bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces
Authors: Schönenbrücher H. , Abdulmawjood A. , Failing K. , Bülte M. 
Published: March 2008

ABSTRACTIn the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2sequences ofMycobacterium aviumsubsp.paratuberculosisfrom bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105M. aviumsubsp.paratuberculosisstrains of bovine, ovine, and human origin and 100 non-M. aviumsubsp.paratuberculosisstrains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogramM. aviumsubsp.paratuberculosisDNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection ofM. aviumsubsp.paratuberculosisDNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgband locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection ofM. aviumsubsp.paratuberculosisfrom bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknownM. aviumsubsp.paratuberculosisstatus resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.
Molecular Epidemiology of
Bacillus anthracis
: Determining the Correct Origin
Authors: Pilo Paola , Perreten Vincent , Frey Joachim 
Published: March 2008

ABSTRACT

We analyzed and compared strains of
Bacillus anthracis
isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human
B. anthracis
strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of
B. anthracis
strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.
Global Response to Desiccation Stress in the Soil Actinomycete
Rhodococcus jostii
RHA1
Authors: LeBlanc Justin C. , Gonçalves Edmilson R. , Mohn William W. 
Published: March 2008

ABSTRACT

Rhodococcus jostii
RHA1 is a soil-residing actinomycete with many favorable metabolic capabilities that make it an ideal candidate for the bioremediation of contaminated soils. Arguably the most basic requirement for life is water, yet some nonsporulating bacteria, like RHA1, can survive lengthy droughts. Here we report the first transcriptomic analysis of a gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air-drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of approximately 8 times more differentially regulated genes than in the control (819 versus 106 genes, respectively). Genes that were differentially expressed during only the desiccation treatment primarily had expression profiles that were maximally up-regulated upon complete drying of the cells. The microarray expression ratios for some of the highly up-regulated genes were verified by reverse transcriptase quantitative PCR. These genes included
dps1
, encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, and the two genes encoding sigma factors SigF1 and SigF3, possibly involved in the regulatory response to desiccation. RHA1 cells also induced the biosynthetic pathway for the compatible solute ectoine. These desiccation-specific responses represent the best candidates for important mechanisms of desiccation resistance in RHA1.
Analysis of Neurotoxin Cluster Genes in
Clostridium botulinum
Strains Producing Botulinum Neurotoxin Serotype A Subtypes
Authors: Jacobson Mark J. , Lin Guangyun , Raphael Brian , Andreadis Joanne , Johnson Eric A. 
Published: March 2008

ABSTRACT

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of
Clostridium botulinum
encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA
−
Orfx
+
A1). These sequences were compared to the known neurotoxin cluster sequences of
C. botulinum
strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA
−
Orfx
+
A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the
ntnh
gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the
ntnh
genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the
ntnh
gene was assigned.
Highly Selective and Rapid Arsenic Removal by Metabolically Engineered
Escherichia coli
Cells Expressing
Fucus vesiculosus
Metallothionein
Authors: Singh Shailendra , Mulchandani Ashok , Chen Wilfred 
Published: March 2008

ABSTRACT

An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga
Fucus vesiculosus
was expressed in
Escherichia coli
, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting
E. coli
cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.
Role of PUG1 in Inducible Porphyrin and Heme Transport in
Saccharomyces cerevisiae
Authors: Protchenko Olga , Shakoury-Elizeh Minoo , Keane Patricia , Storey Joshua , Androphy Rachel , Philpott Caroline C. 
Published: March 2008

ABSTRACT

Unlike pathogenic fungi, the budding yeast
Saccharomyces cerevisiae
is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of
PUG1
(protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing
PUG1
exhibited decreased accumulation of [
55
Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of
PUG1
overexpression on intracellular heme pools, we used a
CYC1
-
lacZ
reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.
DictyosteliumAurora Kinase Has Properties of both Aurora A and Aurora B Kinases
Authors: Li Hui , Chen Qian , Kaller Markus , Nellen Wolfgang , Gräf Ralph , De Lozanne Arturo 
Published: March 2008

ABSTRACTAurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show thatDictyostelium discoideumcontains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.
RcoM: A New Single-Component Transcriptional Regulator of CO Metabolism in Bacteria
Authors: Kerby Robert L. , Youn Hwan , Roberts Gary P. 
Published: March 2008

ABSTRACT

Genomic analysis suggested the existence of a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain. UV/visible-light spectral analyses of heterologously expressed, purified full-length proteins indicated that they contained a hexacoordinated
b
-type heme moiety that avidly binds CO and NO. Studies of protein variants strongly suggested that the PAS domain residues His74 and Met104 serve as the heme Fe(II) axial ligands, with displacement of Met104 upon binding of the gaseous effectors. Two RcoM (
r
egulator of
CO
m
etabolism) homologs were shown to function in vivo as CO sensors capable of regulating an aerobic CO oxidation (
cox
) regulon. The genetic linkage of
rcoM
with both aerobic (
cox
) and anaerobic (
coo
) CO oxidation systems suggests that in different organisms RcoM proteins may control either regulon type.
The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an
Escherichia coli arcA
Mutant
Authors: Nikel Pablo I. , de Almeida Alejandra , Pettinari M. Julia , Méndez Beatriz S. 
Published: March 2008

ABSTRACT

Strains derived from HfrH carrying the
arcA2
null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than
arcA
deletion mutants of a different lineage. The phenotype of the
arcA2
mutants was due to the presence of a
creC
constitutive mutation introduced by P1 transduction.
RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids inPseudomonas aeruginosa
Authors: Zhu Kun , Rock Charles O. 
Published: March 2008

ABSTRACTPseudomonas aeruginosasecretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of β-hydroxydecanoyl-β-hydroxydecanoate from two molecules of β-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily β-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity inP. aeruginosaincreases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for β-hydroxydecanoyl-ACP. InEscherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.
ORF334 in
Vibrio
Phage KVP40 Plays the Role of gp27 in T4 Phage To Form a Heterohexameric Complex
Authors: Nemoto Mai , Mio Kazuhiro , Kanamaru Shuji , Arisaka Fumio 
Published: March 2008

ABSTRACT
KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.
Glutamate Metabolism inBacillus subtilis: Gene Expression and Enzyme Activities Evolved To Avoid Futile Cycles and To Allow Rapid Responses to Perturbations of the System
Authors: Commichau Fabian M. , Gunka Katrin , Landmann Jens J. , Stülke Jörg 
Published: March 2008

ABSTRACTGlutamate is a central metabolite in all organisms since it provides the link between carbon and nitrogen metabolism. InBacillus subtilis, glutamate is synthesized exclusively by the glutamate synthase, and it can be degraded by the glutamate dehydrogenase. InB. subtilis, the major glutamate dehydrogenase RocG is expressed only in the presence of arginine, and the bacteria are unable to utilize glutamate as the only carbon source. In addition torocG, a second cryptic gene (gudB) encodes an inactive glutamate dehydrogenase. Mutations inrocGresult in the rapid accumulation ofgudB1suppressor mutations that code for an active enzyme. In this work, we analyzed the physiological significance of this constellation of genes and enzymes involved in glutamate metabolism. We found that the weak expression ofrocGin the absence of the inducer arginine is limiting for glutamate utilization. Moreover, we addressed the potential ability of the active glutamate dehydrogenases ofB. subtilisto synthesize glutamate. Both RocG and GudB1 were unable to catalyze the anabolic reaction, most probably because of their very highKmvalues for ammonium. In contrast, theEscherichia coliglutamate dehydrogenase is able to produce glutamate even in the background of aB. subtiliscell.B. subtilisresponds to any mutation that interferes with glutamate metabolism with the rapid accumulation of extragenic or intragenic suppressor mutations, bringing the glutamate supply into balance. Similarly, with the presence of a cryptic gene, the system can flexibly respond to changes in the external glutamate supply by the selection of mutations.
Genomic and Functional Analysis of ICE
Pda
Spa1, a Fish-Pathogen-Derived SXT-Related Integrating Conjugative Element That Can Mobilize a Virulence Plasmid
Authors: Osorio Carlos R. , Marrero Joeli , Wozniak Rachel A. F. , Lemos Manuel L. , Burrus Vincent , Waldor Matthew K. 
Published: March 2008

ABSTRACT

Integrating conjugative elements (ICEs) are self-transmissible mobile elements that transfer between bacteria via conjugation and integrate into the host chromosome. SXT and related ICEs became prevalent in Asian
Vibrio cholerae
populations in the 1990s and play an important role in the dissemination of antibiotic resistance genes in
V. cholerae
. Here, we carried out genomic and functional analyses of ICE
Pda
Spa1, an SXT-related ICE derived from a Spanish isolate of
Photobacterium damselae
subsp.
piscicida
, the causative agent of fish pasteurellosis. The ∼102-kb DNA sequence of ICE
Pda
Spa1 shows nearly 97% DNA sequence identity to SXT in genes that encode essential ICE functions, including integration and excision, conjugal transfer, and regulation. However, ∼25 kb of ICE
Pda
Spa1 DNA, including a tetracycline resistance locus, is not present in SXT. Most ICE
Pda
Spa1-specific DNA is inserted at loci where other SXT-related ICEs harbor element-specific DNA. ICE
Pda
Spa1 excises itself from the chromosome and is transmissible to other
Photobacterium
strains, as well as to
Escherichia coli
, in which it integrates into
prfC
. Interestingly, the
P. damselae
virulence plasmid pPHDP10 could be mobilized from
E. coli
in an ICE
Pda
Spa1-dependent fashion via the formation of a cointegrate between pPHDP10 and ICE
Pda
Spa1. pPHDP10-Cm integrated into ICE
Pda
Spa1 in a non-site-specific fashion independently of RecA. The ICE
Pda
Spa1::pPHDP10 cointegrates were stable, and markers from both elements became transmissible at frequencies similar to those observed for the transfer of ICE
Pda
Spa1 alone. Our findings reveal the plasticity of ICE genomes and demonstrate that ICEs can enable virulence gene transfer.
Topology and Function of CcmD in Cytochrome
c
Maturation
Authors: Richard-Fogal Cynthia L. , Frawley Elaine R. , Kranz Robert G. 
Published: March 2008

ABSTRACT

The system I cytochrome
c
biogenesis pathway requires CcmD, a small polypeptide of 69 residues in
Escherichia coli
. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.
Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na
+
-Driven Flagellar Motor
Authors: Obara Madoka , Yakushi Toshiharu , Kojima Seiji , Homma Michio 
Published: March 2008

ABSTRACT

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in
Escherichia coli
. Mutational studies have indicated that the Na
+
-driven motor of
Vibrio alginolyticus
may incorporate interactions similar to those of the
E. coli
motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na
+
-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na
+
-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of
V. alginolyticus
or with the Na
+
-driven chimeric motor of
E. coli
. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type
V. alginolyticus
. The R207E mutant conferred motility with the motor of
V. alginolyticus
but not with the chimeric motor of
E. coli
. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in
V. alginolyticus
but was greatly reduced in
E. coli
. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.
Structural Analysis and Biosynthesis Gene Cluster of an Antigenic Glycopeptidolipid fromMycobacterium intracellulare
Authors: Fujiwara Nagatoshi , Nakata Noboru , Naka Takashi , Yano Ikuya , Doe Matsumi , Chatterjee Delphi , McNeil Michael , Brennan Patrick J. , Kobayashi Kazuo , Makino Masahiko , Matsumoto Sohkichi , Ogura Hisashi , Maeda Shinji 
Published: March 2008

ABSTRACTMycobacterium avium-Mycobacterium intracellularecomplex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at theallo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived fromM. intracellularewas determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novelN-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2′-methyl-3′-hydroxy-4′-methoxy-pentanoyl-amido-3,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-6-deoxy-α-l-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. AnM. aviumserotype 1 strain was transformed with cosmid clone no. 253 containinggtfB-drrCofM. intracellulareserotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.