bluuebunny/crossref_metadata_2025_split huggingface dataset:
l
-Threonine Export: Use of Peptides To Identify a New Translocator from
Corynebacterium glutamicum
Authors: Simic Petra , Sahm Hermann , Eggeling Lothar 
Published: July 2002

ABSTRACT

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of
l
-threonine-containing di- or tripeptides results in reduction of the growth of
Corynebacterium glutamicum
, with concomitant high intracellular accumulation of
l
-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of
thrE
. Upon
thrE
overexpression, growth is no longer sensitive to the presence of the Thr peptide, and
l
-threonine is exported at a rate of 3.8 nmol min
−1
mg of dry weight
−1
, whereas the rate of export of a
thrE
inactivation mutant is reduced to 1.1 nmol min
−1
mg of dry weight
−1
. In addition to
l
-threonine,
l-
serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by
thrE
serves to export small molecules such as
l
-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in
Mycobacterium tuberculosis
and
Streptomyces coelicolor
.
Characterization of Mutations in the
metY
-
nusA
-
infB
Operon That Suppress the Slow Growth of a Δ
rimM
Mutant
Authors: Bylund Göran O. , Lövgren J. Mattias , Wikström P. Mikael 
Published: July 2002

ABSTRACT

The RimM protein in
Escherichia coli
is associated with free 30S ribosomal subunits but not with 70S ribosomes. A Δ
rimM
mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a Δ
rimM
mutant by increasing the expression of
rbfA
. Nine of these mutations were in the
nusA
gene, which is located upstream from
rbfA
in the
metY-nusA-infB
operon; three mutations deleted the transcriptional terminator between
infB
and
rbfA
; one was an insertion of IS
2
in
infB
, creating a new promoter for
rbfA
; and one was a duplication, placing a second copy of
rbfA
downstream from a promoter for the
yhbM
gene. Two of the
nusA
mutations were identical, while another mutation (
nusA98
) was identical to a previously isolated mutation,
nusA11
, shown to decrease termination of transcription. The different
nusA
mutations were found to increase the expression of
rbfA
by increasing the read-through of two internal transcriptional terminators located just downstream from the
metY
gene and that of the internal terminator preceding
rbfA
. Induced expression of the
nusA
+
gene from a plasmid in a
nusA
+
strain decreased the read-through of the two terminators just downstream from
metY
, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the
metY-nusA-infB
operon expression is these terminators. All of the
nusA
mutations produced temperature-sensitive phenotypes of
rimM
+
strains. The
nusA
gene has previously been shown to be essential at 42°C and below 32°C. Here, we show that
nusA
is also essential at 37°C.
Novel Form of ClpB/HSP100 Protein in the Cyanobacterium
Synechococcus
Authors: Eriksson Mats-Jerry , Schelin Jenny , Miskiewicz Ewa , Clarke Adrian K. 
Published: July 2002

ABSTRACT

Synechococcus
sp. strain PCC 7942 has a second
clpB
gene that encodes a 97-kDa protein with novel features. ClpBII is the first ClpB not induced by heat shock or other stresses; it is instead an essential, constitutive protein. ClpBII is unable to complement ClpBI function for acquired thermotolerance. No truncated ClpBII version is normally produced, unlike other bacterial forms, while ectopic synthesis of a putative truncated ClpBII dramatically decreased cell viability.
Genomic Sequence and Transcriptional Analysis of a 23-Kilobase Mycobacterial Linear Plasmid: Evidence for Horizontal Transfer and Identification of Plasmid Maintenance Systems
Authors: Le Dantec Corinne , Winter Nathalie , Gicquel Brigitte , Vincent Véronique , Picardeau Mathieu 
Published: July 2002

ABSTRACT

Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from
Mycobacterium celatum
, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both
M. celatum
pCLP and the
Mycobacterium tuberculosis
chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (
par
operon) and postsegregational (
pem
operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.
The
Ralstonia eutropha
PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production
Authors: York Gregory M. , Stubbe JoAnne , Sinskey Anthony J. 
Published: July 2002

ABSTRACT

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the
Ralstonia eutropha
PhaR protein on the regulation of PhaP accumulation.
R. eutropha
strains with
phaR
,
phaC
, and/or
phaP
deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the
phaC
deletion strain accumulated no PhaP, as expected. In contrast, both the
phaR
and the
phaR phaC
deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the
R. eutropha phaR
,
phaP
, and PHA biosynthesis (
phaCAB
) genes into a heterologous system,
Escherichia coli
, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of
phaR
caused a decrease in PHA yields, and a
phaR phaP
deletion strain exhibited a more severe PHA defect than a
phaP
deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.
An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions inEscherichia coli
Authors: Brantl Sabine , Wagner E. Gerhart H. 
Published: July 2002

ABSTRACTAntisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and theinc18family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported byEscherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivolacZreporter gene assays showed that the antisense RNAs of these plasmids can inhibitlacZexpression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in anE. coliin vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g.,Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.
A Membrane-Bound Archaeal Lon Protease Displays ATP-Independent Proteolytic Activity towards Unfolded Proteins and ATP-Dependent Activity for Folded Proteins
Authors: Fukui Toshiaki , Eguchi Tomohiro , Atomi Haruyuki , Imanaka Tadayuki 
Published: July 2002

ABSTRACTIn contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain,Archaea. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon fromThermococcus kodakaraensisKOD1 (LonTk) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA+superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that LonTkwas actually a membrane-bound protein. The recombinant LonTkpossessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70°C, respectively. Unlike the enzyme fromEscherichia coli, we found that LonTkshowed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that LonTkrequired ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, LonTkdegraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in LonTkdifferent from that of its bacterial counterpart.
Amino Acid Substitution within the VP7 Protein of G2 Rotavirus Strains Associated with Failure To Serotype
Authors: Gómara Miren Iturriza , Cubitt David , Desselberger Ulrich , Gray Jim 
Published: July 2002

ABSTRACT
Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp→Asn) correlated with the change in ability to serotype these G2 strains.
Detection of Rifampin Resistance in
Mycobacterium tuberculosis
in a Single Tube with Molecular Beacons
Authors: El-Hajj Hiyam H. , Marras Salvatore A. E. , Tyagi Sanjay , Kramer Fred Russell , Alland David 
Published: July 2002

ABSTRACT

Current clinical assays for determining antibiotic susceptibility in
Mycobacterium tuberculosis
require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant
M. tuberculosis
in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene,
rpoB
. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the
rpoB
gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible
M. tuberculosis
is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148
M. tuberculosis
clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.
Case of Catheter Sepsis with
Ralstonia gilardii
in a Child with Acute Lymphoblastic Leukemia
Authors: Wauters Georges , Claeys Geert , Verschraegen Gerda , De Baere Thierry , Vandecruys Els , Van Simaey Leen , De Ganck Catharine , Vaneechoutte Mario 
Published: July 2002

ABSTRACT

Acute lymphoblastic leukemia was diagnosed in a 7-year-old girl. Two months after insertion of a central venous catheter, she developed fever and complained of headache and abdominal pain. Physical examination revealed no focus of infection. A gram-negative nonfermenting bacillus was recurrently cultured from blood. Extensive biochemical testing and 16S ribosomal DNA sequencing led to the identification of
Ralstonia gilardii
.
Comparison of Serogroup W-135 Meningococci Isolated in Sweden during a 23-Year Period and Those Associated with a Recent Hajj Pilgrimage
Authors: Mölling Paula , Bäckman Anders , Olcén Per , Fredlund Hans 
Published: July 2002

ABSTRACT
An outbreak of serogroup W-135 meningococcal disease was reported among pilgrims returning from the annual hajj (pilgrimage to Mecca) in mid-March 2000. Molecular characterization was used to investigate the similarity of the hajj-associated W-135 strains with those isolated in Sweden during a 23-year period (1978 to 2000). The same hajj-associated genosubtype, genosubtype P1.5,2,36b, has been documented in Sweden since 1979, while pulsed-field gel electrophoresis and the sulfadiazine resistance of the W-135 isolates indicated that the outbreak was probably due to a new clone of W-135 meningococci.
Laboratory Diagnosis of Human Neurocysticercosis: Double-Blind Comparison of Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Assay
Authors: Proaño-Narvaez J. V. , Meza-Lucas Antonio , Mata-Ruiz Olga , García-Jerónimo Roberto Carlos , Correa Dolores 
Published: July 2002

ABSTRACT

Neurocysticercosis is a common disease in underdeveloped countries. Its diagnosis is based on clinical, imaging (tomography or magnetic resonance), epidemiological, and laboratory data. Several methods based on the detection of antibodies against cysticerci in cerebrospinal fluid or serum have been tested. Among them, an enzyme-linked immunosorbent assay (ELISA) based on the use of a crude parasite antigen has been used by the laboratory network of cysticercosis in Mexico, which has given support to clinicians for up to 7 years. A
Taenia solium
-specific glycoprotein-based electroimmunotransfer blot (EITB) assay was reported to be highly sensitive and specific for this purpose. In order to compare both techniques, we studied 100 neurocysticercosis patients and 70 neurological noncysticercosis controls and searched for specific antibodies in paired samples of serum and cerebrospinal fluid using both techniques. We found that the EITB assay is more sensitive than the ELISA, especially when serum is being tested. Both techniques are more sensitive in cases with multiple living cysts than in cases with single cysts or calcified lesions. No global differences among cases with parasites located in different parts of the central nervous system were found. In the patients with cysts within the parenchyma, the sensitivity of the EITB assay was higher with serum than with cerebrospinal fluid. The immunodominant bands were found to be the same as those previously reported, i.e., GP-39 to -42, GP-24, and GP-13. Based on these results, we suggest the use of the EITB assay in routine diagnosis of cysticercosis for clinical cases.
Transient Fungemia Caused by an Amphotericin B-Resistant Isolate of
Candida haemulonii
Authors: Rodero Laura , Cuenca-Estrella Manuel , Córdoba Susana , Cahn Pedro , Davel Graciela , Kaufman Sara , Guelfand Liliana , Rodríguez-Tudela Juan L. 
Published: July 2002

ABSTRACT

A bloodstream infection due to
Candida haemulonii
afflicting a patient with fever and a medical history of megaloblastic anemia is reported. The clinical isolate was misidentified by the API 20C and VITEK identification systems. The results of susceptibility tests showed that the MIC of amphotericin B for
C. haemulonii
was 4 μg/ml. Additional susceptibility testing procedures based on the use of antibiotic medium 3 and Iso-Sensitest broth were performed, and killing curves were determined. Two collection strains of
C. haemulonii
were employed as controls. The three isolates exhibited resistance to amphotericin B in vitro regardless of the antifungal susceptibility testing method employed. In addition, the MICs of fluconazole for the three isolates were high. Further studies are needed in order to ascertain whether this species exhibits innate or acquired resistance to amphotericin B and other antifungal agents.
Effect of the Murine Leukemia Virus Extended Packaging Signal on the Rates and Locations of Retroviral Recombination
Authors: Anderson Jeffrey A. , Pathak Vinay K. , Hu Wei-Shau 
Published: July 2002

ABSTRACT

Reverse transcriptase (RT) switches templates frequently during DNA synthesis; the acceptor template can be the same RNA (intramolecular) or the copackaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular template switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA was not accessible to the RT. To test our hypothesis, the murine leukemia virus (MLV) extended packaging signal (Ψ
+
) containing a dimer linkage structure (DLS) was relocated from the 5′ untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in this region. It was found that the overall maximum recombination rates of vectors with Ψ
+
in the 5′ UTR or Ψ
+
between selectable markers were not drastically different. However, vectors with Ψ
+
located between selectable markers reached a plateau of recombination rate at a shorter distance. This suggested a limited enhancement of recombination by Ψ
+
. The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5′ Ψ
+
including DLS did not undergo more recombination than expected from the size of the region. These experiments indicated that although the accessibility of the copackaged RNA was important in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addition, recombinants with multiple template switches were observed at a frequency much higher than expected, indicating the presence of high negative interference in the MLV-based system. This extends our observation with the spleen necrosis virus system and suggests that high negative interference may be a common phenomenon in retroviral recombination.
Human Immunodeficiency Virus-Specific Circulating CD8 T Lymphocytes Have Down-Modulated CD3ζ and CD28, Key Signaling Molecules for T-Cell Activation
Authors: Trimble Linda A. , Shankar Premlata , Patterson Mark , Daily Johanna P. , Lieberman Judy 
Published: July 2002

ABSTRACTAlthough human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3ζ, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3ζ down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3ζ-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3ζ−. CD8 T cells with down-modulated CD3ζ also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR+CD62L−). After T-cell activation, CD3ζ-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor α-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3ζ is not reexpressed even after IL-2 exposure.
Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies
Authors: Pfeiffer Julie K. , Georgiadis Millie M. , Telesnitsky Alice 
Published: July 2002

ABSTRACT

Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functional
lacZ
gene generation from vectors in which
lacZ
was disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.