bluuebunny/crossref_metadata_2025_split huggingface dataset:
Toxicogenomic Response of
Rhodospirillum rubrum
S1H to the Micropollutant Triclosan
Authors: Pycke Benny F. G. , Vanermen Guido , Monsieurs Pieter , De Wever Heleen , Mergeay Max , Verstraete Willy , Leys Natalie 
Published: April 2010

ABSTRACT

In the framework of the Micro-Ecological Life Support System Alternative (MELiSSA) project, a pilot study was performed to identify the effects of triclosan on the MELiSSA carbon-mineralizing microorganism
Rhodospirillum rubrum
S1H. Triclosan is a biocide that is commonly found in human excrement and is considered an emerging pollutant in wastewater and the environment. Chronic exposure to MELiSSA-relevant concentrations (≥25 μg liter
−1
) of triclosan resulted in a significant extension of the lag phase of this organism but hardly affected the growth rate. Analytical determinations gave no indication of triclosan biodegradation during the growth experiment, and flow cytometric viability analyses revealed that triclosan is bacteriostatic and only slightly toxic to
R. rubrum
S1H. Using microarray analyses, the genetic mechanisms supporting the reversibility of triclosan-induced inhibition were scrutinized. An extremely triclosan-responsive cluster of four small adjacent genes was identified, for which there was up to 34-fold induction with 25 μg liter
−1
triclosan. These four genes, for which the designation
muf
(micropollutant-upregulated factor) is proposed, appear to be unique to
R. rubrum
and are shown here for the first time to be involved in the response to stress. Moreover, numerous other systems that are associated with the proton motive force were shown to be responsive to triclosan, but they were never as highly upregulated as the
muf
genes. In response to triclosan,
R. rubrum
S1H induced transcription of the phage shock protein operon (
pspABC
), numerous efflux systems, cell envelope consolidation mechanisms, the oxidative stress response, beta-oxidation, and carbonic anhydrase, while there was downregulation of bacterial conjugation and carboxysome synthesis genes. The
muf
genes and three efflux-related genes showed the most potential to be low-dose biomarkers.
Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of
Salmonella enterica
Serovar Enteritidis Phage Types
Authors: Preisner Ornella , Guiomar Raquel , Machado Jorge , Menezes José Cardoso , Lopes João Almeida 
Published: April 2010

ABSTRACT

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related
Salmonella enterica
serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm
−1
. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the
Salmonella
Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different
Salmonella
Enteritidis phage types.
Novel Technique for Quantifying Adhesion of
Metarhizium anisopliae
Conidia to the Tick Cuticle
Authors: Ment Dana , Gindin Galina , Rot Asael , Soroker Victoria , Glazer Itamar , Barel Shimon , Samish Michael 
Published: April 2010

ABSTRACT

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of
Metarhizium anisopliae
var.
anisopliae
(Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged
Rhipicephalus annulatus
(Say) (Arachnida: Ixodidae) females and was also verified for
M. anisopliae
var.
acridum
Driver et Milner (Hypocreales: Clavicipitaceae) and
Alphitobius diaperinus
(Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female
R. annulatus
and tick mortality. However, no correlation was observed between a tick's susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.
Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study
Listeria monocytogenes
Infection Processes
Authors: Balestrino Damien , Hamon Mélanie Anne , Dortet Laurent , Nahori Marie-Anne , Pizarro-Cerda Javier , Alignani Diego , Dussurget Olivier , Cossart Pascale , Toledo-Arana Alejandro 
Published: April 2010

ABSTRACT

Listeria monocytogenes
is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect
L. monocytogenes in vitro
and
in vivo
. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring
in vivo
infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells
in vitro
and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-
Listeria
interactions in a fast, robust, and high-throughput manner.
Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters
Authors: Radomski Nicolas , Cambau Emmanuelle , Moulin Laurent , Haenn Sophie , Moilleron Régis , Lucas Françoise S. 
Published: April 2010

ABSTRACT

The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of
Mycobacterium chelonae
and
M. avium
with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 μg/ml amphotericin B, 16 μg/ml nalidixic acid, 4 μg/ml trimethoprim, and 4 μg/ml azlocillin) or PANTAV (PANTA plus 10 μg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 ± 0.4 log
10
CFU/liter on Middlebrook 7H11j medium and 4.2 ± 0.2 log
10
CFU/liter on Middlebrook 7H11j medium containing PANTA [
P
< 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of
M. chelonae
on the two media were 7.0 ± 0.0 log
10
CFU/liter and 6.9 ± 0.0 log
10
CFU/liter, respectively, and the concentrations of
M. avium
were 9.1 ± 0.0 log
10
CFU/liter and 8.9 ± 0.0 log
10
CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.
A Predominant Multidrug-Resistant
Salmonella enterica
Serovar Saintpaul Clonal Line in German Turkey and Related Food Products
Authors: Beutlich Janine , Rodríguez Irene , Schroeter Andreas , Käsbohrer Annemarie , Helmuth Reiner , Guerra Beatriz 
Published: April 2010

ABSTRACT

Recently,
Salmonella enterica
subsp.
enterica
serovar Saintpaul has increasingly been observed in several countries, including Germany. However, the pathogenic potential and epidemiology of this serovar are not very well known. This study describes biological attributes of
S
. Saintpaul isolates obtained from turkeys in Germany based on characterization of their pheno- and genotypic properties. Fifty-five
S
. Saintpaul isolates from German turkeys and turkey-derived food products isolated from 2000 to 2007 were analyzed by using antimicrobial agent, organic solvent, and disinfectant susceptibility tests, isoelectric focusing, detection of resistance determinants, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization experiments. These isolates were compared to an outgroup consisting of 24
S
. Saintpaul isolates obtained from humans and chickens in Germany and from poultry and poultry products (including turkeys) in Netherlands. A common core resistance pattern was detected for 27 German turkey and turkey product isolates. This pattern included resistance (full or intermediate) to ampicillin, amoxicillin-clavulanic acid, gentamicin, kanamycin, nalidixic acid, streptomycin, spectinomycin, and sulfamethoxazole and intermediate resistance or decreased susceptibility to ciprofloxacin (MIC, 2 or 1 μg/ml, respectively) and several third-generation cephalosporins (including ceftiofur and cefoxitin [MIC, 4 to 2 and 16 to 2 μg/ml, respectively]). These isolates had the same core resistance genotype, with
bla
TEM-1
,
aadB
,
aadA2
,
sul1
, a Ser83→Glu83 mutation in the
gyrA
gene, and a chromosomal class 1 integron carrying the
aadB-aadA2
gene cassette. Their XbaI, BlnI, and combined XbaI-BlnI PFGE patterns revealed levels of genetic similarity of 93, 75, and 90%, respectively. This study revealed that a multiresistant
S
. Saintpaul clonal line is widespread in turkeys and turkey products in Germany and was also detected among German human fecal and Dutch poultry isolates.
Exploiting the Natural Diversity of Microviridin Gene Clusters for Discovery of Novel Tricyclic Depsipeptides
Authors: Ziemert Nadine , Ishida Keishi , Weiz Annika , Hertweck Christian , Dittmann Elke 
Published: April 2010

ABSTRACT

Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium
Microcystis
via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene
mdnA
encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in
M
icrocystis aeruginosa
strain NIES843 and heterologously produced variants.
The Case of Botulinum Toxin in Milk: Experimental Data
Authors: Weingart Oliver G. , Schreiber Tanja , Mascher Conny , Pauly Diana , Dorner Martin B. , Berger Thomas F. H. , Egger Charlotte , Gessler Frank , Loessner Martin J. , Avondet Marc-Andre , Dorner Brigitte G. 
Published: April 2010

ABSTRACT

Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism. Due to its high toxicity and the availability of the producing organism
Clostridium botulinum
, BoNT is regarded as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product distribution and consumption, the milk supply chain has long been considered a potential target of a bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B, as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pasteurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We demonstrated that standard pasteurization at 72°C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed “BoNT in milk” scenario.
Partial Chromosome Sequence of
Spiroplasma citri
Reveals Extensive Viral Invasion and Important Gene Decay
Authors: Carle Patricia , Saillard Colette , Carrère Nathalie , Carrère Sébastien , Duret Sybille , Eveillard Sandrine , Gaurivaud Patrice , Gourgues Géraldine , Gouzy Jérome , Salar Pascal , Verdin Eric , Breton Marc , Blanchard Alain , Laigret Frédéric , Bové Joseph-Marie , Renaudin Joel , Foissac Xavier 
Published: April 2010

ABSTRACT

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the
Spiroplasma citri
genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS
30
, IS
481
, IS
3
, and IS
NCY
) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins.
S. citri
has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that
S. citri
possesses a nearly complete pathway for the biosynthesis of a terpenoid.
Comparison of Genotypes of
Salmonella enterica
Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds
Authors: Parker Craig T. , Huynh Steven , Quiñones Beatriz , Harris Linda J. , Mandrell Robert E. 
Published: April 2010

ABSTRACT

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of
Salmonella enterica
serovar Enteritidis were linked with the consumption of raw almonds. The
S
. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental
S
. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these
S
. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the
S
. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of
S
. Enteritidis PT4 and
S. enterica
serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between
S
. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that
S
. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized
l
-aspartic acid,
l
-glutamic acid,
l
-proline,
l
-alanine, and
d
-alanine amino acids more efficiently than
S
. Enteritidis PT30 strains isolated from orchards. These data indicate that
S
. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.
Interplay between Iron Homeostasis and the Osmotic Stress Response in the Halophilic Bacterium
Chromohalobacter salexigens
Authors: Argandoña Montserrat , Nieto Joaquín J. , Iglesias-Guerra Fernando , Calderón Maria Isabel , García-Estepa Raúl , Vargas Carmen 
Published: April 2010

ABSTRACT

In this study, the connection between iron homeostasis and the osmostress response in the halophile
Chromohalobacter salexigens
was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (
cfuABC
-
fur
-
hisI
-
orf6
operon) located downstream of the
ectABC
ectoine synthesis genes was characterized. A
fur
strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of
fur
mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that
fur
encodes a functional iron regulator.
hisI
was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the
cfuABC
-
fur
-
hisI
-
orf6
and
ectABC
operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of
cfuABC
-
fur
-
hisI
-
orf6
operon expression by iron and functioned as a positive regulator of the
ectABC
genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria.
Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies
Authors: Schöbel Felicitas , Jacobsen Ilse D. , Brock Matthias 
Published: April 2010

ABSTRACT

Aspergillus fumigatus
is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal α-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence.
A. fumigatus
HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and α-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An
hcsA
deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified
A. fumigatus
proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.
Single-Step Affinity Purification for Fungal Proteomics
Authors: Liu Hui-Lin , Osmani Aysha H. , Ukil Leena , Son Sunghun , Markossian Sarine , Shen Kuo-Fang , Govindaraghavan Meera , Varadaraj Archana , Hashmi Shahr B. , De Souza Colin P. , Osmani Stephen A. 
Published: April 2010

ABSTRACT

A single-step protein affinity purification protocol using
Aspergillus nidulans
is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.