bluuebunny/crossref_metadata_2025 huggingface dataset:
In
Lactobacillus plantarum
, Carbamoyl Phosphate Is Synthesized by Two Carbamoyl-Phosphate Synthetases (CPS): Carbon Dioxide Differentiates the Arginine-Repressed from the Pyrimidine-Regulated CPS
Authors: Nicoloff Hervé , Hubert Jean-Claude , Bringel Françoise
Published: July 2002

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO
2
.
Lactobacillus plantarum
, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37–43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by
carA
(GLN) and
carB
(SYN). Transcriptional studies have demonstrated that
carB
is transcribed both monocistronically and in the
carAB
arginine-repressed operon. CP biosynthesis in
L. plantarum
was studied with three mutants (ΔCPS-P, ΔCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO
2
-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type
L. plantarum
without CO
2
enrichment may be due to the low affinity of CPS-A for its substrate CO
2
or to regulation of the CP pool by the cellular CO
2
/bicarbonate level.
Detection of mRNA Transcripts and Active Transcription in Persistent
Mycobacterium tuberculosis
Induced by Exposure to Rifampin or Pyrazinamide
Authors: Hu Yanmin , Mangan Joseph A. , Dhillon Jasvir , Sole Kath M. , Mitchison Denis A. , Butcher Philip D. , Coates Anthony R. M.
Published: July 2002

Mycobacterium tuberculosis
can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase
M. tuberculosis
treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent
M. tuberculosis
has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.
Maturation of IncP Pilin Precursors Resembles the Catalytic Dyad-Like Mechanism of Leader Peptidases
Authors: Eisenbrandt Ralf , Kalkum Markus , Lurz Rudi , Lanka Erich
Published: July 2002

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the
trbC
gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548–22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of
Escherichia coli
for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as “prepilin cyclization.” We have used site-directed mutagenesis of
trbC
and
traF
to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization–mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.
Transcription from Fusion Promoters Generated during Transposition of Transposon Tn
4652
Is Positively Affected by Integration Host Factor in
Pseudomonas putida
Authors: Teras Riho , Hõrak Rita , Kivisaar Maia
Published: July 2002

We have previously shown that both ends of the Tn
3
family transposon Tn
4652
contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn
4652
transposase gene
tnpA
in
Pseudomonas putida
(R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822–2829, 1998). Tn
4652
can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the −35 hexamer provided by the terminal inverted repeats of Tn
4652
and the −10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn
4652
, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts from
P. putida
cells allowed us to detect specific binding of
P. putida
IHF to the ends of the transposon Tn
4652
. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of
P. putida
reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in
P. putida
. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.
Expression of a New Operon from
Bacillus subtilis
,
ykzB-ykoL
, under the Control of the TnrA and PhoP-PhoR Global Regulators
Authors: Robichon Denis , Arnaud Maryvonne , Gardan Rozenn , Pragai Zoltan , O'Reilly Mary , Rapoport Georges , Débarbouillé Michel
Published: July 2002

The
ykzB
and
ykoL
genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The
ykzB
and
tnrA
genes are contiguous and transcribed divergently. Expression of
ykzB
and
ykoL
is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between
tnrA
and
ykzB
. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the
ykzB
promoter. In addition, transcription of
tnrA
depends on the presence of the TnrAB1 binding site. The
ykzB
and
ykoL
genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension.
ykoL
expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between
ykoL
and
ykzB
. Four boxes similar to the PhoP binding site are present upstream from the
ykoL
promoter. These boxes are probably recognized by PhoP∼P during the activation of transcription in phosphate limitation conditions.
Differential Distribution of Novel Restriction-Modification Systems in Clonal Lineages of
Neisseria meningitidis
Authors: Claus Heike , Friedrich Alexander , Frosch Matthias , Vogel Ulrich
Published: July 2002

Using representational difference analysis, we isolated novel meningococcal restriction-modification (R-M) systems.
Nme
BI, which is a homologue of the R-M system
Hga
I of
Pasteurella volantium
, was present in meningococci of the ET-5 complex and of lineage III.
Nme
AI was found in serogroup A, ET-37 complex, and cluster A4 meningococci.
Nme
DI was harbored by meningococci of the ET-37 complex and of cluster A4, but not by serogroup A meningococci. Two of the R-M systems,
Nme
BI and
Nme
DI, were located at homologous positions between the phenylalanyl-tRNA synthetase genes
pheS
and
pheT
, which appeared to be a preferential target for the insertion of foreign DNA in meningococci. The distribution of the three R-M systems was tested with 103 meningococcal strains comprising 49 sequence types. The vast majority of the strains had either
Nme
BI,
Nme
AI, or both
Nme
AI and
Nme
DI. Using cocultivation experiments, we could demonstrate that
Nme
BI, which was present in ET-5 complex meningococci, was responsible for a partial restriction of DNA transfer from meningococci of the ET-37 complex to meningococci of the ET-5 complex.
Identification of a
UPC2
Homolog in
Saccharomyces cerevisiae
and Its Involvement in Aerobic Sterol Uptake
Authors: Shianna Kevin V. , Dotson W. David , Tove Shirley , Parks Leo W.
Published: July 2002

Saccharomyces cerevisiae
normally will not take up sterols from the environment under aerobic conditions. A specific mutant,
upc2
-
1
, of the predicted transcriptional activator
UPC2
(YDR213w) has been recognized as a strain that allows a high level of aerobic sterol uptake. Another predicted transcriptional activator, the YLR228c gene product, is highly homologous to Upc2p. In fact, at the carboxy terminus 130 of the last 139 amino acids are similar between the two proteins. Since these proteins are very similar, the effect of mutations in the YLR228c open reading frame (ORF) was compared with like alterations in
UPC2
. First, the YLR228c ORF was insertionally inactivated and crossed with various
UPC2
constructs. Deletion of YLR228c and
UPC2
in combination resulted in nonviability, suggesting that the two proteins have some essential overlapping function. The
upc2
-
1
point mutation responsible for aerobic sterol uptake was duplicated in the homologous carboxy region of the YLR228c ORF using site-directed mutagenesis. This mutation on a high-copy vector resulted in an increase in sterol uptake compared to an isogenic wild-type strain. The combination of both point mutations resulted in the greatest level of aerobic sterol uptake. When the YLR228c point mutation was expressed from a low-copy vector there was little if any effect on sterol uptake. Gas chromatographic analysis of the nonsaponifiable fractions of the various strains showed that the major sterol for all YLR228c and
UPC2
combinations was ergosterol, the consensus yeast sterol.
Bacillus anthracis
pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species
Authors: Pannucci James , Okinaka Richard T. , Sabin Robert , Kuske Cheryl R.
Published: July 2002

The complete sequencing and annotation of the 181.7-kb
Bacillus anthracis
virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen
Bacillus
species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related
Bacillus
species, and some were detected only in
B. anthracis
isolates. Three isolates,
Bacillus cereus
D-17,
B. cereus
43881, and
Bacillus thuringiensis
33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both
B. cereus
43881 (341 kb) and
B. thuringiensis
ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.
Anaerobic Respiration Using Fe
3+
, S
0
, and H
2
in the Chemolithoautotrophic Bacterium
Acidithiobacillus ferrooxidans
Authors: Ohmura Naoya , Sasaki Kazuhiro , Matsumoto Norio , Saiki Hiroshi
Published: July 2002

The chemolithoautotrophic bacterium
Acidithiobacillus ferrooxidans
has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H
2
/O
2
under aerobic conditions but also on H
2
/Fe
3+
, H
2
/S
0
, or S
0
/Fe
3+
under anaerobic conditions. Anaerobic respiration using Fe
3+
or S
0
as an electron acceptor and H
2
or S
0
as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe
3+
induced the bacterium to synthesize significant amounts of a
c-
type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe
3+
. Respiration based on reduction of Fe
3+
coupled to oxidation of a
c-
type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.
Distinct Roles of P
II
-Like Signal Transmitter Proteins and
amtB
in Regulation of
nif
Gene Expression, Nitrogenase Activity, and Posttranslational Modification of NifH in
Azoarcus
sp. Strain BH72
Authors: Martin Dietmar E. , Reinhold-Hurek Barbara
Published: July 2002

P
II
-like signal transmitter proteins, found in
Bacteria
,
Archaea
, and plants, are known to mediate control of carbon and nitrogen assimilation. They indirectly regulate the activity of key metabolic enzymes and transcription factors by protein-protein interactions with signal transduction proteins. Many
Proteobacteria
harbor two paralogous P
II
-like proteins, GlnB and GlnK, whereas a novel third P
II
paralogue (GlnY) was recently identified in
Azoarcus
sp. strain BH72, a diazotrophic endophyte of grasses. In the present study, evidence was obtained that the P
II
-like proteins have distinct roles in mediating nitrogen and oxygen control of
nif
gene transcription and nitrogenase activity. Full repression of
nif
gene transcription in the presence of a combined nitrogen source or high oxygen concentrations was observed in wild-type and
glnB
and
glnK
knockout mutants, revealing that GlnB and GlnK can complement each other in mediating the repression. In contrast, in a
glnBK
double mutant strain in the presence of only GlnY,
nif
gene transcription was still detectable, albeit at a lower level, on nitrate or 20% oxygen. As another level of control, nitrogenase activity was regulated by at least three types of mechanisms in strain BH72: covalent modification of dinitrogenase reductase (NifH), probably by ADP-ribosylation, and two other, unknown means. Functional inactivation upon ammonium addition (switch-off) required the putative high-affinity ammonium transporter AmtB and GlnK, but not GlnB or GlnY. Functional inactivation in response to anaerobiosis did not depend on AmtB, GlnK, or GlnB. In contrast, covalent modification of NifH required both GlnB and GlnK and AmtB as response to ammonium addition, whereas it required either GlnB or GlnK and not AmtB when cells were shifted to anaerobiosis. In a
glnBK
double mutant expressing only GlnY, NifH modification was completely abolished, further revealing functional differences between the three P
II
paralogues.
Transmission Dynamics and Molecular Characterization of
Mycobacterium tuberculosis
Isolates with Low Copy Numbers of IS
6110
Authors: Soini Hanna , Pan Xi , Teeter Larry , Musser James M. , Graviss Edward A.
Published: July 2002

Population-based analysis of
Mycobacterium tuberculosis
transmission in Houston, Tex., over 5 years identified 377 patients infected with an isolate containing one to four copies of IS
6110
. The isolates were analyzed by spoligotyping and assigned to one of three major genetic groups based on nucleotide polymorphisms in codons
katG
463 and
gyrA
95. Prospectively obtained patient interviews were reviewed to assess epidemiologic links between apparently clustered patients. A total of 13 groups of isolates with the same IS
6110
profile were identified, representing 326 of the 377 patients (86.5%; range 2 to 113 patients). In contrast, 28 groups of isolates containing 334 patients (88.6%) had the same spoligotype (range, 2 to 143 patients). Combination of IS
6110
profile and spoligotype data identified 31 clusters with 300 patients (79.6%; range, 2 to 82 patients). All 377 isolates belonged to major genetic group 1 (77 patients) or genetic group 2 (300 patients); no major genetic group 3 isolates were identified. Among the 228 patients interviewed, 33 patients (14.5%) were directly linked to another patient in the same cluster. Possible epidemiologic links were also found among 11 patients. Moreover, many clusters consisted of individuals with the same ethnicity. In conclusion, we confirmed that IS
6110
profiling and spoligotyping together provide enhanced molecular discrimination of
M. tuberculosis
isolates with low copy numbers of IS
6110
. Identification of epidemiologic links among some of the patients verified that the combination of these two methods reliably indexes tuberculosis transmission.
Rapid Identification of Virulent Type I Strains of the Protozoan Pathogen
Toxoplasma gondii
by PCR-Restriction Fragment Length Polymorphism Analysis at the
B1
Gene
Authors: Grigg Michael E. , Boothroyd John C.
Published: July 2002

Sequence analysis at the 35-fold-repetitive
B1
locus identified three restriction sites capable of discriminating type I (mouse-virulent) from type II or III (mouse-avirulent) strains of
Toxoplasma gondii. B1
PCR-restriction fragment length polymorphism analysis of 8 type I, 17 type II, and 8 type III strains confirms the specificity of the assay. It should now be possible to ask whether strain genotype affects the severity and type of clinical disease in humans.
Quantification of Proviral Load of Human Immunodeficiency Virus Type 2 Subtypes A and B Using Real-Time PCR
Authors: Damond Florence , Descamps Diane , Farfara Isabelle , Telles Jean Noël , Puyeo Sophie , Campa Pauline , Leprêtre Annie , Matheron Sophie , Brun-Vezinet Françoise , Simon François
Published: July 2002

We have developed and evaluated a new method to quantify human immunodeficiency virus type 2 (HIV-2) proviral DNA based on LightCycler real-time PCR. The assay has a detection limit of 5 copies/10
5
peripheral blood mononuclear cells (PBMC) and is insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are both recognized and quantified. The intra- and interassay coefficients of variation range from 16 to 40% for high provirus concentrations (5 × 10
5
copies) and from 41 to 39% for low concentrations (5 copies). We used this method to compare the proviral DNA load and viral RNA load in plasma with clinical and immunological status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B in 12). The proviral load (median, 201 copies/10
5
PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate with the CD4
+
cell count categories and were as follows for CD4
+
cell counts of >400, 200 to 400, and <200 cells/mm
3
, respectively: 121 copies/10
5
PBMC (
n
= 8; range, <5 to 712 copies/10
5
PBMC); 114 copies/10
5
PBMC (
n
= 9; range, <5 to 1,907 copies/10
5
PBMC); and 285 copies/10
5
PBMC (
n
= 12; range, 53 to 2,524 copies/10
5
PBMC). Proviral load did not correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered to replicate less efficiently than HIV-1, these high proviral loads might be explained by the proliferation of infected cells.
Viral Load of Human Herpesvirus 8 in Peripheral Blood of Human Immunodeficiency Virus-Infected Patients with Kaposi's Sarcoma
Authors: Tedeschi Rosamaria , Enbom Malin , Bidoli Ettore , Linde Annika , De Paoli Paolo , Dillner Joakim
Published: July 2002

Viral load is an important marker of activity of viral diseases for a number of viruses. We wished to evaluate whether the viral load of human herpesvirus 8 (HHV-8) in peripheral blood was a consistent feature of Kaposi's sarcoma (KS) patients and whether the viral load correlated with human immunodeficiency virus (HIV) RNA levels, CD4 counts, and/or the HHV-8 seroreactivity. Fifty-four consecutive plasma samples from 14 patients with KS were evaluated for HHV-8 viral load by quantitative real-time PCR. Samples were analyzed at the start of highly active antiretroviral therapy (HAART) and at different intervals during treatments. The median HHV-8 DNA load before HAART treatment was 8,998 (ranging from 170 to 40,100) copies/ml and 12,270 (ranging from 40 to 142,575) copies/ml during HAART. There were both increasing and decreasing trends. There was an association between HHV-8 DNA and HIV RNA viral loads (odds ratio [OR] = 5.40; 95% confidence interval [95% CI], 1.54 to 18.98) and between HHV-8 viral load and CD4 cell counts (OR = 7.24; 95% CI, 1.30 to 40.35). High HHV-8 viral load was also correlated with the titers of antibodies to the lytic HHV-8 antigen detected with immunofluorescence (
P
< 0.01), but not with antibodies to the latent HHV-8 antigen. In conclusion, we found that HHV-8 viremia in KS is associated with HIV viral load, CD4 cell counts, and lytic HHV-8 serological reactivity. HHV-8 viral load monitored by real time PCR might be useful for determination HHV-8 viral load during the follow-up of KS patients.
Molecular Investigation of the Aum Shinrikyo Anthrax Release in Kameido, Japan
Authors: Keim Paul , Smith Kimothy L. , Keys Christine , Takahashi Hiroshi , Kurata Takeshi , Kaufmann Arnold
Published: July 2002

In 1993, the Aum Shinrikyo cult aerosolized
Bacillus anthracis
spores over Kameido, Japan. Spore samples were obtained from the release site, cultured, and characterized by molecular genetic typing. The isolates were consistent with strain Sterne 34F2, which is used in Japan for animal prophylaxis against anthrax.
Phenotypic and Genotypic Approaches to Characterization of Isolates of
Neisseria meningitidis
from Patients and Their Close Family Contacts
Authors: Tzanakaki G. , Urwin R. , Musilek M. , Kriz P. , Kremastinou J. , Pangalis A. , Blackwell C. C. , Maiden M. C. J.
Published: July 2002

Characterization of isolates of
Neisseria meningitidis
obtained from patients with meningococcal disease or from pharyngeal swabs of asymptomatic carriers can be achieved by several approaches which provide different levels of discrimination. A total of 45 gram negative, oxidase-positive diplococcus strains isolated from 15 individuals with meningococcal disease and 30 of their family contacts were examined by three approaches: serological typing, multilocus enzyme electrophoresis (MLEE), and multilocus sequence typing (MLST). For 10 of the 15 patient and contact groups, all of the isolates were confirmed as meningococci, and the bacteria obtained from the patients and contacts, including their mother or principal caregiver in the case of children, were indistinguishable by all three methods. In the remaining five groups the isolates from the patients were distinct from those recovered from the contacts, and in three examples, in two separate groups, the contacts were shown by MLST to be carrying strains of
Neisseria lactamica
. The data obtained from the three techniques were consistent, although complete serological typing was possible for only a minority of isolates. Both MLEE and MLST established the genetic relationships of the isolates and identified members of known hypervirulent lineages, but MLST was faster than MLEE and had the additional advantages that it could be performed on noninfective material distributed by mail and that the results from different laboratories could be compared via the internet (
http://mlst.zoo.ox.ac.uk
).
Effects of Storage and Type of Blood Collection Tubes on Hepatitis C Virus Level in Whole Blood Samples
Authors: Kessler Harald H. , Stelzl Evelyn , Raggam Reinhard B. , Haas Josef , Kirchmeir Franz , Hegenbarth Karin , Daghofer Elisabeth , Santner Brigitte I. , Marth Egon , Stauber Rudolf E.
Published: July 2002

In this study, we compared serum hepatitis C virus (HCV) RNA concentrations with HCV RNA concentrations in whole blood collection tubes, including two different types of EDTA tubes and nucleic acid stabilization tubes (NASTs). We also investigated the impact of a processing delay on HCV RNA concentration in these tubes. In NASTs, the mean HCV RNA concentration was comparable to the mean serum HCV RNA concentration at “date zero.” In EDTA tubes, mean baseline HCV RNA concentrations were higher. Storage at room temperature up to 96 h did not result in a decline of HCV RNA concentration in any of the whole blood collection tubes. In NASTs, HCV RNA concentrations remained stable during the whole study period, whereas a significant increase of HCV RNA was observed in both types of EDTA tubes at 96 h compared to date zero. We concluded that HCV RNA remains stable in NASTs at room temperature for at least 96 h, allowing greater flexibility in sample collection and transport.
Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of
Mycoplasma agalactiae
Authors: Fleury Bénédicte , Bergonier Dominique , Berthelot Xavier , Schlatter Yvonne , Frey Joachim , Vilei Edy M.
Published: July 2002

The gene for a 30-kDa immunodominant antigen, P30, of
Mycoplasma agalactiae
was cloned from type strain PG2 and expressed in
Escherichia coli
. P30 is encoded on a monocistronic operon determined by two −10 boxes and a possible −35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of
M. agalactiae
PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to
M. agalactiae
. Furthermore, PCR amplification with specific primers for
p30
and Southern blot analysis revealed the presence of the gene in all
M. agalactiae
strains tested and its absence in the other mycoplasma species. Among 27 strains of
M. agalactiae
studied
,
20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The
p30
gene was sequenced in 15 strains of
M. agalactiae
, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both −10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with
M. agalactiae
revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.
Atypical,
fljB
-Negative
Salmonella enterica
subsp.
enterica
Strain of Serovar 4,5,12:i:− Appears To Be a Monophasic Variant of Serovar Typhimurium
Authors: Echeita M. Aurora , Herrera Silvia , Usera Miguel A.
Published: July 2002

An
fljB
-negative, multidrug-resistant
Salmonella enterica
serovar 4,5,12:i:− phage type DT U302 strain (resistant to ampicillin, chloramphenicol, sulfonamide, gentamicin, streptomycin, tetracycline, and sulfamethoxazole-trimethoprim) emerged and spread in Spain in 1997. Sequences specific for
Salmonella
serovar Typhimurium and phage type DT 104 and U302 were present in this atypical
Salmonella
strain, suggesting that it is a monophasic
Salmonella
serovar Typhimurium variant.
International Surveillance of Bloodstream Infections Due to
Candida
Species: Frequency of Occurrence and In Vitro Susceptibilities to Fluconazole, Ravuconazole, and Voriconazole of Isolates Collected from 1997 through 1999 in the SENTRY Antimicrobial Surveillance Program
Authors: Pfaller M. A. , Diekema D. J. , Jones R. N. , Sader H. S. , Fluit A. C. , Hollis R. J. , Messer S. A. ,   The SENTRY Participant Group
Published: July 2002

A surveillance program (SENTRY) of bloodstream infections (BSI) in the United States, Canada, Latin America, and Europe from 1997 through 1999 detected 1,184 episodes of candidemia in 71 medical centers (32 in the United States, 23 in Europe, 9 in Latin America, and 7 in Canada). Overall, 55% of the yeast BSIs were due to
Candida albicans
, followed by
Candida glabrata
and
Candida parapsilosis
(15%),
Candida tropicalis
(9%), and miscellaneous
Candida
spp. (6%). In the United States, 45% of candidemias were due to non-
C. albicans
species.
C. glabrata
(21%) was the most common non-
C. albicans
species in the United States, and the proportion of non-
C. albicans
BSIs was highest in Latin America (55%).
C. albicans
accounted for 60% of BSI in Canada and 58% in Europe.
C. parapsilosis
was the most common non-
C. albicans
species in Latin America (25%), Canada (16%), and Europe (17%). Isolates of
C. albicans, C. parapsilosis
, and
C. tropicalis
were all highly susceptible to fluconazole (97 to 100% at ≤8 μg/ml). Likewise, 97 to 100% of these species were inhibited by ≤1 μg/ml of ravuconazole (concentration at which 50% were inhibited [MIC
50
], 0.007 to 0.03 μg/ml) or voriconazole (MIC
50
, 0.007 to 0.06 μg/ml). Both ravuconazole and voriconazole were significantly more active than fluconazole against
C. glabrata
(MIC
90
s of 0.5 to 1.0 μg/ml versus 16 to 32 μg/ml, respectively). A trend of increased susceptibility of
C. glabrata
to fluconazole was noted over the three-year period. The percentage of
C. glabrata
isolates susceptible to fluconazole increased from 48% in 1997 to 84% in 1999, and MIC
50
s decreased from 16 to 4 μg/ml. A similar trend was documented in both the Americas (57 to 84% susceptible) and Europe (22 to 80% susceptible). Some geographic differences in susceptibility to triazole were observed with Canadian isolates generally more susceptible than isolates from the United States and Europe. These observations suggest susceptibility patterns and trends among yeast isolates from BSI and raise additional questions that can be answered only by continued surveillance and clinical investigations of the type reported here (SENTRY Program).
Evaluation of a New Combined Antigen and Antibody Human Immunodeficiency Virus Screening Assay, VIDAS HIV DUO Ultra
Authors: Weber Bernard , Berger Annemarie , Rabenau Holger , Doerr Hans Wilhelm
Published: July 2002

Combined antigen and antibody screening (fourth-generation) assays reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis by 4 days, on average, in comparison to antibody-only (third generation) enzyme immunoassays (EIAs). The aim of the present study was to assess whether the new VIDAS HIV DUO Ultra (Biomérieux, Marcy-l'Etoile, France) showed an improved sensitivity and specificity in comparison to licensed fourth-generation assays. A total of 16 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 257 potentially cross-reactive serum samples were tested with VIDAS DUO HIV Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen HIV 1/2, version 2 (third-generation EIA), and Genetic Systems HIV-1 Ag EIA (p24 antigen assay). VIDAS HIV DUO Ultra showed a comparable sensitivity to the single p24 antigen assay in seroconversion panels and a dilution series of virus lysates. The diagnostic window was reduced with VIDAS HIV DUO Ultra by 3.82 days, on average, in comparison with the fourth-generation assay with the lowest sensitivity of the antigen detection module. HIV-1 infection was detected 5.88 days earlier than with third-generation EIA. The mean time delay between reverse transcription-PCR and VIDAS HIV DUO Ultra was only 2.31 days. The specificity of fourth-generation assays after retesting ranged between 98.1 and 100%. In conclusion, VIDAS HIV DUO Ultra can replace single-antigen screening for laboratory diagnosis and screening of HIV infection in blood donors. There was no evidence for a second diagnostic window due to impaired sensitivity of the antibody detection module of all the fourth-generation EIAs evaluated in the present study. The specificity after initial and/or repeated testing of VIDAS HIV DUO Ultra was equivalent to that of a third-generation assay.
Epidemiologic Genotyping of Methicillin-Resistant
Staphylococcus aureus
by Pulsed-Field Gel Electrophoresis at a University Hospital and Comparison with Antibiotyping and Protein A and Coagulase Gene Polymorphisms
Authors: Montesinos I. , Salido E. , Delgado T. , Cuervo M. , Sierra A.
Published: July 2002

A total of 124 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates were ascertained at the University Hospital of the Canary Islands between January 1997 and April 2000. Genotyping included pulsed-field gel electrophoresis (PFGE) (
Sma
I digestion) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for the coagulase (
coa
) and protein A (
spa
) genes. Antibiotic resistance was the main phenotypic marker correlated with genotyping results. Three main PFGE types were detected: A (with 12 subtypes), B (with 2 subtypes), and C. PFGE type A1 was the most commonly found (61% of isolates) and the one responsible for all the epidemic outbreaks. Other genetics markers used (
coa
and
spa
RFLPs) were significantly correlated with the PFGE types detected (
P
< 0.001). These PCR-RFLP assays were useful as molecular markers for a quick, preliminary study of MRSA outbreaks.
Prolonged Dominance of Clonally Restricted CD4+T Cells in Macaques Infected with Simian Immunodeficiency Viruses
Authors: Chen Zheng W. , Shen Yun , Kou Zhongchen , Ibegbu Chris , Zhou Dejiang , Shen Ling , Morrison Paul , Bogle Christine , McClure Harold M. , Nahmias Andre J. , Sehgal Prahbat K. , Letvin Norman L.
Published: July 2002

The repertoire of functional CD4+T lymphocytes in human immunodeficiency virus type 1-infected individuals remains poorly understood. To explore this issue, we have examined the clonality of CD4+T cells in simian immunodeficiency virus (SIV)-infected macaques by assessing T-cell receptor complementarity-determining region 3 (CDR3) profiles and sequences. A dominance of CD4+T cells expressing particular CDR3 sequences was identified within certain Vβ-expressing peripheral blood lymphocyte subpopulations in the infected monkeys. Studies were then done to explore whether these dominant CD4+T cells represented expanded antigen-specific cell subpopulations or residual cells remaining in the course of virus-induced CD4+T-cell depletion. Sequence analysis revealed that these selected CDR3-bearing CD4+T-cell clones emerged soon after infection and dominated the CD4+T-cell repertoire for up to 14 months. Moreover, inoculation of chronically infected macaques with autologous SIV-infected cell lines to transiently increase plasma viral loads in the monkeys resulted in the dominance of these selected CDR3-bearing CD4+T cells. Both the temporal association of the detection of these clonal cell populations with infection and the dominance of these cell populations following superinfection with SIV suggest that these cells may be SIV specific. Finally, the inoculation of staphylococcal enterotoxin B superantigen into SIV-infected macaques uncovered a polyclonal background underlying the few dominant CDR3-bearing CD4+T cells, demonstrating that expandable polyclonal CD4+T-cell subpopulations persist in these animals. These results support the notions that a chronic AIDS virus infection can induce clonal expansion, in addition to depletion of CD4+T cells, and that some of these clones may be SIV specific.
Adeno-Associated Virus Type 2 Rep78 Induces Apoptosis through Caspase Activation Independently of p53
Authors: Schmidt Michael , Afione Sandra , Kotin Robert M.
Published: July 2002

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G
1
and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.
In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90
Authors: Hu Jianming , Anselmo Dana
Published: July 2002

Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.
Characterization of Gammaherpesvirus 68 Gene 50 Transcription
Authors: Liu Shaofan , Pavlova Iglika V. , Virgin Herbert W. , Speck Samuel H.
Published: July 2002

Gene 50 is the only immediate-early gene that appears to be conserved among the characterized gammaherpesviruses. It has recently been demonstrated for the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) that ectopic expression of the gene 50-encoded product in some latently infected cell lines can lead to the induction of virus replication, indicating that gene 50 is likely to play a pivotal role in regulating gammaherpesvirus reactivation. Here we demonstrate that the murine gammaherpesvirus 68 (γHV68) gene 50 is an immediate-early gene and that transcription of γHV68 gene 50 leads to the production of both spliced and unspliced forms of the gene 50 transcript. Splicing of the transcript near the 5′ end serves to extend the gene 50 open reading frame, as has been observed for the gene 50 transcripts encoded by KSHV and herpesvirus saimiri (Whitehouse et al., J. Virol. 71:2550–2554, 1997; Lukac et al., Virology 252:304–312, 1998; Sun et al., Proc. Natl. Acad. Sci. USA 95:10866–10871, 1998). Reverse transcription-PCR analyses, coupled with S1 nuclease protection assays, provided evidence that gene 50 transcripts initiate at several sites within the region from bp 66468 to 66502 in the γHV68 genome. Functional characterization of the region upstream of the putative gene 50 transcription initiation site demonstrated orientation-dependent promoter activity and identified a 110-bp region (bp 66442 to 66552) encoding the putative gene 50 promoter. Finally, we demonstrate that the γHV68 gene 50 can transactivate the γHV68 gene 57 promoter, a known early gene target of the gene 50-encoded transactivator in other gammaherpesviruses. These studies show that the γHV68 gene 50 shares several important molecular similarities with the gene 50 homologs in other gammaherpesviruses and thus provides an impetus for future studies analyzing the role of the γHV68 gene 50-encoded protein in acute virus replication and reactivation from latency in vivo.
Detection of Hepatitis B Virus Infection in Wild-Born Chimpanzees (
Pan troglodytes verus
): Phylogenetic Relationships with Human and Other Primate Genotypes
Authors: MacDonald D. M. , Holmes E. C. , Lewis J. C. M. , Simmonds P.
Published: July 2002

Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (
Pan troglodytes
subsp.
verus
) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3,182 nucleotides in length, and the surface gene sequence predicted the existence of
a
,
d
, and
w
serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652–654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.
Regulation of Human Immunodeficiency Virus Type 1 Replication in Human T Lymphocytes by Nitric Oxide
Authors: Jiménez Jose Luis , González-Nicolás Josefa , Alvarez Susana , Fresno Manuel , Muñoz-Fernández M. Angeles
Published: July 2002

Addition of nitric oxide (NO) donors to mitogen-activated human immunodeficiency virus type 1 (HIV-1)-infected peripheral blood mononuclear cultures produced a significant increase in virus replication, and this effect was not associated with a change in cell proliferation. This effect was only observed with T-tropic X4 or X4R5 virus but not with R5 virus. Moreover, HIV-1 replication in mitogen-stimulated cultures was partially prevented by the specific inhibitors of the inducible nitric oxide synthase (iNOS). NO donors also enhanced HIV-1 infection of the human T-cell lines, Jurkat and MT-2. We have also observed that NO leads to an enhancement of HIV-1 replication in resting human T cells transfected with a plasmid carrying the entire HIV-1 genome and activated with phorbol ester plus ionomycin. Thus, in those cultures NO donors strongly potentiated HIV-1 replication in a dose-dependent manner, up to levels comparable to those with tumor necrosis factor alpha (TNF-α) stimulation. Furthermore, iNOS inhibitors decreased HIV-1 replication in HIV-1-transfected T cells to levels similar to those obtained with neutralizing anti-TNF-α antibodies. Moreover, HIV-1 replication induced iNOS and TNF-α transcription in T cells and T-cell lines. Interestingly, NO donors also stimulated long terminal repeat (LTR)-driven transcription whereas iNOS inhibitors partially blocked TNF-α-induced LTR transcription. Therefore, our results suggest that NO is involved in HIV-1 replication, especially that induced by TNF-α.
Mapping the Determinants of the CCR5 Amino-Terminal Sulfopeptide Interaction with Soluble Human Immunodeficiency Virus Type 1 gp120-CD4 Complexes
Authors: Cormier Emmanuel G. , Tran Diep N. H. , Yukhayeva Liyana , Olson William C. , Dragic Tatjana
Published: July 2002

CD4 and CCR5 mediate fusion and entry of R5 human immunodeficiency virus type 1 (HIV-1) strains. Sulfotyrosine and other negatively charged residues in the CCR5 amino-terminal domain (Nt) are crucial for gp120 binding and viral entry. We previously showed that a soluble gp120-CD4 complex specifically binds to a peptide corresponding to CCR5 Nt residues 2 to 18, with sulfotyrosines in positions 10 and 14. This sulfopeptide also inhibits soluble gp120-CD4 binding to cell surface CCR5 as well as infection by an R5 virus. Here we show that residues 10 to 18 constitute the minimal domain of the CCR5 Nt that is able to specifically interact with soluble gp120-CD4 complexes. In addition to sulfotyrosines in positions 10 and 14, negatively charged residues in positions 11 and 18 participate in this interaction. Furthermore, the CCR5 Nt binds to a CD4-induced surface on gp120 that is composed of conserved residues in the V3 loop stem and the C4 domain. Binding of gp120 to cell surface CCR5 is further influenced by residues in the crown of the V3 loop, C1, C2, and C3. Our data suggest that gp120 docking to CCR5 is a multistep process involving several independent regions of the envelope glycoprotein and the coreceptor.