bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects ofAnaplasma phagocytophilaon NADPH Oxidase Components in Human Neutrophils and HL-60 Cells
Authors: Mott Jason , Rikihisa Yasuko , Tsunawaki Shohko 
Published: July 2002

ABSTRACTThe human granulocytic ehrlichiosis agent,Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes.A. phagocytophilarapidly inhibits the superoxide anion (O2−) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence ofA. phagocytophilaon protein levels and localization of components of the NADPH oxidase were examined.A. phagocytophiladecreased levels of p22phox, but not gp91phox, p47phox, p67phox, or P40phoxreactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47phox, p67phox, Rac2, and p22phoxdid not colocalize withA. phagocytophilainclusions in neutrophils or HL-60 cells, and p22phoxlevels were also reduced.A. phagocytophiladid not prevent either membrane translocation of cytoplasmic p47phoxand p67phoxor phosphorylation of p47phoxupon stimulation by phorbol myristate acetate. The inhibitory signals for O2−generation was independent of several signals required forA. phagocytophilainternalization. These results suggest that rapid alteration in p22phoxinduced by binding ofA. phagocytophilato neutrophils is involved in the inhibition of O2−generation. Absence of colocalization of NADPH oxidase components with the inclusion further protectsA. phagocytophilafrom oxidative damage.
The Cytolethal Distending Toxin of
Haemophilus ducreyi
Inhibits Endothelial Cell Proliferation
Authors: Svensson Liselott A. , Henning Petra , Lagergård Teresa 
Published: July 2002

ABSTRACT

Haemophilus ducreyi,
the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits mammalian cell proliferation. We investigated the effects of HdCDT on normal human endothelial cells and on tubule formation in an in vitro model of angiogenesis. Endothelial cells were arrested in the G
2
phase of the cell cycle, and tubule formation was inhibited in a dose-dependent manner. The antiproliferative activities of HdCDT on endothelial cells might contribute to the characteristic slow healing and persistence of chancroid ulcers.
Assessment of
flhDC
mRNA Levels in
Serratia liquefaciens
Swarm Cells
Authors: Tolker-Nielsen Tim , Christensen Allan Beck , Holmstrøm Kim , Eberl Leo , Rasmussen Thomas Bovbjerg , Sternberg Claus , Heydorn Arne , Molin Søren , Givskov Michael 
Published: July 2002

ABSTRACT

We reported previously that artificial overexpression of the
flhDC
operon in liquid-grown
Serratia liquefaciens
resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, and M. Givskov, J. Bacteriol. 178:554–559, 1996). In the present report we show by means of reporter gene measurements, Northern analysis, and in situ reverse transcription-PCR that the amount of
flhDC
mRNA in surface-grown swarm cells does not exceed the maximum level found in nondifferentiated, vegetative cells. This suggests that surface-induced
S. liquefaciens
swarm cell differentiation, although dependent on
flhDC
gene expression, does not occur through elevated
flhDC
mRNA levels.
Deletion Analysis of the Flagellar Switch Protein FliG of
Salmonella
Authors: Kihara May , Miller Gabriele U. , Macnab Robert M. 
Published: July 2002

ABSTRACT
The flagellar motor/switch complex, consisting of the three proteins FliG, FliM, and FliN, plays a central role in bacterial motility and chemotaxis. We have analyzed FliG, using 10-amino-acid deletions throughout the protein and testing the deletion clones for their motility and dominance properties and for interaction of the deletion proteins with the MS ring protein FliF. Only the N-terminal 46 amino acids of FliG (segments 1 to 4) were important for binding to FliF; consistent with this, an N-terminal fragment consisting of residues 1 to 108 bound FliF strongly, whereas a C-terminal fragment consisting of residues 109 to 331 did not bind FliF at all. Deletions in the region from residues 37 to 96 (segments 4 to 9), 297 to 306 (segment 30), and 317 to 326 (segment 32) permitted swarming, though not at wild-type levels; all other deletions caused paralyzed or, more commonly, nonflagellate phenotype. Except for those near the N terminus, deletions had a dominant negative effect on wild-type cells.
Mutations in
oxyR
Resulting in Peroxide Resistance in
Xanthomonas campestris
Authors: Mongkolsuk Skorn , Whangsuk Wirongrong , Fuangthong Mayuree , Loprasert Suvit 
Published: July 2002

ABSTRACT

A spontaneous
Xanthomonas campestris
pv. phaseoli H
2
O
2
-resistant mutant emerged upon selection with 1 mM H
2
O
2
. In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H
2
O
2
resistance phenotype was abolished in
oxyR
-minus derivatives of the mutant, suggesting that elevated levels and mutations in
oxyR
were responsible for the phenotype. Nucleotide sequence analysis of the
oxyR
mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in
oxyR
affected expression of genes in the
oxyR
regulon. Expression of an
oxyR
-regulated gene,
ahpC
, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed
ahpC
expression. By contrast, expression of
oxyR5
from the
X. campestris
pv. phaseoli H
2
O
2
-resistant mutant and its derivative
oxyR5
G197D with a single-amino-acid change on expression vectors activated
ahpC
expression in the absence of inducer. The other single-amino-acid mutant derivative of
oxyR5
L301R had effects on
ahpC
expression similar to those of the wild-type
oxyR
. However, when the two single mutations were combined, as in
oxyR5
, these mutations had an additive effect on activation of
ahpC
expression.
The Chromosomal Location of the
Bacillus subtilis
Sporulation Gene
spoIIR
Is Important for Its Function
Authors: Khvorova Anastasia , Chary Vasant K. , Hilbert David W. , Piggot Patrick J. 
Published: July 2002

ABSTRACT

Formation of the asymmetrically located septum during sporulation of
Bacillus subtilis
results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment. The rest of the chromosome is then translocated into the prespore from the mother cell. Transcription of
spoIIR
is initiated in the prespore by RNA polymerase containing ς
F
soon after the septum is formed. The SpoIIR protein is required for the activation of the transcription program directed by ς
E
in the mother cell. The
spoIIR
locus is located at 324°, near the origin of replication (0/360°). We show here that movement of
spoIIR
to 28° had little effect on sporulation. However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency. At 283° sporulation was reduced to less than 20% of the level obtained when
spoIIR
was at its natural location, and movement to 190° reduced sporulation to about 6% of that level. These positional effects were also seen in the transcription of a
spoIIR-lacZ
fusion. In contrast, movement of other
spo-lacZ
fusions from 28° to 190° had little effect on their expression. These results suggest that
spoIIR
is the subject of “positional regulation,” in the sense that the chromosomal position of
spoIIR
is important for its expression and function.
Structural Analysis of
Bacillus subtilis
Spore Peptidoglycan during Sporulation
Authors: Meador-Parton Jennifer , Popham David L. 
Published: July 2002

ABSTRACT

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing
Bacillus subtilis
forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.
Two Conserved Glutamates in the Bacterial Nitric Oxide Reductase Are Essential for Activity but Not Assembly of the Enzyme
Authors: Butland Gareth , Spiro Stephen , Watmough Nicholas J. , Richardson David J. 
Published: July 2002

ABSTRACT

The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe
B
–heme-Fe dinuclear catalytic center rather than a Cu
B
–heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in
Paracoccus denitrificans
NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the
cycA
promoter to drive aerobic expression of NOR in
P. denitrificans
and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome
bc
complex in
Escherichia coli
. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in
P. denitrificans
and
E. coli
. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.
Regulatory Functions of Serine-46-Phosphorylated HPr in
Lactococcus lactis
Authors: Monedero Vicente , Kuipers Oscar P. , Jamet Emmanuel , Deutscher Josef 
Published: July 2002

ABSTRACT

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of
Lactococcus lactis
participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a
ptsH1
mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with
L. lactis
vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as
methyl
β-
d
-thiogalactoside (TMG) and 2-deoxy-
d
-glucose (2-DG). In vivo experiments with the
ptsH1
mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and
ptsH1
mutant strains, whereas TMG expulsion was slowed in the
ptsH1
mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.
Amplification of Mutator Cells in a Population as a Result of Horizontal Transfer
Authors: Funchain Pauline , Yeung Annie , Stewart Jean , Clendenin Wendy M. , Miller Jeffrey H. 
Published: July 2002

ABSTRACT

Mutator cells that lack the mismatch repair system (MMR
−
) occur at rates of 10
−5
or less in laboratory populations started from wild-type cells. We show that after selection for recombinants in an interspecies mating between
Salmonella enterica
serovar Typhimurium and
Escherichia coli
, the percentage of MMR
−
cells rises to several percent of the recombinant population, and after a second successive mating and selection, greater than 95% of the recombinants are MMR
−
. Coupling a single cross and selection with either mutagenesis or selection for spontaneous mutants also results in a dramatic increase in MMR
−
cells. We discuss how horizontal transfer can result in mutator strains during adaptive evolution.
Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis
Authors: Jobling Michael G. , Holmes Randall K. 
Published: July 2002

ABSTRACTCholera toxin (CT) is the prototype for theVibrio cholerae-Escherichia colifamily of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.
Eukaryotic Translation Initiation Factor 4E-Dependent Translation Is Not Essential for Survival of Starved Yeast Cells
Authors: Paz Irit , Choder Mordechai 
Published: July 2002

ABSTRACT

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5′ cap structure (m
7
GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast
Saccharomyces cerevisiae CDC33
gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the
cdc33
temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved
cdc33
mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.
Differential Regulation of
ftsZ
Transcription during Septation of
Streptomyces griseus
Authors: Kwak Jangyul , Dharmatilake Amitha J. , Jiang Hao , Kendrick Kathleen E. 
Published: July 2002

ABSTRACT

Streptomyces
has been known to form two types of septa. The data in this research demonstrated that
Streptomyces griseus
forms another type of septum near the base of sporogenic hyphae (basal septum). To understand the regulation of the septation machinery in
S. griseus
, we investigated the expression of the
ftsZ
gene. S1 nuclease protection assays revealed that four
ftsZ
transcripts were differentially expressed during morphological differentiation. The vegetative transcript (emanating from P
veg
) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation. Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form. Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely. Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5′ end point of the transcripts. The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays. The aseptate phenotype of the P
spo
mutant indicated that the increased transcription from P
spo
is required for sporulation septation, but not for vegetative or basal septum formation.
Initiation of Anaerobic Degradation of
p
-Cresol by Formation of 4-Hydroxybenzylsuccinate in
Desulfobacterium cetonicum
Authors: Müller Jochen A. , Galushko Alexander S. , Kappler Andreas , Schink Bernhard 
Published: July 2002

ABSTRACT

The anaerobic bacterium
Desulfobacterium cetonicum
oxidized
p
-cresol completely to CO
2
with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of
p
-cresol is activated by addition to fumarate, analogous to anaerobic toluene,
m
-xylene, and
m
-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from
p
-cresol and fumarate was detected at an initial rate of 0.57 nmol min
−1
(mg of protein)
−1
. This activity was specific for extracts of
p
-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via β-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of
p
-cresol via methyl group oxidation by
p
-cresol-methylhydroxylase in this bacterium.
SmcR-Dependent Regulation of Adaptive Phenotypes in
Vibrio vulnificus
Authors: McDougald Diane , Rice Scott A. , Kjelleberg Staffan 
Published: July 2002

ABSTRACT

Vibrio vulnificus
contains homologues of the
V. harveyi luxR
and
luxS
genes. A null mutation in
smcR
(
luxR
) resulted in a defect in starvation survival, inhibition of starvation-induced maintenance of culturability that occurs when
V. vulnificus
is starved prior to low-temperature incubation, and increased expression of stationary-phase phenotypes.
Analysis of a Complete Library of Putative Drug Transporter Genes in
Escherichia coli
Authors: Nishino Kunihiko , Yamaguchi Akihito 
Published: July 2002

ABSTRACT

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In
Escherichia coli
, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in
E. coli
and investigated their drug resistance phenotypes using an
E. coli
drug-sensitive mutant as a host.
E. coli
cells transformed with a plasmid carrying one of 20 ORFs, i.e.,
fsr
,
mdfA
,
yceE
,
yceL
,
bcr
,
emrKY
,
emrAB
,
emrD
,
yidY
,
yjiO
,
ydhE
,
acrAB
,
cusA
(formerly
ybdE
),
yegMNO
,
acrD
,
acrEF
,
yhiUV
,
emrE
,
ydgFE
, and
ybjYZ
, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs,
cusA
,
yegMNO
,
ydgFE
,
yceE
,
yceL
,
yidY,
and
ybjYZ
are novel drug resistance genes. The
fsr, bcr, yjiO
,
ydhE, acrD,
and
yhiUV
genes gave broader resistance spectra than previously reported.
GerN, an Endospore Germination Protein of
Bacillus cereus
, Is an Na
+
/H
+
-K
+
Antiporter
Authors: Southworth Thomas W. , Guffanti Arthur A. , Moir Anne , Krulwich Terry A. 
Published: July 2002

ABSTRACT

GerN, a
Bacillus cereus
spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na
+
-sensitive phenotype of an
Escherichia coli
mutant that is deficient in Na
+
/H
+
antiport activity (strain KNabc). GerN also reduced the concentration of K
+
required to support growth of an
E. coli
mutant deficient in K
+
uptake (strain TK2420). In a fluorescence-based assay of everted
E. coli
KNabc membrane vesicles, GerN exhibited robust Na
+
/H
+
antiport activity, with a

K
m

for Na
+
estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li
+
, but not K
+
, served as a substrate. GerN-mediated Na
+
/H
+
antiport was further demonstrated in everted vesicles as energy-dependent accumulation of
22
Na
+
. GerN also used K
+
as a coupling ion without completely replacing H
+
, as indicated by partial inhibition by K
+
of H
+
uptake into right-side-out vesicles loaded with Na
+
. K
+
translocation as part of the antiport was supported by the stimulatory effect of intravesicular K
+
on
22
Na
+
uptake by everted vesicles and the dependence of GerN-mediated
86
Rb
+
efflux on the presence of Na
+
in
trans
. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na
+
/H
+
-K
+
antiport. GerN-mediated Na
+
/H
+
-K
+
antiport was much more rapid than GerN-mediated Na
+
/H
+
antiport.