bluuebunny/crossref_metadata_2025_split huggingface dataset:
Size Dependence of Protein Diffusion in the Cytoplasm of
Escherichia coli
Authors: Nenninger Anja , Mastroianni Giulia , Mullineaux Conrad W. 
Published: June 2010

ABSTRACT

Diffusion in the bacterial cytoplasm is regarded as the primary method of intracellular protein movement and must play a major role in controlling the rates of cell processes. A number of recent studies have used green fluorescent protein (GFP) tagging and fluorescence microscopy to probe the movement and distribution of proteins in the bacterial cytoplasm. However, the dynamic behavior of indigenous proteins must be controlled by a complex mixture of specific interactions, combined with the basic physical constraints imposed by the viscosity and macromolecular crowding of the cytoplasm. These factors are difficult to unravel in studies with indigenous proteins. To what extent the addition of a GFP tag might affect the movement of a protein through the cytoplasm has also remained unknown. To resolve these problems, we have carried out a systematic study of the size dependence of protein diffusion coefficients in the
Escherichia coli
cytoplasm, using engineered GFP multimers (from 2 to 6 covalently linked GFP molecules). Diffusion coefficients were measured using confocal fluorescence recovery after photobleaching (FRAP). At least up to 110 kDa (four linked GFP molecules), the diffusion coefficient varies with size roughly as would be predicted from the Einstein-Stokes equation for a classical (Newtonian) fluid. Thus, protein diffusion coefficients are predictable over this range. GFP tagging of proteins has little impact on the diffusion coefficient over this size range and therefore need not significantly perturb protein movement. Two indigenous
E. coli
proteins were used to show that their specific interactions within the cell are the main controllers of the diffusion rate.
Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC
Authors: Kim Hong-Man , Xu Yongbin , Lee Minho , Piao Shunfu , Sim Se-Hoon , Ha Nam-Chul , Lee Kangseok 
Published: June 2010

ABSTRACTTripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the α-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we providein vivofunctional data showing that both the length and the amino acid sequence of the α-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.
Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the
Bacillus megaterium
QM B1551 Spore Germination Response to Glucose
Authors: Christie Graham , Götzke Hansjörg , Lowe Christopher R. 
Published: June 2010

ABSTRACT

The molecular basis for the recognition of glucose as a germinant molecule by spores of
Bacillus megaterium
QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed
gerWB
) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.
Complete Nucleotide Sequence of TOL Plasmid pDK1 Provides Evidence for Evolutionary History of IncP-7 Catabolic Plasmids
Authors: Yano Hirokazu , Miyakoshi Masatoshi , Ohshima Kenshiro , Tabata Michiro , Nagata Yuji , Hattori Masahira , Tsuda Masataka 
Published: June 2010

ABSTRACTTo understand the mechanisms for structural diversification ofPseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp fromPseudomonasputidaHS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOBHgroup of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from thexylgene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of sixPseudomonasstrains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to twoPseudomonasstrains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOBHgroup-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.
(p)ppGpp Inhibits Polynucleotide Phosphorylase from
Streptomyces
but Not from
Escherichia coli
and Increases the Stability of Bulk mRNA in
Streptomyces coelicolor
Authors: Gatewood Marcha L. , Jones George H. 
Published: June 2010

ABSTRACT

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in
Streptomyces
species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from
Streptomyces coelicolor
,
Streptomyces antibioticus
, and
Escherichia coli
. We have shown that (p)ppGpp inhibits the activities of both
Streptomyces
PNPases but not the
E. coli
enzyme. The inhibition kinetics for polymerization using the
Streptomyces
enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the
rpsO-pnp
operon of
S. coelicolor
. We have shown further that the chemical stability of mRNA increases during the stationary phase in
S. coelicolor
and that induction of a plasmid-borne copy of
relA
in a
relA
-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition
in vitro
may reflect a role of ppGpp in the regulation of antibiotic production
in vivo
.
Differences in Cold Adaptation of
Bacillus subtilis
under Anaerobic and Aerobic Conditions
Authors: Beranová Jana , Mansilla María C. , de Mendoza Diego , Elhottová Dana , Konopásek Ivo 
Published: June 2010

ABSTRACT

Bacillus subtilis
, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of
des
is induced by the temperature downshift and is decreased when membrane fluidity is restored.
B. subtilis
is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in
B. subtilis
under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of
des
expression after temperature downshift (from 37°C to 25°C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of
des
expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after
des
induction under anaerobic conditions despite the presence of high levels of the
des
protein product, which was shown by immunoblot analysis. The cold adaptation of
B. subtilis
in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.
Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in
Thermoanaerobacter tengcongensis
Authors: Qian Zhong , Wang Quanhui , Tong Wei , Zhou Chuanqi , Wang Qian , Liu Siqi 
Published: June 2010

ABSTRACT

Thermoanaerobacter tengcongensis
could utilize galactose as a carbon source via the enzymes encoded by a novel
gal
operon, whose regulation mechanism has yet to be elucidated. We propose here that the
gal
operon in
T. tengcongensis
is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus
in vivo
. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA

O
2

, whereas the unphosphorylated GalR to

O
1

. A model of HisK:GalR is proposed to explain how galactose mediates the expression of the
gal
operon in
T. tengcongensis.
Rickettsia prowazekiiUses ansn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis
Authors: Frohlich Kyla M. , Roberts Rosemary A. W. , Housley Nicole A. , Audia Jonathon P. 
Published: June 2010

ABSTRACTRickettsia prowazekiiis an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion tosn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway isR. prowazekiiopen reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3Pin vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of theR. prowazekii gpsAgene functioned to complement anEscherichia coli gpsAmutant. Furthermore,gpsAmRNA was detected inR. prowazekiipurified from hen egg yolk sacs, and G3PDH activity was assayable inR. prowazekiilysed-cell extracts. Together, these data strongly suggested thatR. prowazekiiencodes and synthesizes a functional GpsA enzyme, yetR. prowazekiiis unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned thatR. prowazekiitransports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purifiedR. prowazekiitransported and incorporated DHAP into phospholipids, thus implicating a role for GpsAin vivoas part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.
Compartmentalized Glucose Metabolism in
Pseudomonas putida
Is Controlled by the PtxS Repressor
Authors: Daddaoua Abdelali , Krell Tino , Alfonso Carlos , Morel Bertrand , Ramos Juan-Luis 
Published: June 2010

ABSTRACT

Metabolic flux analysis revealed that in
Pseudomonas putida
KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced
in vivo
by glucose, ketogluconate, and 2-ketogluconate,
in vitro
we found that only 2-ketogluconate binds to the regulator with an apparent

K
D

(equilibrium dissociation constant) of 15 μM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 μM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5′-TGAAACCGGTTTCA-3′. This palindrome overlaps with the RNA polymerase binding site.
The
pdh
Operon Is Expressed in a Subpopulation of Stationary-Phase Bacteria and Is Important for Survival of Sugar-Starved
Streptococcus mutans
Authors: Busuioc Monica , Buttaro Bettina A. , Piggot Patrick J. 
Published: June 2010

ABSTRACT

Streptococcus mutans
is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. We show here that inactivation of
pdhD
, putatively encoding a subunit of the pyruvate dehydrogenase complex, impairs survival of both batch cultures and biofilms. We show that
pdhD
and the downstream genes
pdhA
,
pdhB
, and
pdhC
form an operon that is predominantly transcribed in stationary phase. Analysis with fluorescent reporters revealed a bimodal expression pattern for the
pdh
promoter, with less than 1% of stationary-phase populations expressing
pdh
. When it was first detected, after 1 day of sugar starvation in batch culture, expression was mostly in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of the chains increased with time. We infer that the
pdh
-expressing subpopulation is able grow and divide and to persist for extended times in stationary phase.
Genome Sequence of the Milbemycin-Producing Bacterium
Streptomyces
bingchenggensis
Authors: Wang Xiang-Jing , Yan Yi-Jun , Zhang Bo , An Jing , Wang Ji-Jia , Tian Jun , Jiang Ling , Chen Yi-Hua , Huang Sheng-Xiong , Yin Min , Zhang Ji , Gao Ai-Li , Liu Chong-Xi , Zhu Zhao-Xiang , Xiang Wen-Sheng 
Published: June 2010

ABSTRACT

Streptomyces
bingchenggensis
is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of
S
.
bingchenggensis
. The availability of the genome sequence of
S
.
bingchenggensis
should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.
Reference Values and Distribution of Conventional Echocardiographic Doppler Measures and Longitudinal Tissue Doppler Velocities in a Population Free From Cardiovascular Disease
Authors: Dalen Havard , Thorstensen Anders , Vatten Lars J. , Aase Svein A. , Stoylen Asbjorn 
Published: June 2010

Background—
This study aimed to investigate the distribution of conventional Doppler measurements, pulsed wave tissue Doppler imaging (pwTDI)- and color tissue Doppler imaging-derived velocities, by age and sex in a healthy population.

Methods and Results—
Longitudinal tissue Doppler velocities were determined in 1266 healthy individuals from standard apical 4- and 2-chamber views. By the pwTDI method, mean±SD systolic mitral annular velocities were 8.2±1.3 cm/s in women and 8.6±1.4 cm/s in men, and by color tissue Doppler, they were 6.6±1.1 cm/s in women and 6.9±1.3 cm/s in men. With pwTDI, diastolic early mitral annular velocities were 11.8±3.2 cm/s in women and 10.8±3.0 cm/s in men, with corresponding ratios between mitral early flow velocity and early diastolic tissue velocity of 6.7±0.8 in women and 6.4±0.8 in men. By pwTDI, tricuspid annular systolic and early diastolic velocities were 12.5±1.9 and 13.3±3.0, respectively, in women and 12.8±2.2 and 12.5±3.3, respectively, in men. There was a significant decrease in left and right ventricular function with increasing age.

Conclusions—
The longitudinal mitral and tricuspid annular velocities from this population study are widely applicable as reference values. Reference values for annular velocities should be specified by sex and age. The average of inferoseptal and anterolateral wall velocities may be the preferred index of left ventricular performance.
Kinetics of Left Ventricular Strains and Torsion During Incremental Exercise in Healthy Subjects
Authors: Doucende Grégory , Schuster Iris , Rupp Thomas , Startun Aliona , Dauzat Michel , Obert Philippe , Nottin Stéphane 
Published: June 2010

Background—
The dynamics of systolic and diastolic strains and torsional mechanics of the left ventricle (LV) and their relation to diastolic filling never have been evaluated at various exercise intensities.

Methods and Results—

Speckle tracking echocardiography was performed in 20 healthy sedentary subjects at rest and during a progressive submaximal exercise test at 20%, 30%, and 40% of maximal aerobic power. LV twist increased progressively with exercise intensity (10.5±3.2 to 15.8±4.5°;
P
<0.001), whereas longitudinal strain remained unchanged after the first workload, underlining the key role of torsional reserve in systolic-diastolic coupling during exercise. The increase in diastolic untwisting (−88.7±34.2 to −182.9±53.5 deg · s
−1
;
P
<0.01) was correlated to enhanced systolic twist (
R
=0.61;
P
<0.001), and its magnitude of increase was significantly higher compared to diastolic longitudinal and circumferential strain rates (119±64% versus 65±44% and 57±24%, respectively), emphasizing its contribution to diastolic filling. The timing of peak untwisting and the chronology of diastolic mechanical events were unchanged during effort. Untwisting was driven mainly by apical rotation and determined mitral opening and isovolumic relaxation time (
R
=0.47 and 0.61, respectively;
P
<0.001), whereas basal rotation and longitudinal and circumferential diastolic strain rates were major determinants of increased early diastolic filling (
R
=0.64, 0.79, and 0.81, respectively;
P
<0.001).

Conclusions—
The use of speckle tracking echocardiography gives new insights into physiological adaptive LV mechanics during incremental exercise in healthy subjects, underlining the key role of torsional mechanics. It might be useful to better understand the mechanisms of diastolic dysfunction and exercise intolerance in various pathological conditions.
An Echocardiographic Model Predicting Severity of Aortic Regurgitation in Congenital Heart Disease
Authors: Beroukhim Rebecca S. , Graham Dionne A. , Margossian Renee , Brown David W. , Geva Tal , Colan Steven D. 
Published: June 2010

Background—
Multiple echocardiographic parameters have been identified to predict the severity of aortic regurgitation (AR) with variable reliability. This study was performed to identify which echocardiographic parameters best predict the severity of AR in a cohort of patients with congenital heart disease, using cardiovascular MRI quantification as a reference standard.

Methods and Results—

The study involved 2 phases. In phase 1, predictive models were developed on the basis of multivariable analysis of various morphometric and Doppler variables obtained from 174 echocardiograms that best predicted the severity of AR as defined by paired cardiovascular MRI examinations. A nonlinear estimate of regurgitation fraction, using the variables parasternal vena contracta-derived area divided by body surface area and abdominal aorta Doppler retrograde velocity-time integral divided by antegrade velocity-time integral, was identified through multivariable analysis as the best predictive model for AR fraction. In phase 2, the predictive models were prospectively tested on 43 echocardiographic examinations for which a paired cardiovascular MRI was performed. The agreement between the observed and predicted AR fraction was assessed using Bland-Altman analysis. For the 30 studies of the validation data set that had adequate quality images of both the parasternal vena contracta width and the abdominal aorta flow profile, the predicted AR values had a mean bias±SD of 0.4±7.3% (
P
=0.80).

Conclusions—
A model using the 2 variables parasternal vena contracta-derived area divided by body surface area and abdominal aorta Doppler retrograde velocity-time integral divided by antegrade velocity-time integral can predict AR severity in patients with a wide variety of congenital heart disease.