bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant
Pseudomonas putida
DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks
Authors: Segura Ana , Duque Estrella , Hurtado Ana , Ramos Juan L. 
Published: July 2002

ABSTRACT

Pseudomonas putida
DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn
5-′phoA
-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn
5-′phoA
-Km was inserted within the
fliP
gene, which is part of the
fliLMNOPQRflhBA
cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the
P. putida fliP
mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An
flhB
knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the
fliL
gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in
P. putida
DOT-T1E to the periplasm.
Computation-Directed Identification of OxyR DNA Binding Sites in
Escherichia coli
Authors: Zheng Ming , Wang Xunde , Doan Bernard , Lewis Karen A. , Schneider Thomas D. , Storz Gisela 
Published: July 2002

ABSTRACT

A computational search was carried out to identify additional targets for the
Escherichia coli
OxyR transcription factor. This approach predicted OxyR binding sites upstream of
dsbG
, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of
fhuF
, encoding a protein required for iron uptake; and within
yfdI
. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the
dsbG
gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed
ahpC
gene. Although the new binding site was near
dsbG
, Northern blotting and primer extension assays showed that OxyR binding to the
dsbG
-proximal site led to the induction of a second
ahpCF
transcript, while OxyR binding to the
ahpCF
-proximal site leads to the induction of both
dsbG
and
ahpC
transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of the
fhuF
gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the
fhuF
promoter. Interestingly, the two OxyR sites in the
fhuF
promoter overlapped with two regions bound by the Fur repressor. Expression analysis revealed that
fhuF
was repressed by hydrogen peroxide in an OxyR-dependent manner. Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of
yfdI
. These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the
E. coli
genome.
Genetic and Physiologic Analysis of the
groE
Operon and Role of the HrcA Repressor in Stress Gene Regulation and Acid Tolerance in
Streptococcus mutans
Authors: Lemos José A. C. , Chen Yi-Ywan M. , Burne Robert A. 
Published: July 2002

ABSTRACT

Our working hypothesis is that the major molecular chaperones DnaK and GroE play central roles in the ability of oral bacteria to cope with the rapid and frequent stresses encountered in oral biofilms, such as acidification and nutrient limitation. Previously, our laboratory partially characterized the
dnaK
operon of
Streptococcus mutans
(
hrcA
-
grpE
-
dnaK
) and demonstrated that
dnaK
is up-regulated in response to acid shock and sustained acidification (G. C. Jayaraman, J. E. Penders, and R. A. Burne, Mol. Microbiol. 25:329–341, 1997). Here, we show that the
groESL
genes of
S. mutans
constitute an operon that is expressed from a stress-inducible ς
A
-type promoter located immediately upstream of a CIRCE element. GroEL protein and mRNA levels were elevated in cells exposed to a variety of stresses, including acid shock. A nonpolar insertion into
hrcA
was created and used to demonstrate that HrcA negatively regulates the expression of the
groEL
and
dnaK
operons. The SM11 mutant, which had constitutively high levels of GroESL and roughly 50% of the DnaK protein found in the wild-type strain, was more sensitive to acid killing and could not lower the pH as effectively as the parent. The acid-sensitive phenotype of SM11 was, at least in part, attributable to lower F
1
F
0
-ATPase activity. A minimum of 10 proteins, in addition to GroES-EL, were found to be up-regulated in SM11. The data clearly indicate that HrcA plays a key role in the regulation of chaperone expression in
S. mutans
and that changes in the levels of the chaperones profoundly influence acid tolerance.
Hemin Reconstitutes Proton Extrusion in an H

+

-ATPase-Negative Mutant of
Lactococcus lactis
Authors: Blank Lars M. , Koebmann Brian J. , Michelsen Ole , Nielsen Lars K. , Jensen Peter R. 
Published: July 2002

ABSTRACT

H
+
-ATPase is considered essential for growth of
Lactococcus lactis.
However, media containing hemin restored the aerobic growth of an H
+
-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown
L. lactis
cells are capable of coupling NADH oxidation to proton translocation.
Conversion of Pipecolic Acid into Lysine in
Penicillium chrysogenum
Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the
lys7
Gene Encoding Saccharopine Reductase
Authors: Naranjo Leopoldo , de Valmaseda Eva Martin , Bañuelos Oscar , Lopez Pilar , Riaño Jorge , Casqueiro Javier , Martin Juan F. 
Published: July 2002

ABSTRACT

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In
Penicillium chrysogenum
pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the
lys1
,
lys2
, or
lys3
genes allowing a slow growth of these auxotrophs. We have isolated two
P. chrysogenum
mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with α-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named
lys7
) revealed that it encoded a protein with high similarity to the saccharopine reductase from
Neurospora crassa
,
Magnaporthe grisea
,
Saccharomyces cerevisiae
, and
Schizosaccharomyces pombe
. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that
lys7
encodes a functional saccharopine reductase. Our data suggest that in
P. chrysogenum
the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.
Nitrogen-Regulated Group 2 Sigma Factor from
Synechocystis
sp. Strain PCC 6803 Involved in Survival under Nitrogen Stress
Authors: Muro-Pastor Alicia Marı́a , Herrero Antonia , Flores Enrique 
Published: July 2002

ABSTRACT

The expression of
sll1689
, an open reading frame from the cyanobacterium
Synechocystis
sp. strain PCC 6803 putatively encoding a member of the ς
70
family of sigma factors, appears to be regulated by the nitrogen control transcription factor NtcA. Disruption of
sll1689
had no noticeable effect on exponential growth, identifying its product as a member of the group 2, nonessential class of ς
70
-like sigma factors; however, this disruption decreased the viability of the cells after long periods of nitrogen starvation. We have named this gene
rpoD2
-
V
. The expression of
glnN
, encoding a type III glutamine synthetase, was impaired in strains bearing an inactivated copy of the
rpoD2
-
V
gene.
Comparative Specificities of Two Evolutionarily Divergent Hydrolases Involved in Microbial Degradation of Polychlorinated Biphenyls
Authors: Seah Stephen Y. K. , Labbé Geneviève , Kaschabek Stefan R. , Reifenrath Frank , Reineke Walter , Eltis Lindsay D. 
Published: July 2002

ABSTRACT

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by
Burkholderia
sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701–15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of
Rhodococcus globerulus
P6, BphD
P6
, was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD
P6
hydrolyzed HOPDA with a
k
cat
/

K
m

of 1.62 (± 0.03) × 10
7
M
−1
s
−1
(100 mM phosphate [pH 7.5], 25°C), which is within 70% of that of BphD
LB400
. BphD
P6
was also similar to BphD
LB400
in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD
P6
more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD
LB400
. These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.
Dissimilatory Sulfite Reductase (Desulfoviridin) of the Taurine-Degrading, Non-Sulfate-Reducing Bacterium
Bilophila wadsworthia
RZATAU Contains a Fused DsrB-DsrD Subunit
Authors: Laue Heike , Friedrich Michael , Ruff Jürgen , Cook Alasdair M. 
Published: July 2002

ABSTRACT

A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium
Bilophila wadsworthia
RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as an α
2
β
2
γ

n

(
n
≥ 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the α and β subunits (
dsrA
and
dsrB
, respectively), which probably constitute one operon.
dsrA
and
dsrB
encode polypeptides of 49 (α) and 54 kDa (β) which show significant similarities to the homologous subunits of other DSRs. The
dsrB
gene product of
B. wadsworthia
is apparently a fusion protein of
dsrB
and
dsrD
. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in
B. wadsworthia
. A phylogenetic analysis using the available Dsr sequences revealed that
B. wadsworthia
grouped with its closest 16S rDNA relative
Desulfovibrio desulfuricans
Essex 6.
The
miaA
Mutator Phenotype of
Escherichia coli
K-12 Requires Recombination Functions
Authors: Zhao Jingyong , Leung Hon-Chiu Eastwood , Winkler Malcolm E. 
Published: July 2002

ABSTRACT

miaA
mutants, which contain A-37 instead of the ms
2
i
6
A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC→TA transversion. We show that the
miaA
mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.
Global Genomic Analysis of AlgU (σ
E
)-Dependent Promoters (Sigmulon) in
Pseudomonas aeruginosa
and Implications for Inflammatory Processes in Cystic Fibrosis
Authors: Firoved Aaron M. , Boucher J. Cliff , Deretic Vojo 
Published: July 2002

ABSTRACT

The conversion of
Pseudomonas aeruginosa
to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF). A major pathway of conversion to mucoidy in clinical strains of
P. aeruginosa
is dependent upon activation of the alternative sigma factor AlgU (
P. aeruginosa
σ
E
). Here we initiated studies of AlgU-dependent global expression patterns in
P. aeruginosa
in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy. Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (σ
E
) promoter sites on the
P. aeruginosa
chromosome. Each candidate promoter was individually tested by reverse transcription and mRNA 5′-end mapping using RNA isolated from
algU
+
and
algU
::Tc
r
mutant cells. A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped. Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes. Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes,
lptA
and
lptB
, had proinflammatory activity. In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung. Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given σ factor (collectively termed here sigmulon) using conventional molecular tools. In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity. This link may be of significance for infections and inflammatory processes in CF.
Further Evidence that a Cell Wall Precursor [C
55
-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction
Authors: Ruzin Alexey , Severin Anatoly , Ritacco Frank , Tabei Keiko , Singh Guy , Bradford Patricia A. , Siegel Marshall M. , Projan Steven J. , Shlaes David M. 
Published: July 2002

ABSTRACT

Previous studies suggested that a Gly-containing branch of cell wall precursor [C
55
-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in
Staphylococcus aureus.
To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of
Streptomyces
spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.
Functional Measurement of Hepatitis C Virus Core-Specific CD8+T-Cell Responses in the Livers or Peripheral Blood of Patients by Using Autologous Peripheral Blood Mononuclear Cells as Targets or Stimulators
Authors: Fang Shih-Hua , Chiang Bor-Luen , Wu Mei-Hua , Iba Hideo , Lai Ming-Yang , Yang Pei-Ming , Chen Ding-Shinn , Hwang Lih-Hwa 
Published: July 2002

ABSTRACTAs is widely recognized, CD8+cytotoxic T lymphocytes (CTLs) play a crucial role in hepatitis C virus (HCV) infection, both in pathogenesis of liver injury and in clearing the virus. CTL studies with HCV-infected patients have been difficult because of the relatively low frequency of CTL precursors in the peripheral blood and because the targeted epitopes vary depending on the human leukocyte antigen (HLA) types of the individuals. This study attempts to overcome these problems by assessing the feasibility of using autologous peripheral blood mononuclear cells (PBMCs) expressing viral antigens as stimulators or targets in order to monitor the CTL responses. Primary PBMCs were transduced using a retroviral vector pseudotyped with a vesicular stomatitis virus G glycoprotein expressing the HCV core gene. Additionally, the vector-transduced PBMCs were used as targets of CTL assays to measure the HCV core-specific CTL activities from the liver-infiltrating lymphocytes of six different HLA-type patients with chronic HCV infection. The core-expressing PBMCs also served as stimulators, allowing us to measure core-specific CD8+T-cell responses by intracellular gamma interferon staining of the peripheral blood of hepatitis C patients who had received treatment with alpha interferon plus ribavirin. This approach provides an efficient means of measuring antigen-specific CTL responses without HLA constraints.
Rotavirus Genotypes P[4]G9, P[6]G9, and P[8]G9 in Hospitalized Children with Acute Gastroenteritis in Rio de Janeiro, Brazil
Authors: Araújo I. T. , Ferreira M. S. R. , Fialho A. M. , Assis R. M. , Cruz C. M. , Rocha M. , Leite J. P. G. 
Published: July 2002

ABSTRACT
Fifty-three rotavirus-positive fecal specimens from children with diarrhea admitted to a Rio de Janeiro, Brazil, children's hospital between January 1997 and December 1998 were characterized for P and G types by using reverse transcription-PCR. Genotype P[4]G2 accounted for 21% of isolates, while uncommon genotypes P[8]G9, P[6]G9, and P[4]G9 accounted for 13% of the isolates.
LightCycler Multiplex PCR for the Laboratory Diagnosis of Common Viral Infections of the Central Nervous System
Authors: Read Steven J. , Mitchell Joanne L. , Fink Colin G. 
Published: July 2002

ABSTRACT
A conventional multiplex PCR assay that detects herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus, and enteroviruses for the diagnosis of central nervous system infections was modified to be performed using the LightCycler system. The sensitivity of detection of each of the viruses using the LightCycler assay was compared to that of the conventional assay using external quality assessment material. The assays had equivalent sensitivities, but the LightCycler assay was more rapid, reduced the risk of contamination, and used an amplicon detection format that demonstrated greater discrimination than a gel electrophoresis method.
Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples from Mothers with Primary Infection
Authors: Gouarin S. , Gault E. , Vabret A. , Cointe D. , Rozenberg F. , Grangeot-Keros L. , Barjot P. , Garbarg-Chenon A. , Lebon P. , Freymuth F. 
Published: July 2002

ABSTRACT

A real-time PCR assay was developed to quantify human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) samples collected from 30 pregnant women with primary HCMV infection as detected either from HCMV-immunoglobulin G (IgG) seroconversion or by the presence of HCMV-specific IgG and IgM associated with a low IgG avidity. Clinical information available for each case included ultrasonographic examination and fetal or newborn outcome. HCMV infection of fetuses or newborns was confirmed for the 30 studied cases. AF samples were subdivided into three groups. In group A (
n
= 13), fetuses presented major ultrasound abnormalities, and pregnancy was terminated. In group B (
n
= 13), fetuses had normal ultrasound findings, the pregnancy went to term, and the newborns were asymptomatic at birth. In group C (
n
= 4), fetuses had no or minor ultrasonographic signs, and pregnancy was terminated. The HCMV DNA load values in AF samples were significantly higher in group A (median, 2.8 × 10
5
genome equivalents [GE]/ml) than in group B (median, 8 × 10
3
GE/ml) (
P
= 0.014). Our findings suggest that HCMV load level in AF samples correlates with fetal clinical outcome but might also be dependent on other factors, such as the gestational age at the time of AF sampling and the time elapsed since maternal infection.
Prevalence and Genetic Characterization of Toxin A Variant Strains of
Clostridium difficile
among Adults and Children with Diarrhea in France
Authors: Barbut Frédéric , Lalande Valérie , Burghoffer Béatrice , Thien Huong Vu , Grimprel Emmanuel , Petit Jean-Claude 
Published: July 2002

ABSTRACT

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of
Clostridium difficile
have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (
tcdA
) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with
C. difficile
-associated diarrhea in France. The repeating segment of the
tcdA
gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol.
36:
2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire
tcdA
gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11,
C. difficile
variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other
tcdA
variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of
C. difficile
-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion
of C. difficile
-associated diarrhea but a negative immunoassay for toxin A.