bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Assay for Low-Activity Mutant Forms of
Escherichia coli
Ribonucleotide Reductase
Authors: Ekberg Monica , Birgander Pernilla , Sjöberg Britt-Marie 
Published: January 2003

ABSTRACT

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic
Escherichia coli
cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays.
E. coli
RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.
Interactions between Phage-Shock Proteins in
Escherichia coli
Authors: Adams Hendrik , Teertstra Wieke , Demmers Jeroen , Boesten Rolf , Tommassen Jan 
Published: January 2003

ABSTRACT

Expression of the
pspABCDE
operon of
Escherichia coli
is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor σ
54
and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical
pspD
gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the
psp
operon is mediated via protein-protein interactions.
CDP-2,3-Di
-O-
Geranylgeranyl-
sn
-Glycerol:
l
-Serine
O
-Archaetidyltransferase (Archaetidylserine Synthase) in the Methanogenic Archaeon
Methanothermobacter thermautotrophicus
Authors: Morii Hiroyuki , Koga Yosuke 
Published: January 2003

ABSTRACT

CDP-2,3-di
-O-
geranylgeranyl-
sn
-glycerol:
l
-serine
O
-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of
Methanothermobacter thermautotrophicus
cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and
l
-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn
2+
and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of
d
-serine with the enzyme was 30% of that observed for
l
-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in
M. thermautotrophicus
genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to
Bacillus subtilis pssA
but not to
Escherichia coli pssA.
The substrate specificity of phosphatidylserine synthase from
B. subtilis
was quite similar to that observed for the
M. thermautotrophicus
archaetidylserine synthase, while the
E. coli
enzyme had a strong preference for CDP-1,2-diacyl-
sn
-glycerol. It was concluded that
M. thermautotrophicus
archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (
B. subtilis
type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.
Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors
Authors: Minamino Tohru , Imae Yasuo , Oosawa Fumio , Kobayashi Yuji , Oosawa Kenji 
Published: January 2003

ABSTRACT

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of
Escherichia coli
and
Salmonella
at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of
E. coli
cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of
E. coli
cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered
E. coli
cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H
+
) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of
Salmonella
also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.
Analysis of Residues Determining Specificity ofVibrio choleraeTonB1 for Its Receptors
Authors: Mey Alexandra R. , Payne Shelley M. 
Published: January 2003

ABSTRACTIn gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex.Vibrio choleraeencodes two TonB proteins, one of which, TonB1, recognizes only a subset ofV.choleraeTonB-dependent receptors and does not facilitate transport throughEscherichia colireceptors. To investigate the receptor specificity exhibited byV.choleraeTonB1, chimeras were created betweenV.choleraeTonB1 andE.coliTonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus ofV.choleraeTonB1 were identified that allowed TonB1 to recognizeE.colireceptors and at least oneV.choleraeTonB2-dependent receptor. This indicates that the very C-terminal end ofV.choleraeTonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching betweenV.choleraeandE.colireceptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box inE.colireceptors with an aromatic residue allowed these receptors to useV.choleraeTonB1. Further, site-directed mutagenesis of the TonB box −1 residue in aV.choleraeTonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition ofV.choleraeTonB1. These data suggest that the TonB box −1 position controls productive interactions withV.choleraeTonB1.
Molecular Genetic Analysis of a Group AStreptococcusOperon Encoding Serum Opacity Factor and a Novel Fibronectin-Binding Protein, SfbX
Authors: Jeng Arthur , Sakota Varja , Li Zhongya , Datta Vivekananda , Beall Bernard , Nizet Victor 
Published: January 2003

ABSTRACTThe group AStreptococcus(GAS)sofgene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. Thesofgene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a typeemm49strain and a typeemm12strain, the sequences upstream of the 5′ end ofsofand downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of theemm1gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specificemmgenotypes. A new fibronectin-binding protein gene,sfbX, was discovered immediately downstream ofsofinemm12andemm49strains and in several othersof-positive strains. ThesofandsfbXgenes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. ThesfbXgenes from differentemmtypes are predicted to encode ∼650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being theStaphylococcus aureuscoagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded bysofalone and thatsfbXencodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. ThesfbXgene was found to be present only insof-positive strains, and together these genes could influence the spectrum of tissues colonized bysof-positive GAS.
In Vitro and In Vivo Functional Activity ofChlamydiaMurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance
Authors: McCoy Andrea J. , Sandlin Robin C. , Maurelli Anthony T. 
Published: January 2003

ABSTRACTOrganisms ofChlamydiaspp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibitChlamydiagrowth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. ThemurAgene fromChlamydiatrachomatisserovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressiblearapromoter and transformed intoEscherichia coli. After transduction of a lethal ΔmurAmutation into the strain, viability of theE. colistrain became dependent upon expression of theC. trachomatis murA.DNA sequence analysis ofmurAfromC. trachomatispredicted a cysteine-to-aspartate change in a key residue within the active site of MurA. InE. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth ofC. trachomatiswas also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to theE. colistrain expressing the chlamydialmurA. Conversion ofC. trachomatiselementary bodies to reticulate bodies and cell division are correlated with expression ofmurAmRNA. mRNA frommurB, the second enzymatic reaction in the PG pathway, was also detected duringC. trachomatisinfection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists inChlamydiaspp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG inChlamydiaspp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.
QscR, a LysR-Type Transcriptional Regulator and CbbR Homolog, Is Involved in Regulation of the Serine Cycle Genes in
Methylobacterium extorquens
AM1
Authors: Kalyuzhnaya Marina G. , Lidstrom Mary E. 
Published: January 2003

ABSTRACT

A new gene,
qscR
, encoding a LysR-type transcriptional regulator that is a homolog of CbbR, has been characterized from the facultative methylotroph
Methylobacterium extorquens
AM1 and shown to be the major regulator of the serine cycle, the specific C
1
assimilation pathway. The
qscR
mutant was shown to be unable to grow on C
1
compounds, and it lacked the activity of serine-glyoxylate aminotransferase, a key enzyme of the serine cycle. Activities of other serine cycle enzymes were decreased during growth on C
1
compounds compared to the activities found in wild-type
M. extorquens
AM1. Promoter fusion assays, as well as reverse transcription-PCR assays, have indicated that the serine cycle genes belong to three separate transcriptional units,
sga-hpr-mtdA-fch
,
mtkA-mtkB-ppc-mcl
, and
gly
. Gel retardation assays involving the purified QscR have demonstrated the specific binding of QscR to the DNA regions upstream of
sga
,
mtkA
,
gly
, and
qscR
. We conclude that QscR acts as a positive transcriptional regulator of most of the serine cycle enzymes and also as an autorepressor.
Roles of NhaA, NhaB, and NhaD Na
+
/H
+
Antiporters in Survival of
Vibrio cholerae
in a Saline Environment
Authors: Herz Katia , Vimont Sophie , Padan Etana , Berche Patrick 
Published: January 2003

ABSTRACT

Vibrio cholerae
, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na
+
/H
+
antiporters on the survival of
V. cholerae
in a saline environment. We have previously cloned the Vc-
nhaA
gene encoding the
V. cholerae
homolog of
Escherichia coli
. Here we identified two additional antiporter genes, designated Vc-
nhaB
and Vc
-nhaD
, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of
Vibrio
species and
E. coli.
We showed that both Vc-NhaA and Vc-NhaB confer Na
+
resistance and that Vc-NhaA displays an antiport activity in
E. coli
, which is similar in magnitude, kinetic parameters, and pH regulation to that of
E. coli
NhaA. To determine the roles of the Na
+
/H
+
antiporters in
V. cholerae
, we constructed
nhaA
,
nhaB
, and
nhaD
mutants (single, double, and triple mutants). In contrast to
E. coli
, the inactivation of the three putative antiporter genes (Vc-
nhaABD)
in
V. cholerae
did not alter the bacterial exponential growth in the presence of high Na
+
concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li
+
-sensitive phenotype was found with all mutants lacking Vc-
nhaA
during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-
n
-nonyl-4-hydroxyquinoline
N
-oxide, a specific inhibitor of the electron-transport-linked Na
+
pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na
+
/H
+
antiporter activity becomes essential for the resistance of
V. cholerae
to Na
+
at alkaline pH. Since the ion pump NQR is Na
+
specific, we suggest that its activity masks the Na
+
/H
+
but not the Li
+
/H
+
antiporter activities. Our results indicate that the Na
+
resistance of the human pathogen
V. cholerae
requires a complex molecular system involving multiple antiporters and the NQR pump.
Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of
Xanthobacter flavus
Authors: van Keulen Geertje , Ridder Anja N. J. A. , Dijkhuizen Lubbert , Meijer Wim G. 
Published: January 2003

ABSTRACT

The LysR-type transcriptional regulator CbbR controls the expression of the
cbb
and
gap-pgk
operons in
Xanthobacter flavus
, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO
2
fixation. The
cbb
operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and
cbb
promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR
1
is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the
cbb
operon promoter. In addition, it is required for negative autoregulation of
cbbR
expression. IR
3
represents the main low-affinity binding site of CbbR. Binding to IR
3
occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR
1
also prevent binding to the low-affinity site. Although mutations in IR
3
have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR
3
partially overlaps with the −35 region of the
cbb
operon promoter. Mutations in IR
2
do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the
cbb
operon promoter. This IR
2
binding site is therefore critical for transcriptional activation by CbbR.
Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites
Authors: Vaillancourt Frédéric H. , Haro María-Amparo , Drouin Nathalie M. , Karim Zamil , Maaroufi Halim , Eltis Lindsay D. 
Published: January 2003

ABSTRACT

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from
Burkholderia
sp. strain LB400 (DHBD
LB400
; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-dichloro-2,3-dihydroxybiphenyl (2′,6′-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBD
P6
-I and DHBD
P6
-III, two evolutionarily divergent isozymes from
Rhodococcus globerulus
strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBD
LB400
, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBD
P6
-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBD
P6
-I were among the best for DHBD
P6
-III (4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB; DHBD
P6
-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBD
P6
-I and 2′-Cl DHB for DHBD
P6
-III. As observed in DHBD
LB400
, chloro substituents influenced the reactivity of the dioxygenases with O
2
. For example, the apparent specificities of DHBD
P6
-I and DHBD
P6
-III for O
2
in the presence of 2′-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBD
P6
-I and DHBD
P6
-III shared the relative inability of DHBD
LB400
to cleave 2′,6′-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s
−1
, respectively). However, these isozymes had remarkably different apparent
K
m
values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 μM for DHBD
LB400
, DHBD
P6
-I, and DHBD
P6
-III, respectively). The markedly different reactivities of DHBD
P6
-I and DHBD
P6
-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of
R. globerulus
P6.
Pyoverdine-Mediated Regulation of FpvA Synthesis in
Pseudomonas aeruginosa
: Involvement of a Probable Extracytoplasmic-Function Sigma Factor, FpvI
Authors: Rédly Gyula Alan , Poole Keith 
Published: January 2003

ABSTRACT

A search of the
pvd
pyoverdine biosynthesis locus of
Pseudomonas aeruginosa
identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in
Escherichia coli
and
Pseudomonas putida
. Deletion of this gene, dubbed
fpvI
, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and
fpvA
gene expression, while the cloned gene stimulated
fpvA
expression. A Fur-binding site was identified immediately upstream of
fpvI
, consistent with the observed iron-regulated expression of
fpvI
and
fpvA
.
Molecular Characteristics of Spontaneous Deletions in the Hyperthermophilic Archaeon
Sulfolobus acidocaldarius
Authors: Grogan Dennis W. , Hansen Josh E. 
Published: January 2003

ABSTRACT

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of
Sulfolobus acidocaldarius
for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous
pyrE
mutations, corresponding to a frequency of about 10
−8
per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the
pyrE
gene and that duplication mutations in
pyrE
reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in
pyrE
, and another pair of deletions shared an octanucleotide at their 3′ ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the
S
.
acidocaldarius pyrE
gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.