bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of CamH from
Methanosarcina thermophila
, Founding Member of a Subclass of the γ Class of Carbonic Anhydrases
Authors: Zimmerman Sabrina A. , Tomb Jean-Francois , Ferry James G. 
Published: December 2009

ABSTRACT

The homotrimeric enzyme Mt-Cam from
Methanosarcina thermophila
is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from
M. thermophila
(Mt-CamH) was overproduced in
Escherichia coli
and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from
E. coli
cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had
k
cat
and

k
cat

/

K
m

values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from
E. coli
cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective
k
cat
(
k
cat
eff
) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the
k
cat
/

K
m

eff
was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the
k
cat
eff
of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H
2
O
2
, which supports the hypothesis that Fe
2+
has a role in the active site. The
k
cat
for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a “ping-pong” mechanism accepting a proton. The
k
cat
/

K
m

was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO
2
and HCO
3
−
is separate from intermolecular proton transfer.
Characterization of a Glutamate Transporter Operon,
glnQHMP
, in
Streptococcus mutans
and Its Role in Acid Tolerance
Authors: Krastel Kirsten , Senadheera Dilani B. , Mair Richard , Downey Jennifer S. , Goodman Steven D. , Cvitkovitch Dennis G. 
Published: December 2009

ABSTRACT

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium
Streptococcus mutans
is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated
glnQHMP
(Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of
glnQHMP
resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the
glnQHMP
mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the
glnQHMP
operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its
glnQHMP
-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon
glnQHMP
in the acid tolerance response in
S. mutans
.
Bacterial Ammeline Metabolism via Guanine Deaminase
Authors: Seffernick Jennifer L. , Dodge Anthony G. , Sadowsky Michael J. , Bumpus John A. , Wackett Lawrence P. 
Published: December 2009

ABSTRACT

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type
Escherichia coli
and
Pseudomonas
strains, including
E. coli
K12 and
Pseudomonas putida
KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An
E. coli
guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (
Bradyrhizobium japonicum
USDA 110 and
Homo sapiens
) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source.
In silico
models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed
in vitro
, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.
A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in
Streptococcus thermophilus
and
Streptococcus salivarius
Authors: Fontaine Laetitia , Boutry Céline , de Frahan Marie Henry , Delplace Brigitte , Fremaux Christophe , Horvath Philippe , Boyaval Patrick , Hols Pascal 
Published: December 2009

ABSTRACT

In streptococcal species, the key step of competence development is the transcriptional induction of
comX
, which encodes the alternative sigma factor σ
X
, which positively regulates genes necessary for DNA transformation. In
Streptococcus
species belonging to the
mitis
and
mutans
groups, induction of
comX
relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In
Streptococcus thermophilus
, a species belonging to the
salivarius
group, the oligopeptide transporter Ami is essential for
comX
expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of
comX
induction in
S. thermophilus
LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for
comX
induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored
comX
induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in
S. thermophilus
and
Streptococcus salivarius
strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for
comX
induction in species from the
salivarius
group, which differs from the classical phosphorelay three-component system identified previously in streptococci.
Deletion of the
ftsZ
-Like Gene Results in the Production of Superparamagnetic Magnetite Magnetosomes in
Magnetospirillum gryphiswaldense
Authors: Ding Yao , Li Jinhua , Liu Jiangning , Yang Jing , Jiang Wei , Tian Jiesheng , Li Ying , Pan Yongxin , Li Jilun 
Published: December 2009

ABSTRACT

Magnetotactic bacteria (MTB) synthesize unique organelles termed “magnetosomes,” which are membrane-enclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus
Magnetospirillum
, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed “
ftsZ
-like,” whose function is unknown. In the present study, we found that the
ftsZ
-like gene of
Magnetospirillum gryphiswaldense
strain MSR-1 belongs to a 4.9-kb
mamXY
polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 μmol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles
in vitro
. To determine the role of the
ftsZ
-like gene, we constructed a
ftsZ
-like mutant (Δ
ftsZ
-like mutant) and its complementation strain (Δ
ftsZ
-like_C strain). Growth of
ΔftsZ
-like cells was similar to that of the wild type, indicating that the Δ
ftsZ
-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the Δ
ftsZ
-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that Δ
ftsZ
-like produced mainly superparamagnetic (SP) magnetite particles, whereas wild-type and Δ
ftsZ
-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in
M. gryphiswaldense
.
MreB Drives
De N
ovo
Rod Morphogenesis in
Caulobacter crescentus
via Remodeling of the Cell Wall
Authors: Takacs Constantin N. , Poggio Sebastian , Charbon Godefroid , Pucheault Mathieu , Vollmer Waldemar , Jacobs-Wagner Christine 
Published: December 2009

ABSTRACT

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped
Caulobacter crescentus
cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of
C. crescentus
peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.
Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative ofBacillus subtilis(natto) Plasmid pLS32
Authors: Tanaka Teruo 
Published: December 2009

ABSTRACTBacillus subtilisplasmid pBET131 is a derivative of pLS32, which was isolated from anattostrain ofBacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of whichalfA, encoding an actin-like ATPase, andalfBare essential for plasmid stability. In this work, thealfBgene product and its target DNA region were studied in detail. Transcription of thealfoperon initiated from a σA-type promoter was repressed by thealfBgene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of thealfoperon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designatedparN), which partially overlaps the −35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression ofalfA-lacZexpression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA andparN, the mode of which is typified by the type II partition mechanism.
Production of the Bsa Lantibiotic by Community-Acquired
Staphylococcus aureus
Strains
Authors: Daly Karen M. , Upton Mathew , Sandiford Stephanie K. , Draper Lorraine A. , Wescombe Philip A. , Jack Ralph W. , O'Connor Paula M. , Rossney Angela , Götz Friedrich , Hill Colin , Cotter Paul D. , Ross R. Paul , Tagg John R. 
Published: December 2009

ABSTRACT

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of
Staphylococcus aureus
), among clinical isolates of
S
.
aureus
and those associated with community-acquired methicillin-resistant
S. aureus
(MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an
S. aureus
strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired
S. aureus
strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired
S. aureus
.
The Genome of the Amoeba Symbiont “CandidatusAmoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria
Authors: Schmitz-Esser Stephan , Tischler Patrick , Arnold Roland , Montanaro Jacqueline , Wagner Michael , Rattei Thomas , Horn Matthias 
Published: December 2009

ABSTRACTProtozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “CandidatusAmoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to theBacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways forde novobiosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae,Legionella pneumophila,Rickettsia bellii,Francisella tularensis, andMycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.
CotE Binds to CotC and CotU and Mediates Their Interaction during Spore Coat Formation in
Bacillus subtilis
Authors: Isticato Rachele , Pelosi Assunta , De Felice Maurilio , Ricca Ezio 
Published: December 2009

ABSTRACT

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of
Bacillus subtilis
. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.
Completed Genome Sequence of the Anaerobic Iron-Oxidizing Bacterium
Acidovorax ebreus
Strain TPSY
Authors: Byrne-Bailey Kathryne G. , Weber Karrie A. , Chair Antinea H. , Bose Saumyaditya , Knox Traci , Spanbauer Trisha L. , Chertkov Olga , Coates John D. 
Published: December 2009

ABSTRACT

Acidovorax ebreus
strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.
Nitrogenase Switch-Off and Regulation of Ammonium Assimilation in Response to Light Deprivation in
Rhodospirillum rubrum
Are Influenced by the Nitrogen Source Used during Growth
Authors: Teixeira Pedro Filipe , Wang He , Nordlund Stefan 
Published: December 2009

ABSTRACT

Nitrogen fixation and ammonium assimilation in
Rhodospirillum rubrum
are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P
II
modification is dependent on the nitrogen source used for growth, N
2
or glutamate, although both lead to nitrogenase derepression.
Is periprocedural sedation during acute stroke therapy associated with poorer functional outcomes?
Authors: Nichols C , Carrozzella J , Yeatts S , Tomsick T , Broderick J , Khatri P 
Published: December 2009

BackgroundTo safely perform acute intra-arterial revascularization procedures, use of sedative medications and paralytics is often necessary. During the conduct of the Interventional Management of Stroke trials (I and II), the level of sedation used periprocedurally varied. At some institutions, patients were paralyzed and intubated as part of the procedural standard of care while at other institutions no routine sedation protocol was followed. The aim of this study was to identify patient characteristics that would correlate with the need for deeper sedation and to explore whether levels of sedation relate to patient outcome.Methods75 of 81 patients in the Interventional Management of Stroke II Study were studied. Patients had anterior circulation strokes and underwent angiography and/or intervention. Four sedation categories were defined and tested for factors potentially associated with the level of sedation. Clinical outcomes were also analyzed, including successful angiographic reperfusion and the occurrence of clinical complications.ResultsOnly baseline National Institutes of Health Stroke Scale varied significantly by sedation category (p=0.01). Patients that were in the lower sedation category fared better, having a higher rate of good outcomes (p<0.01), lower death rates (p=0.02) and higher successful angiographic reperfusion rates (p=0.01). There was a significantly higher infection rate in patients receiving heavy sedation or pharmacologic paralysis (p=0.02) and a trend towards fewer groin related complications.ConclusionIn this small sample, patients not receiving sedation fared better, had higher rates of successful angiographic reperfusion and had fewer complications. Further examination of the indications for procedural sedation or paralysis and their effect on outcome is warranted.
Prospects for a genetic management strategy to control Gyrodactylus salaris infection in wild Atlantic salmon (Salmo salar) stocks
Authors: Salte Ragnar , Bentsen Hans Bernhard , Moen Thomas , Tripathy Smita , Bakke Tor Andreas , Ødegård Jørgen , Omholt Stig , Hansen Lars Petter 
Published: December 2009

We estimated additive genetic variation and heritability of survival after Gyrodactylus salaris infection from survival records in a pedigreed family material of wild Atlantic salmon ( Salmo salar ) in a controlled challenge test. We used a statistical model that distinguishes between survival time for the fish that died and the ability to survive the entire test as two separate traits. Eleven of the 49 full-sib families suffered 100% mortality, 15 families had between 10% and 25% survival, and the four least affected families had survival rates between 36% and 48%. Estimated heritability of survival on the liability scale was 0.32 ± 0.10. Time until death for fish that died during the test and the ability to survive the entire test were not expressions of the same genetic trait. Simply selecting survivors as parents for the next generation is expected to more than double the overall survival rate in only one generation, given similar exposure to the parasite. Improving the genetic capacity to survive the infection will probably not eradicate the parasite, but when used as a disease control measure, such improvement may contain the infection at a level where the parasite ceases to be a major problem.