bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment
Authors: Woessner David J. , Dawson Scott C. 
Published: January 2012

ABSTRACT

Giardia
has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables
Giardia
to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.
Mutations at Several Loci Cause Increased Expression of Ribonucleotide Reductase in Escherichia coli
Authors: Feeney Morgan Anne , Ke Na , Beckwith Jon 
Published: January 2012

ABSTRACT

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the
nrdAB
genes, is required for aerobic growth of
Escherichia coli
. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the
dnaA
and
dnaN
genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the
hda
gene and insertions in the
nrdAB
promoter region of insertion elements IS
1
and IS
5
. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in
nrdAB
regulation in
E. coli
and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before.
Characterization of Escherichia coli
dinJ-yafQ
Toxin-Antitoxin System Using Insights from Mutagenesis Data
Authors: Armalytė Julija , Jurėnaitė Milda , Beinoravičiūtė Gina , Teišerskas Justinas , Sužiedėlienė Edita 
Published: January 2012

ABSTRACT

Escherichia coli
dinJ-yafQ
operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage
in vivo
, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5′ to A between the second and third nucleotides in the codon
in vivo
. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules
in vitro
, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin
in vitro
. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the
dinJ-yafQ
operon, suggesting the autoregulation mode of this TA system.
AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis
Authors: Mayer Florian , Küper Ulf , Meyer Carolin , Daxer Stefanie , Müller Volker , Rachel Reinhard , Huber Harald 
Published: January 2012

ABSTRACT

Ignicoccus hospitalis
, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO
2
fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of
I. hospitalis
exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of
I. hospitalis
and other described
Ignicoccus
species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H
2
:sulfur oxidoreductase complexes are also located in the outermost membrane of
I. hospitalis
. These results imply that the intermembrane compartment of
I. hospitalis
is not only the site of ATP synthesis but may also be involved in the primary steps of CO
2
fixation.
Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of σ Factors
Authors: Basu Arnab , Chatterjee Sujoy , Chatterjee Soniya , Das Gupta Sujoy K. 
Published: January 2012

ABSTRACT

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the
Actinobacteria
. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10
−3
) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.
Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB
Authors: Lieberman Joshua A. , Frost Nicholas A. , Hoppert Michael , Fernandes Paula J. , Vogt Stefanie L. , Raivio Tracy L. , Blanpied Thomas A. , Donnenberg Michael S. 
Published: January 2012

ABSTRACT

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic
Escherichia coli
(EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.
J-Western Forms of Helicobacter pylori cagA Constitute a Distinct Phylogenetic Group with a Widespread Geographic Distribution
Authors: Duncan Stacy S. , Valk Pieter L. , Shaffer Carrie L. , Bordenstein Seth R. , Cover Timothy L. 
Published: January 2012

ABSTRACT

Chronic infection with
Helicobacter pylori
strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian
cagA
sequences, relatively little is understood about a fourth group, known as “J-Western,” which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western
cagA
sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on multilocus sequence typing [MLST] analysis), and did not arise recently. As shown by comparisons of Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (π
s
) is slightly lower in the J-Western group than in the Western and East Asian groups (0.066, 0.086, and 0.083, respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced π
s
of the J-Western group is attributable to ancestral recombination events within the 5′ region of
cagA.
Population genetic analyses suggest that within the
cagA
region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J-Western groups, in association with positive selection. Finally, we show that J-Western
cagA
sequences are found mainly in strains producing m2 forms of the secreted VacA toxin and propose that these functionally interacting proteins coevolved to optimize the gastric colonization capacity of
H. pylori
.
Structure, Diversity, and Mobility of the Salmonella Pathogenicity Island 7 Family of Integrative and Conjugative Elements within Enterobacteriaceae
Authors: Seth-Smith Helena M. B. , Fookes Maria C. , Okoro Chinyere K. , Baker Stephen , Harris Simon R. , Scott Paul , Pickard Derek , Quail Michael A. , Churcher Carol , Sanders Mandy , Harmse Johan , Dougan Gordon , Parkhill Julian , Thomson Nicholas R. 
Published: January 2012

ABSTRACT

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry.
Salmonella
pathogenicity island 7 (SPI-7), found within some pathogenic strains of
Salmonella enterica
, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus
Salmonella
and other
Enterobacteriaceae
. We report the sequence of two novel SPI-7-like elements, found within strains of
Salmonella bongori
, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within
Salmonella enterica
serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these
Salmonella
mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of
Salmonella
as well as within
Citrobacter
,
Erwinia
,
Escherichia
,
Photorhabdus
, and
Yersinia
species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the
Enterobacteriaceae
.
The
O
-Methyltransferase SrsB Catalyzes the Decarboxylative Methylation of Alkylresorcylic Acid during Phenolic Lipid Biosynthesis by Streptomyces griseus
Authors: Nakano Chiaki , Funa Nobutaka , Ohnishi Yasuo , Horinouchi Sueharu 
Published: January 2012

ABSTRACT

Streptomyces griseus
contains the
srs
operon, which is required for phenolic lipid biosynthesis. The operon consists of
srsA
,
srsB
, and
srsC
, which encode a type III polyketide synthase, an
O
-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13′-methyltetradecyl)-4-methylresorcinol, using
iso
-C
16
fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An
in vitro
SrsA reaction using [
13
C
3
]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in
Streptomyces lividans
and used for the
in vitro
SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of
S
-adenosyl-
l
-methionine (SAM). SrsB was incapable of catalyzing the
O
-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the
O
-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1).
O
-methylated alkylresorcylic acid was not detected in the
in vitro
SrsAB reaction, although it was presumably stable, indicating that
O
-methylation did not precede decarboxylation. We therefore postulated that
O
-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.
Further Unraveling the Regulatory Twist by Elucidating Metabolic Coinducer-Mediated CbbR-
cbb
I
Promoter Interactions in Rhodopseudomonas palustris CGA010
Authors: Joshi Gauri S. , Zianni Michael , Bobst Cedric E. , Tabita F. Robert 
Published: January 2012

ABSTRACT

The
cbb
I
region of
Rhodopseudomonas palustris
(
Rp. palustris
) contains the
cbbLS
genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene,
cbbR
. Juxtaposed between
cbbR
and
cbbLS
are the
cbbRRS
genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence
cbbLS
expression. The nature of the metabolic signals that
Rp. palustris
CbbR perceives to regulate
cbbLS
transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the
cbbLS
promoter by the use of gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the
cbbLS
promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate, and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints, indicating that the coinducers caused significant changes in DNA structure. These
in vitro
studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived by and transduced to the
cbbLS
promoter via the master regulator CbbR.
Nitric Oxide Stress Resistance in Porphyromonas gingivalis Is Mediated by a Putative Hydroxylamine Reductase
Authors: Boutrin Marie-Claire , Wang Charles , Aruni Wilson , Li Xiaojin , Fletcher Hansel M. 
Published: January 2012

ABSTRACT

Porphyromonas gingivalis
, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in
P. gingivalis
W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants
P. gingivalis
FLL455 (
PG0893
∷
ermF
) and FLL456 (
PG2213
∷
ermF
) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain.
P. gingivalis
FLL455 was more sensitive to NO than the wild type. Complementation of
P. gingivalis
FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain.
P. gingivalis
FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that
PG0893
and
PG2213
were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function.
PG1181
, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome
in silico
analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that
PG0893
and several other genes may play an important role in
P. gingivalis
NO stress resistance.
CtsR Regulation in
mcsAB
-Deficient Gram-Positive Bacteria
Authors: Tao Liang , Chattoraj Partho , Biswas Indranil 
Published: January 2012

ABSTRACT

CtsR is an important repressor that modulates the transcription of class III stress genes in Gram-positive bacteria. In
Bacillus subtilis
, a model Gram-positive organism, the DNA binding activity of CtsR is regulated by McsAB-mediated phosphorylation of the protein where phosphorylated CtsR is a substrate for degradation by the ClpCP complex. Surprisingly, the
mcsAB
genes are absent from many Gram-positive bacteria, including streptococci; therefore, how CtsR activity is modulated in those bacteria remains unknown. Here we show that the posttranslational modulation of CtsR activity is different in
Streptococcus mutans
, a dental pathogen. We observed that of all of the Clp-related proteins, only ClpL is involved in the degradation of CtsR. Neither ClpP nor ClpC had any effect on the degradation of CtsR. We also found that phosphorylation of CtsR on a conserved arginine residue within the winged helix-turn-helix domain is necessary for modulation of the repressor activity of CtsR, as demonstrated by both
in vitro
and
in vivo
assays. We speculate that CtsR is regulated posttranslationally by a different mechanism in
S. mutans
and possibly in other streptococci.
Improvement of knowledge, attitude and perception of healthcare workers about ADR, a pre- and post-clinical pharmacists' interventional study
Authors: Khalili Hossein , Mohebbi Niayesh , Hendoiee Narjes , Keshtkar Abbas-Ali , Dashti-Khavidaki Simin 
Published: January 2012

PurposeHealthcare workers have a main role in detection, assessment and spontaneous reporting of adverse drug reactions (ADRs), and improvement of their related knowledge, attitude and perception is essential. The goal of this study was evaluation of clinical pharmacists' interventions in improvement of knowledge, attitude and perception of healthcare workers about ADRs in a teaching referral hospital, Tehran, Iran.MethodChanges in knowledge, attitude and perception of healthcare workers of Imam teaching hospital about ADRs were evaluated before and after clinical pharmacists' interventions including workshops, meetings and presentations.ResultsFrom the 100 participated subjects, 82 of them completed the study. 51% of the health workers have been aware of the Iranian Pharmacovigilance Center at the ministry of health before intervention and after that all the participants knew this centre. About awareness and detection of ADRs in patients, 69 (84.1%) healthcare workers recognised at least one, and following interventions, it was improved to 73 (89%). Only seven (8.5%) subjects have reported ADRs in before intervention phase that were increased significantly to 18 (22%) after intervention.ConclusionClinical pharmacists' interventions were successful in improvement of healthcare workers' knowledge, attitude and perception about ADRs and spontaneous reporting in our hospital.