodontitis than with oral health, and to visualize unculturable “
Synergistetes
” in situ. Sixty samples (saliva, dental plaque, and mucosal swabs) were collected from five subjects with periodontitis and five periodontally healthy controls. Using phylum-specific 16S rRNA gene primers, “
Synergistetes
” were identified by PCR, cloning, and sequencing of 48 clones per PCR-positive sample. Subgingival plaque samples were labeled with probes targeting rRNA of unculturable oral “
Synergistetes
” using fluorescent in situ hybridization (FISH). Analysis of 1,664 clones revealed 12 “
Synergistetes
” operational taxonomic units (OTUs) at the 99% sequence identity level, 5 of which were novel. “
Synergistetes
” OTU 4.2 was found in significantly more subjects with periodontitis than controls (
P
= 0.048) and was more abundant in subgingival plaque at diseased sites than at healthy sites in subjects with periodontitis (
P
= 0.019) or controls (
P
= 0.019). FISH analysis revealed that unculturable oral “
Synergistetes
” cells were large curved bacilli. The human oral cavity harbors a diverse population of “
Synergistetes.
” “
Synergistetes
” OTU 4.2 is associated with periodontitis and may have a pathogenic role.
Identification and Functional Analysis of the Gene Cluster for
l
-Arabinose Utilization in
Corynebacterium glutamicum
Authors: Kawaguchi Hideo , Sasaki Miho , Vertès Alain A. , Inui Masayuki , Yukawa Hideaki
Published: April 2009

Corynebacterium glutamicum
ATCC 31831 grew on
l
-arabinose as the sole carbon source at a specific growth rate that was twice that on
d
-glucose. The gene cluster responsible for
l
-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three
l
-arabinose-catabolizing genes,
araA
(encoding
l
-arabinose isomerase),
araB
(
l
-ribulokinase), and
araD
(
l
-ribulose-5-phosphate 4-epimerase), comprised the
araBDA
operon, upstream of which three other genes,
araR
(LacI-type transcriptional regulator),
araE
(
l
-arabinose transporter), and
galM
(putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the
araA
,
araB
, or
araD
gene eliminated growth on
l
-arabinose, and each of the gene products was functionally homologous to its
Escherichia coli
counterpart. Moreover, compared to the wild-type strain, an
araE
disruptant exhibited a >80% decrease in the growth rate at a lower concentration of
l
-arabinose (3.6 g liter
−1
) but not at a higher concentration of
l
-arabinose (40 g liter
−1
). The expression of the
araBDA
operon and the
araE
gene was
l
-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of
araR
eliminated the repression in the absence of
l
-arabinose. Expression of the regulon was not repressed by
d
-glucose, and simultaneous utilization of
l
-arabinose and
d
-glucose was observed in aerobically growing wild-type and
araR
deletion mutant cells. The regulatory mechanism of the
l
-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.
Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in
Lactobacillus rhamnosus
GG and Functional Analysis of the Priming Glycosyltransferase
Authors: Lebeer Sarah , Verhoeven Tine L. A. , Francius Grégory , Schoofs Geert , Lambrichts Ivo , Dufrêne Yves , Vanderleyden Jos , De Keersmaecker Sigrid C. J.
Published: April 2009

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain
Lactobacillus rhamnosus
GG. Subsequent mutation of the
welE
gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the
welE
mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of
L. rhamnosus
GG, possibly because of less shielding of adhesins such as fimbria-like structures.
Endoplasmic Reticulum-Associated Secretory Proteins Sec20p, Sec39p, and Dsl1p Are Involved in Peroxisome Biogenesis
Authors: Perry Ryan J. , Mast Fred D. , Rachubinski Richard A.
Published: April 2009

Two pathways have been identified for peroxisome formation: (i) growth and division and (ii) de novo synthesis. Recent experiments determined that peroxisomes originate at the endoplasmic reticulum (ER). Although many proteins have been implicated in the peroxisome biogenic program, no proteins in the eukaryotic secretory pathway have been identified as having roles in peroxisome formation. Using the yeast
Saccharomyces cerevisiae
regulatable
Tet
promoter Hughes clone collection, we found that repression of the ER-associated secretory proteins Sec20p and Sec39p resulted in mislocalization of the peroxisomal matrix protein chimera Pot1p-green fluorescent protein (GFP) to the cytosol. Likewise, the peroxisomal membrane protein chimera Pex3p-GFP localized to tubular-vesicular structures in cells suppressed for Sec20p, Sec39p, and Dsl1p, which form a complex at the ER. Loss of Sec39p attenuated formation of Pex3p-derived peroxisomal structures following galactose induction of Pex3p-GFP expression from the
GAL1
promoter. Expression of Sec20p, Sec39p, and Dsl1p was moderately increased in yeast grown under conditions that proliferate peroxisomes, and Sec20p, Sec39p, and Dsl1p were found to cofractionate with peroxisomes and colocalize with Pex3p-monomeric red fluorescent protein under these conditions. Our results show that
SEC20
,
SEC39
, and
DSL1
are essential secretory genes involved in the early stages of peroxisome assembly, and this work is the first to identify and characterize an ER-associated secretory machinery involved in peroxisome biogenesis.
Role of the AcsF Protein in
Chloroflexus aurantiacus
Authors: Tang Kuo-Hsiang , Wen Jianzhong , Li Xianglu , Blankenship Robert E.
Published: April 2009

The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have a primarily structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophylls
c
,
d
, and
e
, is not well understood. In this report, we used spectroscopic, proteomic, and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium
Chloroflexus aurantiacus
. Surprisingly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, was identified under anaerobic growth conditions. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestion. Additionally, quantitative real-time PCR studies showed that the transcript level of the
acsF
gene is not lower in anaerobic growth than in semiaerobic growth. Since the proposed enzymatic activity of AcsF requires molecular oxygen, our studies suggest that the roles of AcsF in
C. aurantiacus
need to be investigated further.
Roles of the Outer Membrane Protein AsmA of
Salmonella enterica
in the Control of
marRAB
Expression and Invasion of Epithelial Cells
Authors: Prieto Ana I. , Hernández Sara B. , Cota Ignacio , Pucciarelli M. Graciela , Orlov Yuri , Ramos-Morales Francisco , García-del Portillo Francisco , Casadesús Josep
Published: April 2009

A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (
dam
) mutants of
Salmonella enterica
serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the
Escherichia coli asmA
gene. Disruption of
asmA
suppressed bile sensitivity also in
phoP
and
wec
mutants of
S. enterica
and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of
marA
mRNA were found in
asmA
,
asmA dam
,
asmA phoP
, and
asmA wec
strains of
S. enterica
, suggesting that lack of AsmA activates expression of the
marRAB
operon. Hence,
asmA
mutations may enhance bile resistance by inducing gene expression changes in the
marRAB
-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the
asmA
gene of
S. enterica
encodes a protein of ∼70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of
marRAB
in
asmA
mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with
asmA
+
and
asmA
isogenic strains indicated that lack of AsmA attenuates
Salmonella
virulence by the oral route but not by the intraperitoneal route. Furthermore,
asmA
mutants showed a reduced ability to invade epithelial cells in vitro.
Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium
Azoarcus
sp. Strain BH72
Authors: Oetjen Janina , Reinhold-Hurek Barbara
Published: April 2009

DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class
Alphaproteobacteria
. Here we present for the first time the DraT/DraG system of a betaproteobacterium,
Azoarcus
sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one
draT
ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of
draT
with two genes encoding hypothetical proteins. Furthermore,
draT
and
draG2
were expressed under all studied conditions, whereas
draG1
expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a
draG1
mutant and a mutant lacking both
draG
alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.