bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptidoglycan from
Staphylococcus aureus
Induces Tissue Factor Expression and Procoagulant Activity in Human Monocytes
Authors: Mattsson Eva , Herwald Heiko , Björck Lars , Egesten Arne 
Published: July 2002

ABSTRACT

Staphylococcus aureus
is one of the most significant pathogens in human sepsis and endocarditis.
S. aureus
can initiate blood coagulation, leading to the formation of microthrombi and multiorgan dysfunction in sepsis, whereas in endocarditis the bacterium induces fibrin clots on the inner surface of the heart, so-called endocardial vegetations. In the present study, we show that live and heat-killed
S. aureus
bacteria are potent inducers of procoagulant activity in human peripheral blood mononuclear cells. Furthermore, purified peptidoglycan, the main cell wall component of
S. aureus
, induced procoagulant activity in mononuclear cells in a concentration-dependent fashion. The procoagulant activity in these cells was dependent on expression of tissue factor, since antibodies to tissue factor inhibited the effect of peptidoglycan. In mononuclear cells stimulated with peptidoglycan, reverse transcription-PCR showed tissue factor gene expression, and the gene product was detected by enzyme-linked immunosorbent assay. Finally, flow cytometry identified tissue factor at the surface of CD14-positive monocytes. Peptidoglycan is known to induce proinflammatory cytokine production in monocytes. The present investigation shows that peptidoglycan also activates the extrinsic pathway of coagulation by inducing the expression of tissue factor in these cells. This mechanism helps to explain the procoagulant activity, which plays such an important role in the pathogenicity of severe
S. aureus
infections.
Identification of a
Neospora caninum
Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans
Authors: Keller Nadine , Naguleswaran Arunasalam , Cannas Angela , Vonlaufen Nathalie , Bienz Marianne , Björkman Camilla , Bohne Wolfgang , Hemphill Andrew 
Published: July 2002

ABSTRACT

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in
Neospora caninum
tachyzoites which, due to its homology to MIC1 in
Toxoplasma gondii
(TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in
Escherichia coli
, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
Identification of a Small RNA within the
pdh
Gene Cluster of
Mycoplasma pneumoniae
and
Mycoplasma genitalium
Authors: Göhlmann Hinrich W. H. , Weiner January , Schön Astrid , Herrmann Richard 
Published: July 2002

ABSTRACT

A highly abundant and heterogenous small RNA about 205 to 210 bases long named MP200 RNA has been identified in
Mycoplasma pneumoniae
. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (
pdh
) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the
pdh
gene cluster of
Mycoplasma genitalium
that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in
Mycoplasma pneumoniae
. The RNAs from both
M. pneumoniae
and
M. genitalium
have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.
Inactivation of the Stress- and Starvation-Inducible
gls24
Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression in
Enterococcus faecalis
Authors: Giard Jean-Christophe , Rince Alain , Capiaux Herve , Auffray Yanick , Hartke Axel 
Published: July 2002

ABSTRACT

Enterococcus faecalis
induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl
2
and bile salts stresses, one of these proteins (Gls24) was qualified as a “general stress protein” and was analyzed at the molecular level. Its corresponding gene,
gls24
, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding
gls24
(
orf4
) showed very strong identity with
gls24
. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from
Lactococcus lactis
and an alkaline stress protein from
Staphylococcus aureus
with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i)
gls24
possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the
gls24
gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in
E. faecalis
. Comparison of two-dimensional protein gels from wild-type cells with those from
gls24
mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for
l
-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.
Expression of
Mycobacterium smegmatis
Pyrazinamidase in
Mycobacterium tuberculosis
Confers Hypersensitivity to Pyrazinamide and Related Amides
Authors: Boshoff Helena I. M. , Mizrahi Valerie 
Published: July 2002

ABSTRACT

A pyrazinamidase (PZase)-deficient
pncA
mutant of
Mycobacterium tuberculosis
, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role of
pncA
in the PZA susceptibility of
M. tuberculosis
(A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the
pzaA
gene encoding the major PZase/nicotinamidase from
Mycobacterium smegmatis
(H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the
M. tuberculosis pncA
gene into the
pncA
mutant complemented its PZase/nicotinamidase defect. In both
pzaA
- and
pncA
-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The
pzaA
-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type and
pncA
-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on
M. smegmatis
(MIC, 150 μg/ml in all cases) and rendered
Escherichia coli
hypersensitive for growth at low pH.
fleN
, a Gene That Regulates Flagellar Number in
Pseudomonas aeruginosa
Authors: Dasgupta Nandini , Arora Shiwani K. , Ramphal Reuben 
Published: July 2002

ABSTRACT

The single polar flagellum of
Pseudomonas aeruginosa
plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the
Pseudomonas
genome database, an open reading frame (ORF), flanked by flagellar genes
flhF
and
fliA
, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in
P. aeruginosa
PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in
P. aeruginosa
. The ORF was named
fleN
. An intact copy of
fleN
on a plasmid complemented the mutant by restoring motility and monoflagellate status. The β-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and
fleN
mutant strains revealed a direct correlation between six promoters that were upregulated in the
fleN
mutant (
fliLMNOPQ
,
flgBCDE
,
fliEFG
,
fliDS orf126
,
fleSR
, and
fliC
) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in
P. aeruginosa
.
The LE1 Bacteriophage Replicates as a Plasmid within
Leptospira biflexa
: Construction of an
L. biflexa-Escherichia coli
Shuttle Vector
Authors: Girons Isabelle Saint , Bourhy Pascale , Ottone Catherine , Picardeau Mathieu , Yelton David , Hendrix Roger W. , Glaser Philippe , Charon Nyles 
Published: July 2002

ABSTRACT

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the
Leptospira biflexa
saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131–1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in
Leptospira
. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the
bla
gene but carrying a kanamycin resistance marker from the gram-positive bacterium
Enterococcus
(
Streptococcus
)
faecalis
. These constructs were transformed into
L. biflexa
strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in
L. biflexa
. However, a larger region including an intact
parA
gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of
Leptospira
species.
Gene Cloning and Molecular Characterization of Lysine Decarboxylase from
Selenomonas ruminantium
Delineate Its Evolutionary Relationship to Ornithine Decarboxylases from Eukaryotes
Authors: Takatsuka Yumiko , Yamaguchi Yoshihiro , Ono Minenobu , Kamio Yoshiyuki 
Published: July 2002

ABSTRACT

Lysine decarboxylase (LDC; EC
4.1.1.18
) from
Selenomonas ruminantium
comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both
l
-lysine and
l
-ornithine with similar

K
m

and

V
max

values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (
ldc
) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of
S. ruminantium
LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC
4.1.1.17
), including the mouse,
Saccharomyces cerevisiae
,
Neurospora crassa
,
Trypanosoma brucei
, and
Caenorhabditis elegans
enzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved in
S. ruminantium
LDC. Computer analysis of the putative secondary structure of
S. ruminantium
LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward both
l
-lysine and
l
-ornithine with a substrate specificity ratio of 0.83 (defined as the
k
cat
/

K
m

ratio obtained with
l
-ornithine relative to that obtained with
l
-lysine). We have succeeded in converting
S. ruminantium
LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in
S. ruminantium
LDC.
A Rac Homolog Is Required for Induction of Hyphal Growth in the Dimorphic Yeast
Yarrowia lipolytica
Authors: Hurtado Cleofe A. R. , Beckerich Jean-Marie , Gaillardin Claude , Rachubinski Richard A. 
Published: July 2002

ABSTRACT

Dimorphism in fungi is believed to constitute a mechanism of response to adverse conditions and represents an important attribute for the development of virulence by a number of pathogenic fungal species. We have isolated
YlRAC1
, a gene encoding a 192-amino-acid protein that is essential for hyphal growth in the dimorphic yeast
Yarrowia lipolytica
and which represents the first Rac homolog described for fungi.
YlRAC1
is not an essential gene, and its deletion does not affect the ability to mate or impair actin polarization in
Y. lipolytica
. However, strains lacking functional
YlRAC1
show alterations in cell morphology, suggesting that the function of
YlRAC1
may be related to some aspect of the polarization of cell growth. Northern blot analysis showed that transcription of
YlRAC1
increases steadily during the yeast-to-hypha transition, while Southern blot analysis of genomic DNA suggested the presence of several
RAC
family members in
Y. lipolytica
. Interestingly, strains lacking functional
YlRAC1
are still able to grow as the pseudohyphal form and to invade agar, thus pointing to a function for
YlRAC1
downstream of
MHY1
, a previously isolated gene encoding a C
2
H
2
-type zinc finger protein with the ability to bind putative stress response elements and whose activity is essential for both hyphal and pseudohyphal growth in
Y. lipolytica.
Isolation of
PDX2
, a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria
Authors: Ehrenshaft Marilyn , Daub Margaret E. 
Published: July 2002

ABSTRACT

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in
Escherichia coli
, recent characterization of the
Cercospora nicotianae
biosynthesis gene
PDX1
led to the discovery that most organisms contain a pyridoxine synthesis gene not found in
E. coli. PDX2
was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to
PDX1
-containing organisms. The role of
PDX2
in pyridoxine biosynthesis was confirmed by complementation of two
C. nicotianae
pyridoxine auxotrophs not mutant in
PDX1
. Also, targeted gene replacement of
PDX2
in
C. nicotianae
results in pyridoxine auxotrophy. Comparable to
PDX1, PDX2
homologues are not found in any of the organisms with homologues to the
E. coli
pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain
PDX1
homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.
SsrA-Mediated Tagging in
Bacillus subtilis
Authors: Wiegert Thomas , Schumann Wolfgang 
Published: July 2002

ABSTRACT

A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the
ssrA
gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this
ssrA
-mediated mechanism in
Bacillus subtilis
, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the
bgaB
reporter gene coding for a heat-stable β-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter β-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the
hrcA
stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by
trans
translational addition of the proteolytic tag. Inactivating the
B. subtilis ssrA
or
smpB
(
yvaI
) gene prevented the
trans
translational tagging reaction. Various protease-deficient strains of
B. subtilis
were tested for proteolysis of tagged HrcA. HrcA remained stable only in
clpX
or
clpP
knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in
B. subtilis
.
ATPase-Defective Derivatives of
Escherichia coli
DnaK That Behave Differently with Respect to ATP-Induced Conformational Change and Peptide Release
Authors: Barthel Thomas K. , Zhang Jundong , Walker Graham C. 
Published: July 2002

ABSTRACT

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of
Escherichia coli
DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The
dnaK
alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a Δ
dnaK
mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.
Carboxy-Terminal Region Involved in Activity of
Escherichia coli
TolC
Authors: Yamanaka Hiroyasu , Izawa Hiroshi , Okamoto Keinosuke 
Published: July 2002

ABSTRACT

The
Escherichia coli
TolC acts as a channel tunnel in the transport of various molecules across the outer membrane. Partial-deletion studies of
tolC
revealed that the region extending from the 50th to the 60th amino acid residue from the carboxy terminus plays an important role in this transport activity of TolC.
MxiM and MxiJ, Base Elements of the Mxi-Spa Type III Secretion System of
Shigella
, Interact with and Stabilize the MxiD Secretin in the Cell Envelope
Authors: Schuch Raymond , Maurelli Anthony T. 
Published: July 2002

ABSTRACT

The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in
Shigella
) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element in
Shigella
, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.