bluuebunny/crossref_metadata_2025_split huggingface dataset:
Results of Continuous Monitoring of the Performance of Rubella Virus IgG and Hepatitis B Virus Surface Antibody Assays Using Trueness Controls in a Multicenter Trial
Authors: Kruk Tamara , Ratnam Sam , Preiksaitis Jutta , Lau Allan , Hatchette Todd , Horsman Greg , Van Caeseele Paul , Timmons Brian , Tipples Graham 
Published: August 2012

ABSTRACTWe conducted a multicenter trial in Canada to assess the value of using trueness controls (TC) for rubella virus IgG and hepatitis B virus surface antibody (anti-HBs) serology to determine test performance across laboratories over time. TC were obtained from a single source with known international units. Seven laboratories using different test systems and kit lots included the TC in routine assay runs of the analytes. TC measurements of 1,095 rubella virus IgG and 1,195 anti-HBs runs were plotted on Levey-Jennings control charts for individual laboratories and analyzed using a multirule quality control (MQC) scheme as well as a single three-standard-deviation (3-SD) rule. All rubella virus IgG TC results were “in control” in only one of the seven laboratories. Among the rest, “out-of-control” results ranged from 5.6% to 10% with an outlier at 20.3% by MQC and from 1.1% to 5.6% with an outlier at 13.4% by the 3-SD rule. All anti-HBs TC results were “in control” in only two laboratories. Among the rest, “out-of-control” results ranged from 3.3% to 7.9% with an outlier at 19.8% by MQC and from 0% to 3.3% with an outlier at 10.5% by the 3-SD rule. In conclusion, through the continuous monitoring of assay performance using TC and quality control rules, our trial detected significant intra- and interlaboratory, test system, and kit lot variations for both analytes. In most cases the assay rejections could be attributable to the laboratories rather than to kit lots. This has implications for routine diagnostic screening and clinical practice guidelines and underscores the value of using an approach as described above for continuous quality improvement in result reporting and harmonization for these analytes.
The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis
Authors: Gallagher Lorna , Owens Rebecca A. , Dolan Stephen K. , O'Keeffe Grainne , Schrettl Markus , Kavanagh Kevin , Jones Gary W. , Doyle Sean 
Published: August 2012

ABSTRACT

The function of a number of genes in the gliotoxin biosynthetic cluster (
gli
) in
Aspergillus fumigatus
remains unknown. Here, we demonstrate that
gliK
deletion from two strains of
A. fumigatus
completely abolished gliotoxin biosynthesis. Furthermore, exogenous H
2
O
2
(1 mM), but not gliotoxin, significantly induced
A. fumigatus gliK
expression (
P
= 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (
P
< 0.001) and H
2
O
2
(
P
< 0.01), unexpectedly, exogenous gliotoxin relieved H
2
O
2
-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from
A. fumigatus
ATCC 26933 significantly inhibited (
P
< 0.05) the growth of the Δ
gliK
26933
deletion mutant. The
A. fumigatus
Δ
gliK
26933
mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with
m/z
394 to 396. These metabolites (
m/z
394 to 396) were present at significantly higher levels in the culture supernatants of the
A. fumigatus
Δ
gliK
26933
mutant than in those of the wild type (
P
= 0.0024 [fold difference, 24] and
P
= 0.0003 [fold difference, 9.6], respectively) and were absent from
A. fumigatus
Δ
gliG
. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the
A. fumigatus
Δ
gliK
26933
mutant compared to the wild type (
P
< 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (
P
= 0.0045) between that of
A. fumigatus
ATCC 46645 (9.3 pg/mg mycelium/min) and the Δ
gliK
46645
mutant (31.4 pg/mg mycelium/min), strongly suggesting that
gliK
absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for
gliK
in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in
A. fumigatus
.
Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa
Authors: Won Susan , Michkov Alexander V. , Krystofova Svetlana , Garud Amruta V. , Borkovich Katherine A. 
Published: August 2012

ABSTRACT

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus
Neurospora crassa
. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three
N. crassa
Gα subunits are analyzed for genetic epistasis with
gnb-1
and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and
gnb-1
and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δ
gnb-1
background. Genetic analysis revealed that
gna-3
is epistatic to
gnb-1
with regard to negative control of submerged conidiation.
gnb-1
is epistatic to
gna-2
and
gna-3
for aerial hyphal height, while
gnb-1
appears to act upstream of
gna-1
and
gna-2
during aerial conidiation. None of the activated Gα alleles restored female fertility to Δ
gnb-1
mutants, and the
gna-3
Q208L
allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the
gng-1
-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between
gnb-1
and
gna-1
,
gna-2
, and
gna-3
for at least one function.
Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus
Authors: Raymond-Bouchard Isabelle , Carroll Cassandra S. , Nesbitt Jason R. , Henry Kevin A. , Pinto Linda J. , Moinzadeh Mina , Scott Jamie K. , Moore Margo M. 
Published: August 2012

ABSTRACT

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen
Aspergillus fumigatus
. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore
N,N
′,
N
″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of
A. fumigatus
MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the
Saccharomyces cerevisiae
strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using
55
Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in
A. fumigatus
showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.
Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica
Authors: Fowler Matthew L. , Ingram-Smith Cheryl , Smith Kerry S. 
Published: August 2012

ABSTRACT

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist
Entamoeba histolytica
. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP
i
)/inorganic phosphate (P
i
) as the sole phosphoryl donor/acceptor. Detection of ACK activity in
E. histolytica
cell extracts in the direction of acetate/PP
i
formation but not in the direction of acetyl phosphate/P
i
formation suggests that the physiological direction of the reaction is toward acetate/PP
i
production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The
E. histolytica
PP
i
-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized
Methanosarcina thermophila
ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the
M. thermophila
ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in
Entamoeba
.
A Distinct Regulatory Sequence Is Essential for the Expression of a Subset of
nle
Genes in Attaching and Effacing Escherichia coli
Authors: García-Angulo Víctor A. , Martínez-Santos Verónica I. , Villaseñor Tomás , Santana Francisco J. , Huerta-Saquero Alejandro , Martínez Luary C. , Jiménez Rafael , Lara-Ochoa Cristina , Téllez-Sosa Juan , Bustamante Víctor H. , Puente José L. 
Published: August 2012

ABSTRACT

Enteropathogenic
Escherichia coli
uses a type III secretion system (T3SS), encoded in the locus of enterocyte effacement (LEE) pathogenicity island, to translocate a wide repertoire of effector proteins into the host cell in order to subvert cell signaling cascades and promote bacterial colonization and survival. Genes encoding type III-secreted effectors are located in the LEE and scattered throughout the chromosome. While LEE gene regulation is better understood, the conditions and factors involved in the expression of effectors encoded outside the LEE are just starting to be elucidated. Here, we identified a highly conserved sequence containing a 13-bp inverted repeat (IR), located upstream of a subset of genes coding for different non-LEE-encoded effectors in A/E pathogens. Site-directed mutagenesis and deletion analysis of the
nleH1
and
nleB2
regulatory regions revealed that this IR is essential for the transcriptional activation of both genes. Growth conditions that favor the expression of LEE genes also facilitate the activation of
nleH1
and
nleB2
; however, their expression is independent of the LEE-encoded positive regulators Ler and GrlA but is repressed by GrlR and the global regulator H-NS. In contrast, GrlA and Ler are required for
nleA
expression, while H-NS silences it. Consistent with their role in the regulation of
nleA
, purified Ler and H-NS bound to the regulatory region of
nleA
upstream of its promoter. This work shows that at least two modes of regulation control the expression of effector genes in attaching and effacing (A/E) pathogens, suggesting that a subset of effector functions may be coordinately expressed in a particular niche or time during infection.
Functional Characterization of the Stringent Response Regulatory GenedksAof Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes
Authors: Pal Ritesh Ranjan , Bag Satyabrata , Dasgupta Shreya , Das Bhabatosh , Bhadra Rupak K. 
Published: August 2012

ABSTRACTIn bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes,relA,spoT, andrelV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogenVibrio cholerae. Here we report functional characterization of thedksAgene ofV. cholerae(dksAVc), coding for the protein DksAVc. Extensive genetic analyses of the ΔdksAVcmutants suggest that DksAVcis an important component involved in the stringent response inV. cholerae. Further analysis of mutants revealed that DksAVcpositively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), underin vitroconditions. We found that DksAVcupregulates expression of the sigma factor FliA (σ28), a critical regulator of motility inV. cholerae. Altogether, it appears that apart from stringent-response regulation, DksAVcalso has important roles in fine regulation of virulence-related phenotypes ofV. cholerae.
Deletion of the Desulfovibrio vulgaris Carbon Monoxide Sensor Invokes Global Changes in Transcription
Authors: Rajeev Lara , Hillesland Kristina L. , Zane Grant M. , Zhou Aifen , Joachimiak Marcin P. , He Zhili , Zhou Jizhong , Arkin Adam P. , Wall Judy D. , Stahl David A. 
Published: August 2012

ABSTRACTThe carbon monoxide-sensing transcriptional factor CooA has been studied only in hydrogenogenic organisms that can grow using CO as the sole source of energy. Homologs for the canonical CO oxidation system, including CooA, CO dehydrogenase (CODH), and a CO-dependent Coo hydrogenase, are present in the sulfate-reducing bacteriumDesulfovibrio vulgaris, although it grows only poorly on CO. We show thatD. vulgarisHildenborough has an active CO dehydrogenase capable of consuming exogenous CO and that the expression of the CO dehydrogenase, but not that of a gene annotated as encoding a Coo hydrogenase, is dependent on both CO and CooA. Carbon monoxide did not act as a general metabolic inhibitor, since growth of a strain deleted forcooAwas inhibited by CO on lactate-sulfate but not pyruvate-sulfate. While the deletion strain did not accumulate CO in excess, as would have been expected if CooA were important in the cycling of CO as a metabolic intermediate, global transcriptional analyses suggested that CooA and CODH are used during normal metabolism.
Direct and Indirect Regulation of the
ycnKJI
Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis
Authors: Hirooka Kazutake , Edahiro Takayosh , Kimura Kosuke , Fujita Yasutaro 
Published: August 2012

ABSTRACT

Northern blot and primer extension analyses revealed that the
ycnKJI
operon and the
ycnL
gene of
Bacillus subtilis
are transcribed from adjacent promoters that are divergently oriented. The
ycnK
and
ycnJ
genes encode a DeoR-type transcriptional regulator and a membrane protein involved in copper uptake, respectively. DNA binding experiments showed that the YcnK protein specifically binds to the
ycnK
-
ycnL
intergenic region, including a 16-bp direct repeat that is essential for the high binding affinity of YcnK, and that a copper-specific chelator significantly inhibits YcnK's DNA binding.
lacZ
reporter analysis showed that the
ycnK
promoter is induced by copper limitation or
ycnK
disruption. These results are consistent with YcnK functioning as a copper-responsive repressor that derepresses
ycnKJI
expression under copper limitation. On the other hand, the
ycnL
promoter was hardly induced by copper limitation, but
ycnK
disruption resulted in a slight induction of the
ycnL
promoter, suggesting that YcnK also represses
ycnL
weakly. Moreover, while the CsoR protein did not bind to the
ycnK
-
ycnL
intergenic region,
lacZ
reporter analysis demonstrated that
csoR
disruption induces the
ycnK
promoter only in the presence of intact
ycnK
and
copZA
genes. Since the
copZA
operon is involved in copper export and repressed by CsoR, it appears that the constitutive
copZA
expression brought by
csoR
disruption causes intracellular copper depletion, which releases the repression of the
ycnKJI
operon by YcnK.
Transcriptional Cross-Regulation between Gram-Negative and Gram-Positive Bacteria, Demonstrated Using ArgP-
argO
of Escherichia coli and LysG-
lysE
of Corynebacterium glutamicum
Authors: Marbaniang Carmelita N. , Gowrishankar J. 
Published: August 2012

ABSTRACT

The protein-gene pairs ArgP-
argO
of
Escherichia coli
and LysG-
lysE
of
Corynebacterium glutamicum
are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in
E. coli
the activation of
lysE
by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP
d
) variants that confer elevated Arg-independent
argO
expression, some (ArgP
d
-P274S, -S94L, and, to a lesser extent, -P108S) activated
lysE
expression in
E. coli
. However, the individual activating effects of LysG and ArgP
d
on
lysE
were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP
d
variants exhibited higher affinity of binding to the
lysE
regulatory region and less DNA bending at both
argO
and
lysE
. We conclude that the transcription factor LysG from a Gram-positive bacterium,
C. glutamicum
, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium,
E. coli
, for activation of its cognate target
lysE in vivo
and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target
lysE
, but by a subtly different mechanism that renders them noninterchangeable with LysG.
Polynucleotide Phosphorylase Plays an Important Role in the Generation of Spontaneous Mutations in Escherichia coli
Authors: Becket Elinne , Tse Lawrence , Yung Madeline , Cosico Alexander , Miller Jeffrey H. 
Published: August 2012

ABSTRACT

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient
Escherichia coli
strain. This is true for base substitution mutations that occur in the
rpoB
gene leading to Rif
r
and the
gyrB
gene leading to Nal
r
and for base substitution and frameshift mutations that occur in the
lacZ
gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the
mutT
mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a
mutY
mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.
Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member
Authors: Martínez-Rodríguez Sergio , García-Pino Abel , Las Heras-Vázquez Francisco Javier , Clemente-Jiménez Josefa María , Rodríguez-Vico Felipe , García-Ruiz Juan M. , Loris Remy , Gavira Jose Antonio 
Published: August 2012

ABSTRACTN-Carbamoyl-l-amino acid amidohydrolases (l-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures ofN-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the firstl-carbamoylase structure belonging toGeobacillus stearothermophilusCECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg234in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a “small-molecule binding domain,” allowing further evolutionary considerations for this enzyme family.
The Spanin Complex Is Essential for Lambda Lysis
Authors: Berry Joel , Rajaure Manoj , Pang Ting , Young Ry 
Published: August 2012

ABSTRACT
Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.