bluuebunny/crossref_metadata_2025_split huggingface dataset:
H-NS Is a Repressor of the
Proteus mirabilis
Urease Transcriptional Activator Gene
ureR
Authors: Coker Christopher , Bakare Olubunmi O. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

Expression of
Proteus mirabilis
urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A
6
TA
2
CA
2
TGGTA
5
GA
6
TGA
5
, is located 16 bp upstream of the ς
70
-like
ureR
promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured β-galactosidase activity of wild-type
Escherichia coli
MC4100 (H-NS
+
) and its isogenic derivative ATM121 (
hns
::Tn
10
) (H-NS
−
) harboring a
ureR-lacZ
operon fusion plasmid (pLC9801). β-Galactosidase activity in the H-NS
−
host strain was constitutive and sevenfold greater (
P
< 0.0001) than that in the H-NS
+
host. A recombinant plasmid containing cloned
P. mirabilis hns
was able to complement and restore repression of the
ureR
promoter in the H-NS
−
host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in β-galactosidase activity in the H-NS
+
host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS
−
host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of
P. mirabilis
) was equivalent in both the H-NS
−
background and the H-NS
+
background in the presence of urea but was eightfold greater (
P
= 0.0001) in the H-NS
−
background in the absence of urea. We conclude that H-NS represses
ureR
expression in the absence of urea induction.
The Phosphoryl Transfer Domain of UhpB Interacts with the Response Regulator UhpA
Authors: Wright Jesse S. , Kadner Robert J. 
Published: July 2002

ABSTRACT

Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription. UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT. Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione
S
-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of
uhpT
expression in a
uhp
+
strain. This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation. The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation. Point mutations which allow GST-Bc to activate
uhpT
expression or which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain. The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR. The GST-Bc protein accelerated dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313. Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred. Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA.
Reconstitution of Acetosyringone-Mediated
Agrobacterium tumefaciens
Virulence Gene Expression in the Heterologous Host
Escherichia coli
Authors: Lohrke Scott M. , Yang Hongjiang , Jin Shouguang 
Published: July 2002

ABSTRACT

The ability to utilize
Escherichia coli
as a heterologous system in which to study the regulation of
Agrobacterium tumefaciens
virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the
rpoA
gene encoding the alpha subunit of RNA polymerase is required for the expression of
lacZ
gene under the control of
virB
promoter (
virBp
::
lacZ
) in
E. coli
containing a constitutively active
virG
gene [
virG
(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from
E. coli
and the C-terminal 89 residues from
A. tumefaciens
was able to significantly express
virBp
::
lacZ
in
E. coli
in a VirG(Con)-dependent manner. Utilization of
lac
promoter-driven
virA
and
virG
in combination with the
A. tumefaciens rpoA
construct resulted in significant inducer-mediated expression of the
virBp
::
lacZ
fusion, and the level of
virBp
::
lacZ
expression was positively correlated to the copy number of the
rpoA
construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in
A. tumefaciens
. Furthermore, the effect of sugars on
vir
gene expression was observed only in the presence of the
chvE
gene, suggesting that the glucose-binding protein of
E. coli
, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in
A. tumefaciens
strain A348 from which the
virA
clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the
vir
genes in
E. coli
is less than what is observed in
A. tumefaciens
, suggesting that additional gene(s) from
A. tumefaciens
may be required for the full expression of virulence genes in
E. coli
.
Regulation of
rpoS
Gene Expression in
Pseudomonas
: Involvement of a TetR Family Regulator
Authors: Kojic Milan , Venturi Vittorio 
Published: July 2002

ABSTRACT

The
rpoS
gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase.
rpoS
gene regulation is known to respond to cell density, showing higher expression in stationary phase. For
Pseudomonas aeruginosa,
it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the
rpoS
promoter of
P. putida,
we identified a genomic Tn
5
insertion mutant of
P. putida
which showed a 90% decrease in
rpoS
promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn
5
insertion in a gene, designated
psrA
(
Pseudomonas
sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the
psrA
gene was identified in
P. aeruginosa
; the protein showed 90% identity to PsrA of
P. putida
. A
psrA
::Tn
5
insertion mutant of
P. aeruginosa
was constructed. In both
Pseudomonas
species,
psrA
was genetically linked to the SOS
lexA
repressor gene. Similar to what was observed for
P. putida
, a
psrA
null mutant of
P. aeruginosa
also showed a 90% reduction in
rpoS
promoter activity; both mutants could be complemented for
rpoS
promoter activity when the
psrA
gene was provided in
trans
.
psrA
mutants of both
Pseudomonas
species lost the ability to induce
rpoS
expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate
psrA
gene expression in
Pseudomonas
and in
Escherichia coli
as well as to be capable of activating the
rpoS
promoter in
E. coli
. Our data suggest that PsrA is an important regulatory protein of
Pseudomonas
spp. involved in the regulatory cascade controlling
rpoS
gene regulation in response to cell density.
Analysis of Phase-Specific Gene Expression at the Single-Cell Level in the White-Opaque Switching System of
Candida albicans
Authors: Strauß Anja , Michel Sonja , Morschhäuser Joachim 
Published: July 2002

ABSTRACT

The opportunistic fungal pathogen
Candida albicans
can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous
ura3
mutants from strain WO-1 by targeted gene deletion. The two
URA3
alleles were sequentially inactivated using the
MPA

R

-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using
URA3
as a selection marker. Fluorescence microscopic analysis of cells in which a
GFP
reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific
SAP1
gene was detectably expressed only in opaque cells and that the white-phase-specific
WH11
gene was detectably expressed only in white cells. When
MPA

R

was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the
SAP1
promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when
MPA

R

was placed under the control of the
WH11
promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the
SAP1
and
WH11
genes did not occur at a detectable frequency. The expression of these phase-specific genes of
C. albicans
in vitro, therefore, is tightly linked to the cell type.
Characterization of an Aminoacylase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Story Sherry V. , Grunden Amy M. , Adams Michael W. W. 
Published: July 2002

ABSTRACT

Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon
Pyrococcus furiosus
by its ability to hydrolyze
N
-acetyl-
l
-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl
2
(and to 74% by the addition of CoCl
2
). After reconstitution with ZnCl
2
, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolar
N
-acylated
l
amino acids (Met, Ala, Val, and Leu), as well as
N
-formyl-
l
-methionine. The high

K
m

values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity with
N
-acetyl-
l
-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in the
P. furiosus
genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in
Escherichia coli
by using two approaches. One involved the T7
lac
promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn
2+
ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of the
P. furiosus
aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.
Properties of a Revertant of
Escherichia coli
Viable in the Presence of Spermidine Accumulation: Increase in
l-
Glycerol 3-Phosphate
Authors: Raj V. Samuel , Tomitori Hideyuki , Yoshida Madoka , Apirakaramwong Auayporn , Kashiwagi Keiko , Takio Koji , Ishihama Akira , Igarashi Kazuei 
Published: July 2002

ABSTRACT

Escherichia coli
CAG2242 cells are deficient in the
speG
gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase ς
38
subunit, factors important for cell viability, was increased in the revertant. Levels of
l
-glycerol 3-phosphate also increased in the revertant. Transformation of
glpFK
, which encodes a glycerol diffusion facilitator (
glpF
) and glycerol kinase (
glpK
), to
E. coli
CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that
l
-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that
l
-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.
Developmental Gene Expression in
Bacillus subtilis crsA47
Mutants Reveals Glucose-Activated Control of the Gene for the Minor Sigma Factor ς
H
Authors: Dixon Laurie G. , Seredick Steve , Richer Martin , Spiegelman George B. 
Published: July 2002

ABSTRACT

The presence of excess glucose in growth media prevents normal sporulation of
Bacillus subtilis
. The
crsA47
mutation, located in the gene for the vegetative phase sigma factor (ς
A
) results in a glucose-resistant sporulation phenotype. As part of a study of the mechanisms whereby the mutation in ς
A
overcomes glucose repression of sporulation, we examined the expression of genes involved in sporulation initiation in the
crsA47
background. The
crsA47
mutation had a significant impact on a variety of genes. Changes to stage II gene expression could be linked to alterations in the expression of the
sinI
and
sinR
genes. In addition, there was a dramatic increase in the expression of genes dependent on the minor sigma factor ς
H
. This latter change was paralleled by the pattern of
spo0H
gene transcription in cells with the
crsA47
mutation. In vitro analysis of RNA polymerase containing ς
A47
indicated that it did not have unusually high affinity for the
spo0H
gene promoter. The in vivo pattern of
spo0H
expression is not predicted by the known regulatory constraints on
spo0H
and suggests novel regulation mechanisms that are revealed in the
crsA47
background.
Identification and Characterization of
fhuD1
and
fhuD2
, Two Genes Involved in Iron-Hydroxamate Uptake in
Staphylococcus aureus
Authors: Sebulsky M. Tom , Heinrichs David E. 
Published: July 2002

ABSTRACT

Staphylococcus aureus
can utilize several hydroxamate siderophores for growth under iron-restricted conditions. Previous findings have shown that
S. aureus
possesses a cytoplasmic membrane-associated traffic ATPase that is involved in the specific transport of iron(III)-hydroxamate complexes. In this study, we have identified two additional genes, termed
fhuD1
and
fhuD2
, whose products are involved in this transport process in
S. aureus
. We have shown that
fhuD2
codes for a posttranslationally modified lipoprotein that is anchored in the cytoplasmic membrane, while the deduced amino acid sequence predicts the same for
fhuD1
. The predicted FhuD1 and FhuD2 proteins share 41.0% identity and 56.4% total similarity with each other, 45.9 and 49.1% total similarity with the FhuD homolog in
Bacillus subtilis
, and 29.3 and 24.6% total similarity with the periplasmic FhuD protein from
Escherichia coli
. Insertional inactivation and gene replacement of both genes showed that while FhuD2 is involved in the transport of iron(III) in complex with ferrichrome, ferrioxamine B, aerobactin, and coprogen, FhuD1 shows a more limited substrate range, capable of only iron(III)-ferrichrome and iron(III)-ferrioxamine B transport in
S. aureus
. Nucleotide sequences present upstream of both
fhuD1
and
fhuD2
predict the presence of consensus Fur binding sequences. In agreement, transcription of both genes was negatively regulated by exogenous iron levels through the activity of the
S. aureus
Fur protein.
Escherichia coli
FadR Positively Regulates Transcription of the
fabB
Fatty Acid Biosynthetic Gene
Authors: Campbell John W. , Cronan John E. 
Published: July 2002

ABSTRACT

In
Escherichia coli
expression of the genes of fatty acid degradation (
fad
) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene,
fabA
, is positively regulated by FadR. We report that
fabB
, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and
fadR
-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of
fabB
was regulated in a manner very similar to that of
fabA
expression. A series of genetic and biochemical tests confirmed these observations. Strains containing both
fabB
and
fadR
mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed in
fabA fadR
mutants. A
fadR
strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the
fabB
promoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within the
fabB
promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of
fabB
is positively regulated by FadR.
The Global Regulators GacA and ς
S
Form Part of a Cascade That Controls Alginate Production in
Azotobacter vinelandii
Authors: Castañeda Miguel , Sánchez Judith , Moreno Soledad , Núñez Cinthia , Espı́n Guadalupe 
Published: July 2002

ABSTRACT

Transcription of the
Azotobacter vinelandii algD
gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of
algD
from its three sites during the stationary phase. Here we show that
algD
is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed
A
.
vinelandii
strains that carried mutations in
gacA
encoding the cognate response regulator of GacS and in
rpoS
coding for the stationary-phase ς
S
factor. The
gacA
mutation impaired alginate production and transcription of
algD
from its three promoters. Transcription of
rpoS
was also abolished by the
gacA
mutation. The
rpoS
mutation impaired transcription of
algD
from the p1 promoter and increased it from the p2 ς
E
promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in
A
.
vinelandii
.
Analysis of Transcription of the
Staphylococcus aureus
Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen
Authors: Masalha Mahmud , Borovok Ilya , Schreiber Rachel , Aharonowitz Yair , Cohen Gerald 
Published: July 2002

ABSTRACT

Staphylococcus aureus
is a gram-positive facultative aerobe that can grow in the absence of oxygen by fermentation or by using an alternative electron acceptor. To investigate the mechanism by which
S. aureus
is able to adapt to changes in oxygen concentration, we analyzed the transcriptional regulation of genes that encode the aerobic class Ib and anaerobic class III ribonucleotide reductase (RNR) systems that are responsible for the synthesis of deoxyribonucleotides needed for DNA synthesis. The
S. aureus
class Ib RNR
nrdIEF
and class III RNR
nrdDG
genes and their regulatory regions were cloned and sequenced. Inactivation of the
nrdDG
genes showed that the class III RNR is essential for anaerobic growth. Inhibition of aerobic growth by hydroxyurea showed that the class Ib RNR is an oxygen-dependent enzyme. Northern blot analysis and primer extension analysis demonstrated that transcription of class III
nrdDG
genes is regulated by oxygen concentration and was at least 10-fold higher under anaerobic than under aerobic conditions. In contrast, no significant effect of oxygen concentration was found on the transcription of class Ib
nrdIEF
genes. Disruption or deletion of
S. aureus nrdDG
genes caused up to a fivefold increase in
nrdDG
and
nrdIEF
transcription under anaerobic conditions but not under aerobic conditions. Similarly, hydroxyurea, an inhibitor of the class I RNRs, resulted in increased transcription of class Ib and class III RNR genes under aerobic conditions. These findings establish that transcription of class Ib and class III RNR genes is upregulated under conditions that cause the depletion of deoxyribonucleotide. Promoter analysis of class Ib and class III RNR operons identified several inverted-repeat elements that may account for the transcriptional response of the
nrdIEF
and
nrdDG
genes to oxygen.
Involvement of ResE Phosphatase Activity in Down-Regulation of ResD-Controlled Genes in
Bacillus subtilis
during Aerobic Growth
Authors: Nakano Michiko M. , Zhu Yi 
Published: July 2002

ABSTRACT

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in
Bacillus subtilis
. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognate response regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD is postulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic conditions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defective in ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes. The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobic induction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaerobiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests that the ResE kinase activity is also subject to control in response to oxygen limitation.