bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein Disulfide Isomerase, a Component of the Estrogen Receptor Complex, Is Associated withChlamydia trachomatisSerovar E Attached to Human Endometrial Epithelial Cells
Authors: Davis C. H. , Raulston J. E. , Wyrick P. B. 
Published: July 2002

ABSTRACTChlamydia trachomatisserovar E, the leading bacterial agent responsible for sexually transmitted diseases, is required to invade genital epithelial cells for its growth and survival, yet little is known about the adhesin-receptor interactions promoting its entry. In contrast, much has been published on the heparan sulfate receptor for bindingC. trachomatisL2 elementary bodies (EBs) prior to entry into HeLa cells. Using a different experimental approach in which a biotinylated apical membrane protein receptor(s) attached to EB at 4°C was stripped off the surface of polarized HEC-1B cells and immunoprecipitated with polyclonal anti-EB antibodies, an ∼55-kDa protein was reproducibly detected by enhanced chemiluminescence and two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization mass-spectrometry sequence analysis revealed the 55-kDa protein to be protein disulfide isomerase (PDI), a member of the estrogen receptor complex which carries out thiol-disulfide exchange reactions at infected host cell surfaces. Exposure of HEC-1B cells during EB attachment (1.5 to 2 h) to three different inhibitors of PDI reductive reactions—(i) the thiol-alkylating reagent DTNB (5,5′-dithiobis[2-nitrobenzoic acid]), (ii) bacitracin, and (iii) anti-PDI antibodies—resulted in reduced chlamydial infectivity. Since (i)C. trachomatisserovar E attachment to estrogen-dominant primary human endometrial epithelial cells is dramatically enhanced and (ii) productive entry into and infectivity of EB in host cells is dependent on reduction of EB cross-linked outer membrane proteins at the host cell surface, these data provide some preliminary evidence for an intriguing new potential receptor candidate for further analysis of luminalC. trachomatisserovar E entry.
A Plasmid Partition System of the P1-P7
par
Family from the pMT1 Virulence Plasmid of
Yersinia pestis
Authors: Youngren Brenda , Radnedge Lyndsay , Hu Ping , Garcia Emilio , Austin Stuart 
Published: July 2002

ABSTRACT

The complete sequence of the virulence plasmid pMT1 of
Yersinia pestis
KIM5 revealed a region homologous to the plasmid partition (
par
) region of the P7 plasmid prophage of
Escherichia coli
. The essential genes
parA
and
parB
and the downstream partition site gene,
parS
, are highly conserved in sequence and organization. The pMT1
parS
site and the
parA-parB
operon were separately inserted into vectors that could be maintained in
E. coli
. A mini-P1 vector containing pMT1
parS
was stably maintained when the pMT1 ParA and ParB proteins were supplied in
trans
, showing that the pMT1
par
system is fully functional for plasmid partition in
E. coli
. The pMT1
par
system exerted a plasmid silencing activity similar to, but weaker than those of P7
par
and P1
par
. In spite of the high degree of similarity, especially to P7
par
, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1
parS
site, and the pMT1 Par proteins failed to support partition with P1
parS
or P7
parS
. Typical of other partition sites, supernumerary copies of pMT1
parS
exerted incompatibility toward plasmids supported by pMT1
par
. However, no interspecies incompatibility effect was observed between pMT1par, P7
par
, and P1
par
.
Effects of Organic Antagonists of Ca
2+
, Na
+
, and K
+
on Chemotaxis and Motility of
Escherichia coli
Authors: Tisa Louis S. , Sekelsky Jeff J. , Adler Julius 
Published: July 2002

ABSTRACT

Various Ca
2+
antagonists used in animal research, many of them known to be Ca
2+
channel blockers, inhibited
Escherichia coli
chemotaxis (measured as entry of cells into a capillary containing attractant). The most effective of these, acting in the nanomolar range, was ω-conotoxin GVIA. The next most effective were gallopamil and verapamil. At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant). Various other Ca
2+
antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility. Cells treated with each of these Ca
2+
antagonists swam with a running bias, i.e., tumbling was inhibited. Similarly, some Na
+
antagonists used in animal research inhibited bacterial chemotaxis.
E. coli
chemotaxis was inhibited by saxitoxin at concentrations above 10
−7
M, while more than 10
−4
M was needed to inhibit motility. Cells treated with saxitoxin swam with a tumbling bias. In the case of other Na
+
antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration. In the case of K
+
antagonists used in animal research, 4-aminopyridine blocked
E. coli
chemotaxis between 10
−3
M and, totally, 10
−2
M, while motility was not affected at 10
−2
M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10
−2
M.
Molecular Cloning and Characterization of Two Genes for the Biotin Carboxylase and Carboxyltransferase Subunits of Acetyl Coenzyme A Carboxylase in
Myxococcus xanthus
Authors: Kimura Yoshio , Miyake Rina , Tokumasu Yushi , Sato Masayuki 
Published: July 2002

ABSTRACT

We have cloned a DNA fragment from a genomic library of
Myxococcus xanthus
using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the
accB
and
accA
genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the
accA
gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO
2
fixation, and biotin binding were found in AccA. The
accB
gene was located upstream of the
accA
gene, and they formed a two-gene operon. The protein (AccB) encoded by the
accB
gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An
accA
disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the
accA
gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this
M. xanthus
acetyl-CoA carboxylase consists of two subunits, which are encoded by the
accB
and
accA
genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.
Developmental Aggregation of
Myxococcus xanthus
Requires
frgA
, an
frz
-Related Gene
Authors: Cho Kyungyun , Treuner-Lange Anke , O'Connor Kathleen A. , Zusman David R. 
Published: July 2002

ABSTRACT

Myxococcus xanthus
is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the
frz
genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called
frgA
(for
frz
-related gene), which confers a subset of the frizzy phenotype when mutated. The
frgA
null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of
frz
mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the
frgA
mutant could be complemented extracellularly by wild-type cells or strains carrying non-
frz
mutations. The phenotype of the
frgA
mutant is similar to that of the
abcA
mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The
frgA
gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The
frgA
gene is part of an operon which includes two additional genes,
frgB
and
frgC
. The
frgB
gene encodes a putative histidine protein kinase, and the
frgC
gene encodes a putative response regulator. The
frgB
and
frgC
null mutants, however, formed wild-type fruiting bodies.
Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, fromThermotoga maritima
Authors: Yamanaka Kunitoshi , Hwang Jihwan , Inouye Masayori 
Published: July 2002

ABSTRACTA gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned fromThermotoga maritima, a hyperthermophilic bacterium.T. maritimaTrmE was overexpressed inEscherichia coliand purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP.Kmandkcatat 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.
Molecular, Antigenic, and Functional Analyses of Omp2b Porin Size Variants of
Brucella
spp
Authors: Paquet Jean-Yves , Diaz Maria A. , Genevrois Stephanie , Grayon Maggy , Verger Jean-Michel , De Bolle Xavier , Lakey Jeremy H. , Letesson Jean-Jacques , Cloeckaert Axel 
Published: July 2002

ABSTRACT

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus
Brucella
, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the
Brucella
genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various
Brucella
species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of
Brucella melitensis
reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus
Brucella
seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of
Brucella suis
biovar 3), the porin variability does not result in major antigenic variability of the
Brucella
surface that could help the bacteria during the reinfection of a host. Porin variation in
Brucella
seems to result mainly in porin conductivity modifications.
Control of a Family of Phosphatase Regulatory Genes (
phr
) by the Alternate Sigma Factor Sigma-H of
Bacillus subtilis
Authors: McQuade Ryan S. , Comella Natalia , Grossman Alan D. 
Published: July 2002

ABSTRACT

A family of 11 phosphatases can help to modulate the activity of response regulator proteins in
Bacillus subtilis
. Downstream of seven of the
rap
(phosphatase) genes are
phr
genes, encoding secreted peptides that function as phosphatase regulators. By using fusions to
lacZ
and primer extension analysis, we found that six of the seven
phr
genes are controlled by the alternate sigma factor sigma-H. These results expand the potential of sigma-H to contribute to the output of several response regulators by controlling expression of inhibitors of phosphatases.
Molecular Evidence for Independent Occurrence of IS
6110
Insertions at the Same Sites of the Genome of
Mycobacterium tuberculosis
in Different Clinical Isolates
Authors: Fang Z. , Kenna D. T. , Doig C. , Smittipat D. N. , Palittapongarnpim P. , Watt B. , Forbes K. J. 
Published: July 2002

ABSTRACT

Several characteristics of
Mycobacterium tuberculosis
(e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS
6110
raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS
6110
insertions in
ipl
loci (
iplA
and
iplB
) in two collections of clinical isolates of
M. tuberculosis
from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS
6110
insertions in the loci were identified:
ipl-4
::IS
6110
,
ipl-5
::IS
6110
,
ipl-11
::IS
6110
,
ipl-12
::IS
6110
, and
ipl-13
::IS
6110
. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS
6110
copies existed in the same site but with different orientations in different isolates, and the insertion site of
ipl-1
::IS
6110
harbored IS
6110
copies in both
iplA
and
iplB
in different strains. All these suggest the independent occurrence of IS
6110
insertions at the same sites of the genome of
M. tuberculosis
in different clinical isolates. The implications of this finding are discussed.
Directed Evolution of Biphenyl Dioxygenase: Emergence of Enhanced Degradation Capacity for Benzene, Toluene, and Alkylbenzenes
Authors: Suenaga Hikaru , Mitsuoka Mariko , Ura Yuko , Watanabe Takahito , Furukawa Kensuke 
Published: July 2002

ABSTRACT

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the
bphA1
gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of
bphA1
of
Pseudomonas pseudoalcaligenes
KF707 and
Burkholderia cepacia
LB400, a number of evolved Bph Dox enzymes were created. Among them, an
Escherichia coli
clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.
Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Adams Michael W. W. , Holden James F. , Menon Angeli Lal , Schut Gerrit J. , Grunden Amy M. , Hou Chun , Hutchins Andrea M. , Jenney Francis E. , Kim Chulhwan , Ma Kesen , Pan Guangliang , Roy Roopali , Sapra Rajat , Story Sherry V. , Verhagen Marc F. J. M. 
Published: July 2002

ABSTRACT

The hyperthermophilic archaeon
Pyrococcus furiosus
grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S
0
). Growth rates were highest on media containing peptides and S
0
, with or without maltose. Growth did not occur on the peptide medium without S
0
. S
0
had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S
0
with or without maltose were the same, suggesting that S
0
is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of
P. furiosus
were shown to be regulated under the five different growth conditions studied. The presence of S
0
in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S
0
-reducing enzyme in this organism and the mechanism of the S
0
dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in
P. furiosus
and a significant regulatory role for elemental sulfur or its metabolites.
Regulation of
Streptococcus pneumoniae clp
Genes and Their Role in Competence Development and Stress Survival
Authors: Chastanet Arnaud , Prudhomme Marc , Claverys Jean-Pierre , Msadek Tarek 
Published: July 2002

ABSTRACT

In vitro
mariner
transposon mutagenesis of
Streptococcus pneumoniae
chromosomal DNA was used to isolate regulatory mutants affecting expression of the
comCDE
operon, encoding the peptide quorum-sensing two-component signal transduction system controlling competence development. A transposon insertion leading to increased
comC
expression was found to lie directly upstream from the
S. pneumoniae clpP
gene, encoding the proteolytic subunit of the Clp ATP-dependent protease, whose expression in
Bacillus subtilis
is controlled by the CtsR repressor. In order to examine
clp
gene regulation in
S. pneumoniae
, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the
clpE
,
clpP
, and
clpL
genes and the
ctsR
-
clpC
and
groESL
operons. The
S. pneumoniae ctsR
gene was cloned under the control of an inducible promoter and used to demonstrate regulation of the
S. pneumoniae clpP
and
clpE
genes and the
clpC
and
groESL
operons by using
B. subtilis
as a heterologous host. The CtsR protein of
S. pneumoniae
was purified and shown to bind specifically to the
clpP, clpC, clpE
, and
groESL
regulatory regions.
S. pneumoniae
Δ
ctsR
, Δ
clpP
, Δ
clpC
, and Δ
clpE
mutants were constructed by gene deletion/replacement. ClpP was shown to act as a negative regulator, preventing competence gene expression under inappropriate conditions. Phenotypic analyses also indicated that ClpP and ClpE are both required for thermotolerance. Contrary to a previous report, we found that ClpC does not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of
S. pneumoniae
.
Genetic Analysis of Assembly of theSalmonella entericaSerovar Typhimurium Type III Secretion-Associated Needle Complex
Authors: Sukhan Anand , Kubori Tomoko , Wilson James , Galán Jorge E. 
Published: July 2002

ABSTRACTSeveral pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells.Salmonella entericaserovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves asec-dependent step leading to the assembly of the base substructure followed by asec-independent process resulting in the assembly of the needle portion.
Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in
Escherichia coli
Authors: Yamanaka Kunitoshi , Inouye Masayori 
Published: July 2002

ABSTRACT

Upon cold shock,
Escherichia coli
cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A
pnp
mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15°C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.