bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycoinositolphospholipids fromTrypanosoma cruziInterfere with Macrophages and Dendritic Cell Responses
Authors: Brodskyn Claudia , Patricio Julie , Oliveira Rubem , Lobo Lucas , Arnholdt Andrea , Mendonça-Previato Lucia , Barral Aldina , Barral-Netto Manoel 
Published: July 2002

ABSTRACTTo investigate the possible effects of glycoinositolphospholipid (GIPL) fromTrypanosoma cruzion human antigen presenting cells, we tested their effects on lipopolysaccharide (LPS)-stimulated human macrophages and dendritic cells (DC). Human macrophages or DC were incubated with GIPL (50 μg/ml) and LPS (500 pg/ml) and tumor necrosis factor alpha (TNF-α), interleukin 8 (IL-8), IL-10, and IL-12p40 levels in supernatants were analyzed by enzyme-linked immunosorbent assay. TNF-α, IL-10, and IL-12 secretion were significantly decreased by GIPL both in macrophages and DC. In contrast, GIPL did not alter IL-8 production. We also analyzed the expression of CD80, CD86, HLA-DR, CD40, and CD57 on the macrophage surface after stimulation with LPS in the presence or absence ofT. cruziGIPL. GIPL led to a down-regulation in the expression of all tested molecules. We additionally examined the influence ofT. cruziGIPL on the response of human DC to LPS. LPS-induced HLA-DR, CD83, and CD86 up-regulation was significantly inhibited by GIPL. A slight down-regulation in CD80 and CD40 expression on DC surfaces in the presence of GIPL was also noticed. Similarly, GIPL led to down-modulation of CD83, CD80, CD86, and HLA-DR surface expression and TNF-α and IL-10 production when DC were stimulated by CD40L. The ceramide portion of GIPL was responsible for most of the activity exhibited by the whole molecule. Considering the important role of the immune response in determining the fate of the host-parasite relationship, the immunoregulatory activities ofT. cruziGIPL are potentially important for parasite evasion and then pathogenesis of infection with protozoan parasites.
Characterization of the
relA
Gene of
Porphyromonas gingivalis
Authors: Sen Keya , Hayashi Jun-ichiro , Kuramitsu Howard K. 
Published: July 2002

ABSTRACT

Based upon the nucleotide sequence of the
relA
gene from
Escherichia coli
, a gene fragment corresponding to the homologous gene from the pathogenic oral bacterium
Porphyromonas gingivalis
381 was isolated by PCR and utilized to construct a
relA
mutant. The mutant, KS7, was defective in ribosome-mediated ppGpp formation and also in the stringent response.
Inactivation of the
inhA
-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of
Mycobacterium smegmatis
Authors: Vilchèze Catherine , Morbidoni Hector R. , Weisbrod Torin R. , Iwamoto Hiroyuki , Kuo Mack , Sacchettini James C. , Jacobs William R. 
Published: July 2002

ABSTRACT

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (
katG
,
inhA
,
ahpC
,
kasA
, and
ndh
) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates
inhA
, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of
Mycobacterium tuberculosis
causes the accumulation of hexacosanoic acid (C
26:0
), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the
inhA
gene of
Mycobacterium smegmatis
that rendered InhA inactive at 42°C. Thermal inactivation of InhA in
M. smegmatis
resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C
16:0
) and a concomitant increase of tetracosanoic acid (C
24:0
) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of
Mycobacterium bovis
BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C
16:0
, and a concomitant accumulation of C
26:0
. Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH.
A
soxA
Gene, Encoding a Diheme Cytochrome
c
, and a
sox
Locus, Essential for Sulfur Oxidation in a New Sulfur Lithotrophic Bacterium
Authors: Mukhopadhyaya Pratap N. , Deb Chirajyoti , Lahiri Chandrajit , Roy Pradosh 
Published: July 2002

ABSTRACT

A mobilizable suicide vector, pSUP5011, was used to introduce Tn
5-mob
in a new facultative sulfur lithotrophic bacterium, KCT001, to generate mutants defective in sulfur oxidation (Sox
−
). The Sox
−
mutants were unable to oxidize thiosulfate while grown mixotrophically in the presence of thiosulfate and succinate. The mutants were also impaired in oxidizing other reduced sulfur compounds and elemental sulfur as evident from the study of substrate oxidation by the whole cells. Sulfite oxidase activity was significantly diminished in the cell extracts of all the mutants. A
soxA
gene was identified from the transposon-adjacent genomic DNA of a Sox
−
mutant strain. The sequence analysis revealed that the
soxA
open reading frame (ORF) is preceded by a potential ribosome binding site and promoter region with −10- and −35-like sequences. The deduced nucleotide sequence of the
soxA
gene was predicted to code for a protein of 286 amino acids. It had a signal peptide of 26 N-terminal amino acids. The amino acid sequence showed similarity with a putative gene product of
Aquifex aeolicus
, soluble cytochrome
c
551
of
Chlorobium limicola
, and the available partial SoxA sequence of
Paracoccus denitrificans
. The
soxA
-encoded product seems to be a diheme cytochrome
c
for KCT001 and
A. aeolicus
, but the amino acid sequence of
C. limicola
cytochrome
c
551
revealed a single heme-binding region. Another transposon insertion mutation was mapped within the
soxA
ORF. Four other independent transposon insertion mutations were mapped in the 4.4-kb
soxA
contiguous genomic DNA region. The results thus suggest that a
sox
locus of KCT001, essential for sulfur oxidation, was affected by all these six independent insertion mutations.
Phosphorylation-Induced Signal Propagation in the Response Regulator NtrC
Authors: Lee Jonghui , Owens Jeffrey T. , Hwang Ingyu , Meares Claude , Kustu Sydney 
Published: July 2002

ABSTRACT

The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the ς
54
holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrC
D86C
, in which the unique cysteine lies near the top of α-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that α-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede—and may give rise to—the oligomerization needed for transcriptional activation.
Mutations in Multidrug Efflux Homologs, Sugar Isomerases, and Antimicrobial Biosynthesis Genes Differentially Elevate Activity of the ς
X
and ς
W
Factors in
Bacillus subtilis
Authors: Turner Mark S. , Helmann John D. 
Published: July 2002

ABSTRACT

The ς
X
and ς
W
extracytoplasmic function sigma factors regulate more than 40 genes in
Bacillus subtilis
. ς
W
activates genes which function in detoxification and the production of antimicrobial compounds, while ς
X
activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate ς
W
or ς
X
as judged by up-regulation from the autoregulatory promoter site P
W
or P
X
. Fourteen insertions that activate P
W
were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (
yqgE
and
yulE
), a component of the oligopeptide uptake system (
oppA
), and two transmembrane proteins with weak similarity to transporters (
yhdP
and
yueF
). Expression from P
W
is also elevated as a result of inactivation of at least one member of the ς
W
regulon (
ysdB
), an ArsR homolog (
yvbA
), a predicted rhamnose isomerase (
yulE
), and a gene (
pksR
) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate P
X
. Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (
yitG
), a mannose-6-phosphate isomerase gene (
yjdE
), and loci involved in antibiotic synthesis (
srfAB
and possibly
yogA
and
yngK
). Significantly, most insertions that activate P
W
have little or no effect on P
X
, and conversely, insertions that activate P
X
have no effect on P
W
. This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.
Transcriptional and Translational Regulation of α-Acetolactate Decarboxylase of
Lactococcus lactis
subsp.
lactis
Authors: Goupil-Feuillerat Nathalie , Corthier Gérard , Godon Jean-Jacques , Ehrlich S. Dusko , Renault Pierre 
Published: July 2002

ABSTRACT

The α-acetolactate decarboxylase (ALDC) gene,
aldB
, is the penultimate gene of the
leu-ilv-ald
operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in
Lactococcus lactis
. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of α-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the
leu
and
ilv
genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although
aldB
is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The
aldB
ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the
aldB
RBS was responsible for a 100-fold decrease in the level of
aldB
translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of
aldB
translation. Finally, the translation of the
ilvA
gene terminating in the latter structure decreased the level of translation of
aldB
fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the
ilvA
product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.
In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of
Pseudomonas
sp. Strain KWI-56 Lipase
Authors: Yang Junhao , Kobayashi Koei , Iwasaki Yugo , Nakano Hideo , Yamane Tsuneo 
Published: July 2002

ABSTRACT

The expression of lipase from
Pseudomonas
sp. strain KWI-56 (recently reclassified as
Burkholderia cepacia
) had been found to be dependent on an activator gene (
act
) downstream of its structural gene (
lip
). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free
Escherichia coli
S30 coupled transcription-translation system by expressing a recombinant lipase gene (r
lip
) encoding the mature lipase in the presence of its purified activator or by coexpression of r
lip
and
act
. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca
2+
binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca
2+
binding site in the activation of the lipase is also discussed.
Characterization of an HPr Kinase Mutant of
Staphylococcus xylosus
Authors: Huynh Phuong Lan , Jankovic Ivana , Schnell Norbert F. , Brückner Reinhold 
Published: July 2002

ABSTRACT

The
Staphylococcus xylosus
gene
hprK
, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His
6
fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of
S. xylosus
resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of
S. xylosus
. The
hprK
mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of
S. xylosus
according to the availability of preferred carbon sources.
Characterization of the
uup
Locus and Its Role in Transposon Excisions and Tandem Repeat Deletions in
Escherichia coli
Authors: Reddy Manjula , Gowrishankar J. 
Published: July 2002

ABSTRACT

Null mutations in the
Escherichia coli uup
locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn
10
and to reduce the plaque size of bacteriophage Mu (Uup
−
phenotype). By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup
−
phenotype is the consequence of the absence of expression of the downstream gene (
uup
) of a two-gene operon, caused either directly by insertions in
uup
or indirectly by the polar effect of insertions in the upstream gene (
ycbY
). The promoter for
uup
was mapped upstream of
ycbY
by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter. The
uup
mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn
10
derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision. Finally, we found that mutations that increase the frequency of precise excision of Tn
10
are divisible into two categories, depending upon whether they did (
uup
,
ssb
,
polA
, and
topA
) or did not (
mutHLS
,
dam
, and
uvrD
) also increase precise excision frequency of the mini-Tn
10
derivatives. It is suggested that the differential response of mini-Tn
10
and Tn
10
to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them.
trans
-Acting Mutations in Loci Other than
kdpDE
That Affect
kdp
Operon Regulation in
Escherichia coli
: Effects of Cytoplasmic Thiol Oxidation Status and Nucleoid Protein H-NS on
kdp
Expression
Authors: Sardesai Abhijit A. , Gowrishankar J. 
Published: July 2002

ABSTRACT

Transcription of the K
+
transport operon
kdp
in
Escherichia coli
is induced during K
+
-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for new
dke
(named
dke
for decreased
kdp
expression) mutations (in loci other than
kdpDE
) that led to substantially decreased
kdp
expression. One
dke
mutation was shown to be in
hns
, encoding the nucleoid protein H-NS. Another
dke
mutation was mapped to
trxB
(encoding thioredoxin reductase), and an equivalent reduction in
kdp
expression was demonstrated also for
trxA
mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the
kdp
expression defect in
trxB
but not
trxA
mutants. Neither
trxB
nor
trxA
affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes
dsbC
and
dsbD
did not affect
kdp
expression, suggesting that the
trx
effects on
kdp
are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced
kdp
expression was observed even in a
trxB
strain that harbored a variant KdpD polypeptide bearing no Cys residues. A
trxB hns
double mutant was even more severely affected for
kdp
expression than either single mutant. The
dke
mutations themselves had no effect on strength of the signal controlling
kdp
expression, and constitutive mutations in
kdpDE
were epistatic to
hns
and
trxB
. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for
kdp
transcription.
trans
-3-Chloroacrylic Acid Dehalogenase from
Pseudomonas pavonaceae
170 Shares Structural and Mechanistic Similarities with 4-Oxalocrotonate Tautomerase
Authors: Poelarends Gerrit J. , Saunier Raymond , Janssen Dick B. 
Published: July 2002

ABSTRACT

The genes (
caaD1
and
caaD2
) encoding the
trans
-3-chloroacrylic acid dehalogenase (CaaD) of the 1,3-dichloropropene-utilizing bacterium
Pseudomonas pavonaceae
170 were cloned and heterologously expressed in
Escherichia coli
and
Pseudomonas
sp. strain GJ1. CaaD is a protein of 50 kDa that is composed of α-subunits of 75 amino acid residues and β-subunits of 70 residues. It catalyzes the hydrolytic cleavage of the β-vinylic carbon-chlorine bond in
trans
-3-chloroacrylic acid with a turnover number of 6.4 s
−1
. On the basis of sequence similarity, oligomeric structure, and subunit size, CaaD appears to be related to 4-oxalocrotonate tautomerase (4-OT). This tautomerase consists of six identical subunits of 62 amino acid residues and catalyzes the isomerization of 2-oxo-4-hexene-1,6-dioate, via hydroxymuconate, to yield 2-oxo-3-hexene-1,6-dioate. In view of the oligomeric architecture of 4-OT, a trimer of homodimers, CaaD is postulated to be a hexameric protein that functions as a trimer of αβ-dimers. The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the β-subunit and Arg-11 of the α-subunit are active-site residues in CaaD. Pro-1 could act as the proton acceptor/donor, and Arg-11 is probably involved in carboxylate binding. Based on these findings, a novel dehalogenation mechanism is proposed for the CaaD-catalyzed reaction which does not involve the formation of a covalent enzyme-substrate intermediate.
Localization of GerAA and GerAC Germination Proteins in the
Bacillus subtilis
Spore
Authors: Hudson Kaye D. , Corfe Bernard M. , Kemp E. Helen , Feavers Ian M. , Coote Peter J. , Moir Anne 
Published: July 2002

ABSTRACT

The GerAA, -AB, and -AC proteins of the
Bacillus subtilis
spore are required for the germination response to
l
-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the
gerA
genes are found in every spore former so far examined. The
gerA
operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of
B. subtilis
were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5∗, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.