bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences
Authors: Agúndez Leticia , Machón Cristina , César Carolina Elvira , Rosa-Garrido Manuel , Delgado M. Dolores , Llosa Matxalen 
Published: October 2010

ABSTRACT

TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the
trwC
gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.
Longitudinal Molecular Epidemiological Study of Thermophilic Campylobacters on One Conventional Broiler Chicken Farm
Authors: Ridley Anne M. , Morris Victoria K. , Cawthraw Shaun A. , Ellis-Iversen Johanne , Harris Jillian A. , Kennedy Emma M. , Newell Diane G. , Allen Vivien M. 
Published: October 2010

ABSTRACT

Improved understanding of the ecology and epidemiology of
Campylobacter
in the poultry farm environment is key to developing appropriate farm-based strategies for preventing flock colonization. The sources of
Campylobacter
causing broiler flock colonization were investigated on one poultry farm and its environment, from which samples were obtained on three occasions during each of 15 crop cycles. The farm was adjacent to a dairy farm, with which there was a shared concreted area and secondary entrance. There was considerable variation in the
Campylobacter
status of flocks at the various sampling times, at median ages of 20, 26, and 35 days, with 3 of the 15 flocks remaining negative at slaughter. Campylobacters were recoverable from various locations around the farm, even while the flock was
Campylobacter
negative, but the degree of environmental contamination increased substantially once the flock was positive. Molecular typing showed that strains from house surroundings and the dairy farm were similar to those subsequently detected in the flock and that several strains intermittently persisted through multiple crop cycles. The longitudinal nature of the study suggested that bovine fecal
Campylobacter
strains, initially recovered from the dairy yard, may subsequently colonize poultry. One such strain, despite being repeatedly recovered from the dairy areas, failed to colonize the concomitant flock during later crop cycles. The possibility of host adaptation of this strain was investigated with 16-day-old chickens experimentally exposed to this strain naturally present in, or spiked into, bovine feces. Although the birds became colonized by this infection model, the strain may preferentially infect cattle. The presence of
Campylobacter
genotypes in the external environment of the poultry farm, prior to their detection in broiler chickens, confirms the horizontal transmission of these bacteria into the flock and highlights the risk from multispecies farms.
Ethanolamine Utilization Contributes to Proliferation of
Salmonella enterica
Serovar Typhimurium in Food and in Nematodes
Authors: Srikumar Shabarinath , Fuchs Thilo M. 
Published: October 2010

ABSTRACT

Only three pathogenic bacterial species,
Salmonella enterica
,
Clostridium perfringens
, and
Listeria monocytogenes
, are able to utilize both ethanolamine and 1,2-propanediol as a sole carbon source. Degradation of these substrates, abundant in food and the gut, depends on cobalamin, which is synthesized
de novo
only under anaerobic conditions. Although the
eut
,
pdu
, and
cob
-
cbi
gene clusters comprise 40 kb, the conditions under which they confer a selection advantage on these food-borne pathogens remain largely unknown. Here we used the luciferase reporter system to determine the response of the
Salmonella enterica
serovar Typhimurium promoters P

eutS

, P

pocR

, P

pduF

, and P

pduA

to a set of carbon sources, to egg yolk, to whole milk, and to milk protein or fat fractions. Depending on the supplements, specific inductions up to 3 orders of magnitude were observed for P

eutS

and P

pduA

, which drive the expression of most
eut
and
pdu
genes. To correlate these significant expression data with growth properties, nonpolar deletions of
pocR
, regulating the
pdu
and
cob
-
cbi
genes, and of
eutR
, involved in
eut
gene activation, were constructed in
S
. Typhimurium strain 14028. During exponential growth of the mutants 14028Δ
pocR
and 14028Δ
eutR
, 2- to 3-fold-reduced proliferation in milk and egg yolk was observed. Using the
Caenorhabditis elegans
infection model, we could also demonstrate that the proliferation of
S
. Typhimurium in the nematode is supported by an active ethanolamine degradation pathway. Taking these findings together, this study quantifies the differential expression of
eut
and
pdu
genes under distinct conditions and provides experimental evidence that the ethanolamine utilization pathway allows salmonellae to occupy specific metabolic niches within food environments and within their host organisms.
Verotoxins in Bovine and Meat Verotoxin-Producing
Escherichia coli
Isolates: Type, Number of Variants, and Relationship to Cytotoxicity
Authors: Krüger Alejandra , Lucchesi Paula M. A. , Parma Alberto E. 
Published: October 2010

ABSTRACT

In this study, we determined
vt
subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing
Escherichia coli
(VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a
vt
2
gene. Moreover, the
vt
2
subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other
vt
subtypes detected were
vt
1
,
vt
1d
,
vt
2vha
,
vt
2vhb
,
vt
2O118
,
vt
2d
(mucus activatable), and
vt
2g
. A total of 41 (22%) of the isolates possessed more than one
vt
subtype in its genome, and among them the most frequent combination was
vt
1
/
vt
2
, but we also observed multiple combinations among
vt
2
subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single
vt
2
variant, those carrying the
vt
2
subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying
vt
2vha
or
vt
2hb
variants. Notably, the isolates carrying the
vt
1
subtype showed a lesser increase than that of most of the
vt
2
-positive VTEC strains. Furthermore, the presence of more than one
vt
gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of
vt
variants more than with the serotype or origin of the isolate.
Raman Spectroscopy of Xylitol Uptake and Metabolism in Gram-Positive and Gram-Negative Bacteria
Authors: Palchaudhuri Sunil , Rehse Steven J. , Hamasha Khozima , Syed Talha , Kurtovic Eldar , Kurtovic Emir , Stenger James 
Published: October 2010

ABSTRACT

Visible-wavelength Raman spectroscopy was used to investigate the uptake and metabolism of the five-carbon sugar alcohol xylitol by Gram-positive viridans group streptococcus and the two extensively used strains of Gram-negative
Escherichia coli
,
E. coli
C and
E. coli
K-12.
E. coli
C, but not
E. coli
K-12, contains a complete xylitol operon, and the viridans group streptococcus contains an incomplete xylitol operon used to metabolize the xylitol. Raman spectra from xylitol-exposed viridans group streptococcus exhibited significant changes that persisted even in progeny grown from the xylitol-exposed mother cells in a xylitol-free medium for 24 h. This behavior was not observed in the
E. coli
K-12. In both viridans group streptococcus and the
E. coli
C derivative HF4714, the metabolic intermediates are stably formed to create an anomaly in bacterial normal survival. The uptake of xylitol by Gram-positive and Gram-negative pathogens occurs even in the presence of other high-calorie sugars, and its stable integration within the bacterial cell wall may discontinue bacterial multiplication. This could be a contributing factor for the known efficacy of xylitol when taken as a prophylactic measure to prevent or reduce occurrences of persistent infection. Specifically, these bacteria are causative agents for several important diseases of children such as pneumonia, otitis media, meningitis, and dental caries. If properly explored, such an inexpensive and harmless sugar-alcohol, alone or used in conjunction with fluoride, would pave the way to an alternative preventive therapy for these childhood diseases when the causative pathogens have become resistant to modern medicines such as antibiotics and vaccine immunotherapy.
Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins
Authors: Gandhi Kamal M. , Mandrell Robert E. , Tian Peng 
Published: October 2010

ABSTRACTNoroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.
Development of a Fatty Acid and RNA Stable Isotope Probing-Based Method for Tracking Protist Grazing on Bacteria in Wastewater
Authors: Kuppardt Steffen , Chatzinotas Antonis , Kästner Matthias 
Published: October 2010

ABSTRACT

Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on
13
C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate
Uronema
sp. and
13
C-labeled
Escherichia coli
as model prey. The transfer of bacterial
13
C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP).
Uronema
sp. showed ingestion rates of ∼390 bacteria protist
−1
h
−1
, and the temporal patterns of
13
C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for
Uronema
sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the
E. coli
-derived
13
C was incorporated into
Uronema
biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (

y
x

/

s

≈ 0.2), and the temporal pattern of
13
C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom%
13
C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of
13
C-labeled protist RNA demonstrated a high incorporation of
13
C, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.
Podospora anserina
Hemicellulases Potentiate the
Trichoderma reesei
Secretome for Saccharification of Lignocellulosic Biomass
Authors: Couturier Marie , Haon Mireille , Coutinho Pedro M. , Henrissat Bernard , Lesage-Meessen Laurence , Berrin Jean-Guy 
Published: October 2010

ABSTRACT

To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by
Trichoderma reesei
, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus
Podospora anserina
using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast
Pichia pastoris
. These novel fungal CAZymes belonging to different glycoside hydrolase families (
Pa
Man5A and
Pa
Man26A mannanases,
Pa
Xyn11A xylanase, and
Pa
Abf51A and
Pa
Abf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although
Pa
Man5A and
Pa
Man26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of
Pa
Man26A displayed dual binding specificity toward xylan and mannan.
Pa
Xyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product.
Pa
Abf51A and
Pa
Abf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each
P. anserina
auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (
Pa
Xyn11A,
Pa
Man5A, and
Pa
Man26A) individually supplemented the secretome of the industrial
T. reesei
CL847 strain. The most striking effect was obtained with
Pa
Man5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.
Same-Day Detection ofEscherichia coliO157:H7 from Spinach by Using Electrochemiluminescent and Cytometric Bead Array Biosensors
Authors: Leach Kelly M. , Stroot Joyce M. , Lim Daniel V. 
Published: October 2010

ABSTRACTContamination of fresh produce withEscherichia coliO157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach forE. coliO157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU ofE. coliO157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection ofE. coliO157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.
Production of Glutaconic Acid in a RecombinantEscherichia coliStrain
Authors: Djurdjevic Ivana , Zelder Oskar , Buckel Wolfgang 
Published: October 2010

ABSTRACTThe assembly of six genes that encode enzymes from glutamate-fermenting bacteria convertedEscherichia coliinto a glutaconate producer when grown anaerobically on a complex medium. The new anaerobic pathway starts with 2-oxoglutarate from general metabolism and proceeds via (R)-2-hydroxyglutarate, (R)-2-hydroxyglutaryl-coenzyme A (CoA), and (E)-glutaconyl-CoA to yield 2.7 ± 0.2 mM (E)-glutaconate in the medium.
Preclinical Class 1 Integron with a Complete Tn
402
-Like Transposition Module
Authors: Sajjad Ammara , Holley Marita P. , Labbate Maurizio , Stokes H. W. , Gillings Michael R. 
Published: October 2010

ABSTRACT

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn
402
-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in
Delftia
tsuruhatensis
.
Diffusion of Nanoparticles in Biofilms Is Altered by Bacterial Cell Wall Hydrophobicity
Authors: Habimana Olivier , Steenkeste Karine , Fontaine-Aupart Marie-Pierre , Bellon-Fontaine Marie-Noëlle , Kulakauskas Saulius , Briandet Romain 
Published: October 2010

ABSTRACT

Diffusion of entities inside biofilm triggers most mechanisms involved in biofilm-specific phenotypes. Using genetically engineered hydrophilic and hydrophobic cells of
Lactococcus lactis
yielding similar biofilm architectures, we demonstrated by fluorescence correlation spectroscopy that bacterial surface properties affect diffusion of nanoparticles through the biofilm matrix.
Expanding the Genetic Toolbox for
Leptospira
Species by Generation of Fluorescent Bacteria
Authors: Aviat Florence , Slamti Leyla , Cerqueira Gustavo M. , Lourdault Kristel , Picardeau Mathieu 
Published: October 2010

ABSTRACT

Our knowledge of the genetics and molecular basis of the pathogenesis associated with
Leptospira
, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of
gfp
and
mRFP1
genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic
Leptospira
strains. We were able to reliably measure the fluorescence of
Leptospira
by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the
gfp
gene had no significant effects on growth
in vivo
and pathogenicity in
L. interrogans
. We constructed an expression vector for
L. biflexa
that contains the
lacI
repressor, an inducible
lac
promoter, and
gfp
as the reporter, demonstrating that the
lac
system is functional in
Leptospira
. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-
d
-thiogalactopyranoside (IPTG) in
L. biflexa
transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of
Leptospira
spp.
Child injury surveillance that guides interventions: the Beijing primary healthcare experience
Authors: Linnan Huan , Duan Jianhua , Chen Xinxin , Yan Shujuan 
Published: October 2010

ObjectivesTo provide feedback to guide injury prevention interventions and to test the feasibility of integrating injury surveillance into the existing primary healthcare service for children under 6 years of age.DesignThis surveillance system was integrated into existing primary healthcare services at the community primary healthcare centre (PHC) and community kindergartens. Parents reported injury incidents during their child's routine health services at scheduled intervals. Kindergarten nurses registered injury incidents at the absentee recording system. A total of 8358 children annually were included into the surveillance in 2006–07.SettingTwo urban communities and three rural communities in Beijing, China.ResultsThe coverage rates were 93.7% in PHCs and 98% in kindergartens. The injury incidence rates in year 1 and year 2 were 2.7% and 1.9% respectively. The leading causes were falls, being hit by blunt object, and animal injury. Major causes of injury were different between urban and rural children. Over the two-year surveillance period, injury incidence rates declined significantly from 2.7% to 1.7% in urban communities. No significant decrease was seen in rural communities.ConclusionSurveillance through the primary healthcare system in Beijing was useful in child injury prevention as data collected allowed managers to design targeted interventions that resulted in decreased injury. In comparison with hospital based surveillance, PHC based surveillance demonstrated an advantage in wide access to children and allowed calculation of injury incidence rates to monitor trends over time. PHC based surveillance can serve as a useful venue to collect child injury data.
Diagnostic reasoning in Nizāmī ‘Arūzī’sFour Discourses
Authors: Aggarwal Neil Krishan 
Published: October 2010

BackgroundMost studies on medical reasoning focus on contemporary allopathic practitioners. Here, the significance of diagnostic sense in Nizāmī ‘Arūzī’sFour Discourses(Chahār Maqāle), an influential text that circulated widely throughout the Islamic world, is explored.MethodsAfter a brief introduction, key passages are translated on how doctors should cultivate analytical skills.ResultsNizāmī ‘Arūzī cites three sources of diagnostic authority: (1) education in the texts of medical experts, (2) formal logic and (3) belief in the power of God.Conclusions‘Arūzī’s methods, though rooted in a religious faith that may seem foreign to contemporary doctors and educators, has much to recommend regarding its clarity of approach, its high ethical standards and expectations of doctors, and its commitment to best practices for the patient. Crosscultural studies of medical education and professionalism can offer valuable insights by demonstrating how doctors across history and geography have grappled with the common concerns of diagnosis and treatment.