bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Glutamine Synthetase in Nitrogen Metabolite Repression in
Aspergillus nidulans
Authors: Margelis Soula , D'Souza Cletus , Small Anna J. , Hynes Michael J. , Adams Thomas H. , Davis Meryl A.
Published: July 2002

Glutamine synthetase (GS), EC
6.3.1.2
, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the
Aspergillus nidulans glnA
gene encoding GS and have shown that
glnA
encodes a highly expressed but not highly regulated mRNA. Inactivation of
glnA
results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional
glnA
gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic
Anabaena glnA
gene from the
A. nidulans alcA
promoter allowed full complementation of the
A. nidulans glnA
Δ mutation. However, the
A. nidulans fluG
gene, which encodes a protein with similarity to prokaryotic GS, did not replace
A. nidulans glnA
function when similarly expressed. Our studies with the
glnA
Δ mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.
Heat-Shock-Induced Proteins from
Myxococcus xanthus
Authors: Otani Mieko , Tabata Junko , Ueki Toshiyuki , Sano Keiji , Inouye Sumiko
Published: July 2002

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from
Myxococcus xanthus
were established. Using these conditions, we analyzed protein patterns of heat-shocked
M. xanthus
cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase α subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in
M. xanthus
(R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357–5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218–224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase α subunit are heat shock inducible.
Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C
1
and C
2
Compounds in the Methylotroph
Methylobacterium extorquens
AM1
Authors: Korotkova Natalia , Lidstrom Mary E.
Published: July 2002

Several DNA regions containing genes involved in poly-β-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph
Methylobacterium extorquens
AM1. Genes involved in PHB biosynthesis include those encoding β-ketothiolase (
phaA
), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (
phaB
), and PHB synthase (
phaC
).
phaA
and
phaB
are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as
orf3. phaC
was unlinked to
phaA
and
phaB
. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (
depA
and
depB
). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding β-hydroxybutyrate dehydrogenase (
hbd
) was identified. Insertion mutations were introduced into
depA, depB, phaA, phaB, phaC
, and
hbd
and also in a gene predicted to encode crotonase (
croA
), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in
depA, depB, hbd
, and
croA
all produced normal levels of PHB, and the only growth phenotype observed was the inability of the
hbd
mutant to grow on β-hydroxybutyrate. However, the
phaA, phaB
, and
phaC
mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C
1
and C
2
compounds and, for
phaB
, these defects were rescued by glyoxylate supplementation. These results suggest that β-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and
Streptomyces
spp.
Identification and Characterization of the
dif
Site from
Bacillus subtilis
Authors: Sciochetti Stephen A. , Piggot Patrick J. , Blakely Garry W.
Published: July 2002

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in
Escherichia coli
is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as
dif
(Ec
dif
). In
Bacillus subtilis
the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bs
dif
), lying 6° counterclockwise from the
B. subtilis
terminus of replication (172°), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bs
dif
in vivo recombination did not require the
B. subtilis
FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the
B. subtilis
SPβ-bacteriophage, and in particular its
yopP
gene product, appears to strongly modulate the extent of the partitioning defects seen in
codV
strains and, to a lesser extent, those seen in
ripX
and
dif
strains.
Analysis of Lipooligosaccharide Biosynthesis in the
Neisseriaceae
Authors: Arking Dan , Tong Yanhong , Stein Daniel C.
Published: July 2002

Neisserial lipooligosaccharide (LOS) contains three oligosaccharide chains, termed the α, β, and γ chains. We used Southern hybridization experiments on DNA isolated from various
Neisseria
spp. to determine if strains considered to be nonpathogenic possessed DNA sequences homologous with genes involved in the biosynthesis of these oligosaccharide chains. The presence or absence of specific genes was compared to the LOS profiles expressed by each strain, as characterized by their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and their reactivities with various LOS-specific monoclonal antibodies. A great deal of heterogeneity was seen with respect to the presence of genes encoding glycosyltransferases in
Neisseria
. All pathogenic species were found to possess DNA sequences homologous with the
lgt
gene cluster, a group of genes needed for the synthesis of the α chain. Some of these genes were also found to be present in strains considered to be nonpathogenic, such as
Neisseria lactamica, N. subflava
, and
N. sicca
. Some nonpathogenic
Neisseria
spp. were able to express high-molecular-mass LOS structures, even though they lacked the DNA sequences homologous with
rfaF
, a gene whose product must act before gonococcal and meningococcal LOS can be elongated. Using a PCR amplification strategy, in combination with DNA sequencing, we demonstrated that
N. subflava
44 possessed
lgtA, lgtB
, and
lgtE
genes. The predicted amino acid sequence encoded by each of these genes suggested that they encoded functional proteins; however, structural analysis of LOS isolated from this strain indicated that the bulk of its LOS was not modified by these gene products. This suggests the existence of an additional regulatory mechanism that is responsible for the limited expression of these genes in this strain.
Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium
Azoarcus evansii
Authors: Mohamed Magdy El-Said , Zaar Annette , Ebenau-Jehle Christa , Fuchs Georg
Published: July 2002

The aerobic metabolism of benzoate in the proteobacterium
Azoarcus evansii
was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.72 mol flavin adenine dinucleotide (FAD), 10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile sulfur per mol of native protein, depending on the method of protein determination. This benzoate-induced enzyme catalyzed a benzoyl-CoA-, FAD-, and O
2
-dependent NADPH oxidation surprisingly without hydroxylation of the aromatic ring; however, H
2
O
2
was formed. The gene (
boxA
, for benzoate oxidation) coding for this protein was cloned and sequenced. It coded for a protein of 46 kDa with two amino acid consensus sequences for two [4Fe-4S] centers at the N terminus. The deduced amino acid sequence showed homology with subunits of ferredoxin-NADP reductase, nitric oxide synthase, NADPH-cytochrome P450 reductase, and phenol hydroxylase. Upstream of the
boxA
gene, another gene,
boxB
, encoding a protein of 55 kDa was found. The
boxB
gene exhibited homology to open reading frames in various other bacteria which code for components of a putative aerobic phenylacetyl-CoA oxidizing system. The
boxB
gene product was one of at least five proteins induced when
A. evansii
was grown on benzoate.
Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49
Authors: Desplats Carine , Dez Christophe , Tétart Françoise , Eleaume Heïdy , Krisch H. M.
Published: July 2002

RB49 is a virulent bacteriophage that infects
Escherichia coli
. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the ≈170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.
Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA
Authors: Garcia Miguel , Pimentel Madalena , Moniz-Pereira José
Published: July 2002

A mycobacteriophage Ms6 strong promoter region (Plys) was isolated by using transcriptional fusions with thelacZreporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin ofLactococcus lactisr1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter regionPlysdrives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments usinglacZfusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.
CcaR Is an Autoregulatory Protein That Binds to the
ccaR
and
cefD-cmcI
Promoters of the Cephamycin C-Clavulanic Acid Cluster in
Streptomyces clavuligerus
Authors: Santamarta Irene , Rodríguez-García Antonio , Pérez-Redondo Rosario , Martín Juan F. , Liras Paloma
Published: July 2002

The putative regulatory CcaR protein, which is encoded in the β-lactam supercluster of
Streptomyces clavuligerus
, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in
Escherichia coli
, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from
S. clavuligerus
was able to bind DNA fragments containing the promoter regions of the
ccaR
gene itself and the bidirectional
cefD-cmcI
promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including
lat
,
blp
,
claR
,
car-cyp
, and the unlinked
argR
gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies.
ccaR
and the bidirectional
cefD-cmcI
promoter region were fused to the
xylE
reporter gene and expressed in
Streptomyces lividans
and
S. clavuligerus
. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the
ccaR
promoter region lacking its coding sequence in a high-copy-number plasmid in
S. clavuligerus
ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the
cefD-cmcI
bidirectional promoter region.
Chloromethane-Induced Genes Define a Third C
1
Utilization Pathway in
Methylobacterium chloromethanicum
CM4
Authors: Studer Alex , McAnulla Craig , Büchele Rainer , Leisinger Thomas , Vuilleumier Stéphane
Published: July 2002

Methylobacterium chloromethanicum
CM4 is an aerobic α-proteobacterium capable of growth with chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B
12
-mediated transfer of the methyl group of chloromethane to tetrahydrofolate. Three genes located near
cmuA
and
cmuB
, designated
metF
,
folD
and
purU
and encoding homologs of methylene tetrahydrofolate (methylene-H
4
folate) reductase, methylene-H
4
folate dehydrogenase-methenyl-H
4
folate cyclohydrolase and formyl-H
4
folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in
Methylobacterium extorquens
AM1, which is unable to grow with chloromethane. Studies with transcriptional
xylE
fusions demonstrated the chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the
Methylobacterium
promoters described thus far. As shown previously for
purU
, inactivation of the
metF
gene resulted in a CM4 mutant unable to grow with chloromethane. Methylene-H
4
folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the
metF
mutant. Taken together, these data provide evidence that
M. chloromethanicum
CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane.
Transcriptional Activation of the
Rhodobacter sphaeroides
Cytochrome
c
2
Gene P2 Promoter by the Response Regulator PrrA
Authors: Comolli James C. , Carl Audrey J. , Hall Christine , Donohue Timothy
Published: July 2002

Anoxygenic photosynthetic growth of
Rhodobacter sphaeroides
, a member of the α subclass of the class
Proteobacteria
, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome
c
2
gene (
cycA
) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of
cycA
P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate
cycA
P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the
prrA
gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.
Early Detection of Acute Rhinovirus Infections by a Rapid Reverse Transcription-PCR Assay
Authors: Steininger Christoph , Aberle Stephan W. , Popow-Kraupp Theresia
Published: July 2002

The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5′-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.
Detection of a New
Mycobacterium
Species in Wild Striped Bass in the Chesapeake Bay
Authors: Heckert Robert A. , Elankumaran S. , Milani Alessandra , Baya Ana
Published: July 2002

Investigation into recent declines in striped bass health in the Chesapeake Bay in Maryland resulted in the isolation of a putative new species of
Mycobacterium
. This isolate was obtained from fish showing skin ulcers and internal granulomas in various organs. The isolate was slow growing at 28°C; was nonchromogenic; showed no activities of nitrate reduction, catalase activity, Tween 80 hydrolysis, tellurite reduction, or arylsulfatase reduction; grew best at low salt concentrations; and was urease and pyrazinamidase positive. By PCR a unique insertional sequence was identified which matched nothing in any database. Analysis of the nearly complete 16S rRNA gene sequence also indicated a unique sequence which had 87.7% sequence homology to
Mycobacterium ulcerans
, 87.6% homology to
Mycobacterium tuberculosis
, and 85.9% homology to
Mycobacterium marinum
. Phylogenetic analysis placed the organism close to the tuberculosis complex. These data support the conclusion that the isolate probably represents a new mycobacterial species.
Three-Year Assessment of Methicillin-Resistant
Staphylococcus aureus
Clones in Latin America from 1996 to 1998
Authors: Aires de Sousa Marta , Miragaia Maria , Santos Sanches Ilda , Ávila Sofia , Adamson Inger , Casagrande Silvana T. , Brandileone Maria Cristina C. , Palacio Rosario , Dell'Acqua Lillia , Hortal Maria , Camou Teresa , Rossi Alicia , Velazquez-Meza Maria Elena , Echaniz-Aviles Gabriela , Solorzano-Santos Fortino , Heitmann Ingrid , de Lencastre Hermı́nia
Published: July 2002

Four hundred ninety-nine methicillin-resistant
Staphylococcus aureus
(MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America—Argentina, Brazil, Chile, Uruguay and Mexico—were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of
Cla
I restriction digests with the
mecA
- and Tn
554
-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal
Sma
I digests (
Cla
I-
mecA
::
Cla
I-Tn
554
::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::κ::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E′::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.
Antibody Response to Shiga Toxins Stx2 and Stx1 in Children with Enteropathic Hemolytic-Uremic Syndrome
Authors: Ludwig Kerstin , Karmali Mohamed A. , Sarkim Volkan , Bobrowski Christoph , Petric Martin , Karch Helge , Müller-Wiefel Dirk E. ,   Arbeitsgemeinschaft für Pädiatrische Nephrologie
Published: July 2002

A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing
Escherichia coli
[STEC] infection) and 110 age-matched controls was established by using a chemiluminescence detection system. Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes. The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10). Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (
P
= 0.0325). For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90%. Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative. Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease. It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.
High-Frequency Recovery of Quinupristin-Dalfopristin-Resistant
Enterococcus faecium
Isolates from the Poultry Production Environment
Authors: Hayes Joshua R. , McIntosh Angela C. , Qaiyumi Sadaf , Johnson Judith A. , English Linda L. , Carr Lewis E. , Wagner David D. , Joseph Sam W.
Published: July 2002

The occurrence of resistance to the streptogramin quinupristin-dalfopristin in
Enterococcus faecium
isolates from chickens on the Eastern Seaboard, was evaluated. Quinupristin-dalfopristin resistance was found in 51 to 78% of
E. faecium
isolates from the food production environment. The high level of resistance in this organism suggests that this reservoir of resistance may compromise the therapeutic potential of quinupristin-dalfopristin.
Antimicrobial Susceptibilities of
Helicobacter pylori
Isolates under Microaerophilic Atmospheres Established by Two Different Methods
Authors: Kobayashi Intetsu , Muraoka Hiroe , Saika Takeshi , Nishida Minoru , Fujioka Toshio , Nasu Masaru
Published: July 2002

The MICs of clarithromycin, amoxicillin, and metronidazole for 150
Helicobacter pylori
isolates were determined using the AnaeroPack system and were compared with those determined using a microaerophilic incubator. The MICs of clarithromycin, amoxicillin, and metronidazole determined under both microaerophilic atmospheres were mostly within one twofold dilution for 146 (97.3%), 150 (100%), and 149 (99.3%) of the isolates, respectively.
16S/23S rRNA Intergenic Spacer Regions for Phylogenetic Analysis, Identification, and Subtyping of
Bartonella
Species
Authors: Houpikian Pierre , Raoult Didier
Published: July 2002

Species of the genus
Bartonella
are currently recognized in growing numbers and are involved in an increasing variety of human diseases, mainly trench fever, Carrion's disease, bacillary angiomatosis, endocarditis, cat scratch disease, neuroretinitis, and asymptomatic bacteremia. Such a wide spectrum of infections makes it necessary to develop species and strain identification tools in order to perform phylogenetic and epidemiological studies. The 16S/23S rRNA intergenic spacer region (ITS) was sequenced for four previously untested species,
B. vinsonii
subsp.
arupensis, B. tribocorum, B. alsatica
, and
B. koehlerae
, as well as for 28 human isolates of
B. quintana
(most of them from French homeless people), six human or cat isolates of
B. henselae
, five cat isolates of
B. clarridgeiae
, and four human isolates of
B. bacilliformis
. Phylogenetic trees inferred from full ITS sequences of the 14 recognized
Bartonella
species using parsimony and distance methods revealed high statistical support, as bootstrap values were higher than those observed with other tested genes. Five well-supported lineages were identified within the genus and the proposed phylogenetic organization was consistent with that resulting from protein-encoding gene sequence comparisons. The ITS-derived phylogeny appears, therefore, to be a useful tool for investigating the evolutionary relationships of
Bartonella
species and to identify
Bartonella
species. Further, partial ITS amplification and sequencing offers a sensitive means of intraspecies differentiation of
B. henselae, B. clarridgeiae
, and
B. bacilliformis
isolates, as each strain had a specific sequence. The usefulness of this approach in epidemiological investigations should be highlighted. Among
B. quintana
strains, however, the genetic heterogenity was low, as only three ITS genotypes were identified. It was nevertheless sufficient to show that the
B. quintana
population infecting homeless people in France was not clonal.
Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis
Authors: Yazdankhah Siamak P. , Sørum Henning , Larsen Hans Jørgen S. , Gogstad Geir
Published: July 2002

A rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 × 106CFU/ml forStaphylococcus aureusand 1 × 106CFU/ml forEscherichia coliin spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.
Colorimetric Assay for Antifungal Susceptibility Testing of
Aspergillus
Species
Authors: Meletiadis Joseph , Mouton Johan W. , Meis Jacques F. G. M. , Bouman Bianca A. , Donnelly J. Peter , Verweij Paul E. ,   EUROFUNG Network
Published: July 2002

A colorimetric assay for antifungal susceptibility testing of
Aspergillus
species (
Aspergillus fumigatus
,
Aspergillus flavus
,
Aspergillus terreus
,
Aspergillus nidulans
, and
Aspergillus ustus
) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 μg of XTT/ml with 25 μM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10
2
CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.
Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by Using TaqMan Technology
Authors: Zhao Yuqi , Yu Min , Miller Johann W. , Chen Mingzhong , Bremer Eric G. , Kabat William , Yogev Ram
Published: July 2002

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.
Endotoxin Stimulates Liver Macrophages To Release Mediators That Inhibit an Early Step in Hepadnavirus Replication
Authors: Klöcker Uta , Schultz Ursula , Schaller Heinz , Protzer Ulrike
Published: July 2002

Hepadnaviruses are known to be sensitive to various extracellular mediators. Therefore, bacterial endotoxin, which induces the secretion of proinflammatory mediators in the liver, was studied for its effect on hepadnavirus infection in vitro using the duck hepatitis B virus (DHBV) model. In initial experiments, endotoxin was shown to inhibit DHBV replication in primary duck hepatocyte cultures prepared by standard collagenase perfusion. As a primary endotoxin target, hepatic nonparenchymal cells (NPC) contaminating primary hepatocyte cultures, and among these probably macrophages (Kupffer cells), were identified to secrete polypeptide mediators into the cell culture medium. When added during DHBV infection, these mediators elicited the principal antiviral effect in a dose-dependent fashion. On the molecular level, they inhibited accumulation of viral proteins as well as amplification of the nuclear extrachromosomal DHBV DNA templates. In hepatocytes with an established DHBV infection, DHBV protein and progeny virus production was inhibited while the levels of established nuclear DHBV DNA templates and viral transcripts remained unaffected. Finally, in hepatocytes infected with a replication-deficient recombinant DHBV-green fluorescent protein (GFP) virus, the endotoxin-induced mediators markedly reduced GFP expression from chimeric DHBV-GFP transcripts, indicating that the major effect is at a level of translation of viral RNAs. Taken together, the data obtained demonstrate that antiviral mediators, and among these the cytokines alpha interferon (IFN-α) and IFN-γ, are released from hepatic NPC, most probably liver macrophages, upon endotoxin stimulation; furthermore, these mediators act at a posttranscriptional step of hepadnavirus replication.
Enhancement of Primary and Secondary Cellular Immune Responses against Human Immunodeficiency Virus Type 1 Gag by Using DNA Expression Vectors That Target Gag Antigen to the Secretory Pathway
Authors: Qiu Jian-Tai , Liu Bindong , Tian Chunjuan , Pavlakis George N. , Yu Xiao-Fang
Published: July 2002

In this study, we have investigated the influence of antigen targeting after DNA vaccination upon the induction of cellular immune responses against human immunodeficiency virus type 1 (HIV-1) Gag. In addition to the standard version of HIV-1 Gag, we constructed Gag expression vectors that encode a secreted (Sc-Gag) and a cytoplasmic (Cy-Gag) Gag molecule. Although all three HIV-1 Gag expression vectors induced detectable humoral and cellular immune responses, after intramuscular injection the DNA vector encoding the Sc-Gag generated the highest primary cytotoxic T-lymphocyte (CTL) and T-helper responses. Mice immunized with one of the HIV-1 Gag DNA vectors (but not with the control vector pcDNA3.1) developed a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag, and this response persisted for 125 days. The magnitude of the protection correlated with the levels of Gag-specific ex vivo CTL activity and the number of CD8+T cells producing gamma interferon. The DNA vector encoding the Sc-Gag induced higher levels of protection and greater secondary CTL responses than did the DNA vector encoding Cy-Gag.
The C-Terminal Proline-Rich Tail of Human Immunodeficiency Virus Type 2 Vpx Is Necessary for Nuclear Localization of the Viral Preintegration Complex in Nondividing Cells
Authors: Pancio Heather A. , Vander Heyden Nancy , Ratner Lee
Published: July 2002

Human immunodeficiency virus type 2 (HIV-2), like other lentiviruses, is capable of infecting nondividing T cells and macrophages. The present work shows that in HIV-2-infected cells, Vpx is necessary for efficient nuclear import of the preintegration complex. In agreement with this finding, the subcellular localization of a GFP-Vpx fusion protein was found to be predominantly nuclear. However, deletion of the proline-rich C-terminal 11 residues of Vpx resulted in a shift of the fusion protein to the cytoplasm. Furthermore, the same deletion in the context of the provirus resulted in a decrease in nuclear import of the preintegration complex and attenuated replication in macrophages.
A New Mutant Class, Made by Targeted Mutagenesis, of Phage PRD1 Reveals That Protein P5 Connects the Receptor Binding Protein to the Vertex
Authors: Bamford Jaana K. H. , Bamford Dennis H.
Published: July 2002

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene
V
revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5
−
particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.
Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1
Authors: Pierson Theodore , Hoffman Trevor L. , Blankson Joel , Finzi Diana , Chadwick Karen , Margolick Joseph B. , Buck Christopher , Siliciano Janet D. , Doms Robert W. , Siliciano Robert F.
Published: July 2002

Latently infected resting CD4+T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1envgenes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.
CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells
Authors: Zhang Yi , Chirmule Narendra , Gao Guang-ping , Wilson James
Published: July 2002

Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding β-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L−/−) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a β-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZtransduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.
Yeast Three-Hybrid Screening of Rous Sarcoma Virus Mutants with Randomly Mutagenized Minimal Packaging Signals Reveals Regions Important for Gag Interactions
Authors: Lee Eun-Gyung , Linial Maxine L.
Published: July 2002

We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define
cis
-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MΨ) made using PCR amplification after cotransformation with GagΔPR protein into yeast cells. Colonies with low β-galactosidase activity were analyzed to locate mutations in MΨ sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.
Human Cytomegalovirus Latency-Associated Protein pORF94 Is Dispensable for Productive and Latent Infection
Authors: White Kirsten Lofgren , Slobedman Barry , Mocarski Edward S.
Published: July 2002

Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the
ie1/ie2
region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated
ie1/ie2
region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.
High-Level Resistance to 3′-Azido-3′-Deoxythimidine due to a Deletion in the Reverse Transcriptase Gene of Human Immunodeficiency Virus Type 1
Authors: Imamichi Tomozumi , Sinha Tanima , Imamichi Hiromi , Zhang Yi-Ming , Metcalf Julie A. , Falloon Judith , Lane H. Clifford
Published: July 2002

A variant of human immunodeficiency virus type 1 (HIV-1) possessing a deletion in the reverse transcriptase (RT) gene at codon 67 was identified in a patient who had failed combination antiretroviral therapy. This deletion initially emerged under the selective pressure of combination therapy with 3′-azido-3′-deoxythymidine (AZT) plus 2′,3′-dideoxyinosine. It has persisted for more than 3 years in association with the accumulation of a variety of other well-described drug resistance mutations and an uncharacterized mutation at RT codon 69 (T69G). Phenotypic studies demonstrated that the codon 67 deletion by itself had little effect on AZT sensitivity. However, in the context of the T69G mutation and three other mutations known to be associated with AZT resistance (K70R, T215F, and K219Q), this deletion led to a increase in AZT resistance from 8.5-fold to 445-fold. A further increase in resistance (up to 1,813-fold) was observed when two mutations associated with nonnucleoside RT inhibitor resistance (K103N and L74I) were added to the deletion T69G K70R T215F K219Q construct. Hence, these results establish that a deletion at RT codon 67 may be selected for in the presence of antiretroviral therapy and may lead to high-level resistance to AZT.