bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phagocytosis of Wild-TypeLegionella pneumophilaOccurs through a Wortmannin-Insensitive Pathway
Authors: Khelef Nadia , Shuman Howard A. , Maxfield Frederick R. 
Published: July 2002

ABSTRACTWild-typeLegionella pneumophilagrows in human macrophages within a replicative phagosome, avoiding lysosomal fusion, while nonreplicative mutants are killed in lysosomes. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocks phagocytosis of an avirulent mutant, but not of wild-typeL. pneumophila, without affecting membrane ruffling and actin polymerization. These results show that wild-type and mutantLegionellastrains use different entry pathways. They suggest that PI3Ks are involved in phagocytosis of an avirulentL. pneumophilamutant and regulate the ability of microorganisms to generate a replicative phagosome.
Role of Endogenous Interleukin-12 in Immune Response to Staphylococcal Enterotoxin B in Mice
Authors: Lauw Fanny N. , Florquin Sandrine , Speelman Peter , van Deventer Sander J. H. , van der Poll Tom 
Published: July 2002

ABSTRACTIn the present study, the roles of interleukin 12 (IL-12) and IL-18 and their possible interaction during superantigen-induced responses were studied by injection of staphylococcal enterotoxin B (SEB) into mice. These data suggest that the role of IL-12 in SEB-induced responses is limited to sustaining gamma interferon release by an IL-18-independent mechanism.
STK Receptor Tyrosine Kinase Regulates Susceptibility to Infection withListeria monocytogenes
Authors: Lutz Michael A. , Gervais Francine , Bernstein Alan , Hattel Arthur L. , Correll Pamela H. 
Published: July 2002

ABSTRACTWe have previously identified the STK receptor tyrosine kinase as a key regulator of macrophage activation and cell-mediated immune responses. Here we demonstrate that, although MSP activation of STK inhibits NO production by macrophages in response to heat-killedListeria monocytogenes,STK-deficient mice exhibit increased susceptibility to infection withListeria.
Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression byPorphyromonas gingivalisMembrane Vesicles
Authors: Srisatjaluk Ratchapin , Kotwal Girish J. , Hunt Lawrence A. , Justus David E. 
Published: July 2002

ABSTRACTGamma interferon (IFN-γ)-induced endothelial cells actively participate in initiating immune responses by interacting with CD4+T cells via class II major histocompatibility complex (MHC) surface glycoproteins. Previously,Porphyromonas gingivalismembrane vesicles were shown to selectively inhibit IFN-γ-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells. In this study, we demonstrated an absence of HLA-DRα mRNA from IFN-γ-induced cells in the presence ofP. gingivalismembrane vesicles by using reverse transcriptase-PCR and Southern blotting. Vesicles also prevented transcription of the gene encoding class II transactivator, a transactivator protein required for IFN-γ-induced expression of MHC class II genes. In addition, the effects of vesicles on IFN-γ signal transduction involving Jak and Stat proteins were characterized by using immunoprecipitation and Western blot analyses. Jak1 and Jak2 proteins could not be detected in endothelial cells treated with membrane vesicles. Consequently, IFN-γ-induced phosphorylation of Jak1, Jak2, and Stat1α proteins was prevented. The class II-inhibitory effect of the membrane vesicles could be eliminated by heating vesicles at 100°C for 30 min or by treating them with a cysteine proteinase inhibitor. This indicates that the cysteine proteinases were most likely responsible for the absence of Jak proteins observed in vesicle-treated cells. The observed increased binding of radiolabeled IFN-γ to vesicle-treated cells suggests that vesicles may also modulate the IFN-γ interactions with the cell surface. However, no evidence was obtained demonstrating that vesicles affected the expression of IFN-γ receptors. Thus,P. gingivalismembrane vesicles apparently inhibited IFN-γ-induced MHC class II by disrupting the IFN-γ signaling transduction pathway. Vesicle-inhibited class II expression also occurred in other IFN-γ-inducible cells. This suggested that the ability ofP. gingivalismembrane vesicles to modulate antigen presentation by key cells may be an important mechanism used by this particular bacterium to escape immunosurveillance, thereby favoring its colonization and invasion of host tissues.
Balance of Pro- and Anti-Inflammatory Cytokines Correlates with Outcome of Acute ExperimentalPseudomonas aeruginosaKeratitis
Authors: Thakur A. , Xue M. , Stapleton F. , Lloyd A. R. , Wakefield D. , Willcox M. D. P. 
Published: July 2002

ABSTRACTThe purpose of this study was to elucidate the expression of pro- and anti-inflammatory cytokines in mouse corneas infected withPseudomonas aeruginosa. Three bacterial strains (invasive, cytotoxic, or CLARE [contact lens-induced acute red eye]) which have recently been shown to produce distinct patterns of corneal disease in the mouse were used. The left mouse (BALB/c) corneas were scarified and infected with 2 × 106CFU of one of the threeP. aeruginosastrains, while right eyes served as controls. Animals were examined at 1, 4, 8, 16, and 24 h with a slit lamp biomicroscope to grade the severity of infection. Following examination, eyes were collected and processed for histopathology, multiprobe RNase protection assay for cytokine mRNA, enzyme-linked immunosorbent assay to quantitate cytokine proteins, and myeloperoxidase activity to quantitate polymorphonuclear leukocytes. The kinetics of appearance and magnitude of expression of key cytokines varied significantly in the three different phenotypes ofP. aeruginosainfection. The predominant cytokines expressed in response to all three phenotypes were interleukin-1β (IL-1β), IL-1Ra, and IL-6. In response to the invasive strain, which induced severe corneal inflammation, significantly lower ratios of IL-1Ra to IL-1β were present at all time points, whereas corneas challenged with the CLARE strain, which induced very mild inflammation, showed a high ratio of IL-1Ra to IL-1β. The outcome of infection in bacterial keratitis correlated with the relative induction of these pro- and anti-inflammatory cytokines, and exogenous administration of recombinant rIL-1Ra (rIL-1Ra) was able to reduce the disease severity significantly. These findings point to the therapeutic potential of rIL-1Ra protein in possible treatment strategies for bacterial keratitis.
Cloning of a Mucin-Desulfating Sulfatase Gene from
Prevotella
Strain RS2 and Its Expression Using a
Bacteroides
Recombinant System
Authors: Wright Damian P. , Knight Catriona G. , Parkar Shanthi G. , Christie David L. , Roberton Anthony M. 
Published: July 2002

ABSTRACT

A gene encoding the mucin-desulfating sulfatase in
Prevotella
strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into
Escherichia coli
expression vectors. However, cloning the gene into a
Bacteroides
expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.
Synechocystis
Strain PCC 6803
cya
2, a Prokaryotic Gene That Encodes a Guanylyl Cyclase
Authors: Ochoa de Alda Jesús A. G. , Ajlani Ghada , Houmard Jean 
Published: July 2002

ABSTRACT

Synechocystis
strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of
cya2
caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.
Transcriptional Control of the Citrate-Inducible
citMCDEFGRP
Operon, Encoding Genes Involved in Citrate Fermentation in
Leuconostoc paramesenteroides
Authors: Martín Mauricio , Magni Christian , López Paloma , de Mendoza Diego 
Published: July 2002

ABSTRACT

In this study we describe the expression pattern of the
Leuconostoc paramesenteroides citMCDEFGRP
operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the
citMCDEFGRP
genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the
cit
operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the
citMCDEFGRP
operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated
citI
. The start and end sites of transcription of the
cit
operon and
citI
gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A+T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3′ end of two secondary structures characteristic of ρ-independent transcriptional terminators. The effect of the
citI
gene on expression of the
cit
operon was studied in
Escherichia coli
. The presence of the
citI
gene in
cis
and in
trans
resulted in increased activity of the
cit
promoter. These data provide the first evidence that citrate fermentation in
Leuconostoc
is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.
Characterization of Spores of
Bacillus subtilis
Which Lack Dipicolinic Acid
Authors: Paidhungat Madan , Setlow Barbara , Driks Adam , Setlow Peter 
Published: July 2002

ABSTRACT

Spores of
Bacillus subtilis
with a mutation in
spoVF
cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Δ
ger3
), as well as
spoVF
, can be isolated, although they spontaneously germinate much more readily than Δ
ger3
spores. The Δ
ger3 spoVF
spores lack DPA and have higher levels of core water than Δ
ger3
spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δ
ger3 spoVF
spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Δ
ger3 spoVF
spores also contain minimal levels of the processed active form (termed P
41
) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P
41
formed in Δ
ger3 spoVF
spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δ
ger3
, and Δ
ger3 spoVF
spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Δ
ger3
spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.
Identification of
Brucella abortus
OxyR and Its Role in Control of Catalase Expression
Authors: Kim Jeong-a , Mayfield John 
Published: July 2002

ABSTRACT

We report the cloning and sequencing of the
Brucella abortus oxyR
homolog and provide evidence that the transcription product of this gene binds to the
B. abortus
catalase promoter region. A gene replacement/deletion
Brucella oxyR
mutant exhibits increased sensitivity to prolonged exposure to H
2
O
2
and is unable to adapt to H
2
O
2
in the environment.
YsxC, a Putative GTP-Binding Protein Essential for Growth of
Bacillus subtilis
168
Authors: Prágai Zoltán , Harwood Colin R. 
Published: July 2002

ABSTRACT

YsxC is a member of a family of GTP-binding proteins carried by a diverse range of organisms from bacteria to yeasts, plants, and humans. To resolve the issue of whether
ysxC
of
Bacillus subtilis
is essential for growth, we attempted to construct mutants in which
ysxC
was either inactivated or placed under the control of an inducible promoter. Viable mutants were obtained only in the latter case, and these were inducer dependent, demonstrating unambiguously that
ysxC
is an essential gene.
Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in
Escherichia coli
Authors: Riondet Christophe , Cachon Rémy , Waché Yves , Alcaraz Gérard , Diviès Charles 
Published: July 2002

ABSTRACT

Wild-type
Escherichia coli
K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (−100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (−320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.
prpR
,
ntrA
, and
ihf
Functions Are Required for Expression of the
prpBCDE
Operon, Encoding Enzymes That Catabolize Propionate in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Palacios Sergio , Escalante-Semerena Jorge C. 
Published: July 2002

ABSTRACT

The genes required for the catabolism of propionate in
Salmonella enterica
serovar Typhimurium are organized as two transcriptional units (
prpR
and
prpBCDE
) that are divergently transcribed from one another. Sequence homology to genes encoding members of the sigma-54 family of transcriptional activators and the identification of a consensus sigma-54 promoter 5′ to the
prpBCDE
operon suggested that PrpR was required to activate expression of this operon. We isolated insertions in
prpR
and showed that
prpR
function was needed for growth on propionate as a carbon and energy source. A medium-copy-number plasmid carrying the
lacZ
gene under the control of the native sigma-54 promoter of
prpBCDE
was used to study
prpBCDE
operon expression. Transcription of the
lacZ
reporter in
prpR
,
ntrA
, and
ihfB
mutants was 85-, 83-, and 15-fold lower than the level of transcription measured in strains carrying the wild-type allele of the gene tested. These data indicated that PrpR, IHF, and transcription sigma factor RpoN were required for the expression of the
prpBCDE
operon. Further analysis of the involvement of the integration host factor (IHF) protein in the expression of this operon is required due to the well-documented pleiotropic effect the lack of this global regulator has on gene expression. Deletion of the 5′ 615-bp portion of the
prpR
gene resulted in a PrpR
c
mutant protein that activated
prpBCDE
transcription regardless of the ability of the strain to synthesize 2-methylcitrate, the putative coactivator of PrpR. These results indicate that the N terminus of PrpR is the coactivator-sensing domain of the protein. When placed under the control of the arabinose-inducible promoter P

araBAD

, expression of

prpR
c

allele by arabinose had a strong negative effect on growth of the cell. It is proposed that this deleterious effect of PrpR
c
may be due to an uncontrolled ATPase activity of PrpR or to cross-activation of genes whose functions negatively affect cell growth under the conditions tested.
The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil
Authors: Reich-Slotky Ronit , Panagiotidis Cynthia , Reyes Moraima , Shuman Howard A. 
Published: July 2002

ABSTRACT

The maltose transporter FGK2 complex of
Escherichia coli
was purified with the aid of a glutathione
S
-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the
V
max
of the ATPase 2.7-fold but had no effect on the

K
m

of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN
3
, as well as a mercurial,
p
-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.
Calcofluor Antifungal Action Depends on Chitin and a Functional High-Osmolarity Glycerol Response (HOG) Pathway: Evidence for a Physiological Role of the
Saccharomyces cerevisiae
HOG Pathway under Noninducing Conditions
Authors: García-Rodriguez L. J. , Durán A. , Roncero C. 
Published: July 2002

ABSTRACT

We have isolated several
Saccharomyces cerevisiae
mutants resistant to calcofluor that contain mutations in the
PBS2
or
HOG1
genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely,
SHO1
and
SLN1
, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated,
pbs2-14
, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.
Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from
Lactobacillus casei
Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion
Authors: Dossonnet Valérie , Monedero Vicente , Zagorec Monique , Galinier Anne , Pérez-Martínez Gaspar , Deutscher Josef 
Published: July 2002

ABSTRACT

We have cloned and sequenced the
Lactobacillus casei hprK
gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant
L. casei
HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When
hprK
was inactivated, carbon catabolite repression of
N
-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the
hprK
mutant. However, inducer expulsion of
methyl
β-
d
-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in
ptsH
mutants altered at Ser-46 of HPr and the
hprK
mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.