bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of
ptsO
in Carbon-Mediated Inhibition of the
Pu
Promoter Belonging to the pWW0
Pseudomonas putida
Plasmid
Authors: Cases Ildefonso , Velázquez Francisco , de Lorenzo Vı́ctor
Published: July 2002

An investigation was made into the role of the
ptsO
gene in carbon source inhibition of the
Pu
promoter belonging to the
Pseudomonas putida
upper TOL (toluene degradation) operon.
ptsO
is coexpressed with
ptsN
, the loss of which is known to render
Pu
unresponsive to glucose. Both
ptsN
and
ptsO
, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA
Ntr
and NPr, respectively, have been mapped adjacent to the
rpoN
gene of
P. putida
. The roles of these two genes in the responses of
Pu
to glucose were monitored by
lacZ
reporter technology with a
P. putida
strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking
ptsO
,
Pu
activity seemed to be inhibited even in the absence of glucose. A functional relationship with
ptsN
was revealed by the phenotype of a double
ptsN ptsO
mutant that was equivalent to the phenotype of a mutant with a single
ptsN
disruption. Moreover, phosphorylation of the product of
ptsO
seemed to be required for C inhibition of
Pu,
since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate
ptsO
revealed the presence of two open reading frames, designated
ptsP
and
mtp
, with the potential to encode PTS type I enzymes in
P. putida.
However, neither an insertion in
ptsP
nor an insertion in
mtp
resulted in a detectable change in inhibition of
Pu
by glucose. These results indicate that some PTS proteins have regulatory functions in
P. putida
that are independent of their recognized role in sugar transport in other bacteria.
Functional Analysis of OleY
l
-Oleandrosyl 3-
O
-Methyltransferase of the Oleandomycin Biosynthetic Pathway in
Streptomyces antibioticus
Authors: Rodrı́guez Leticia , Rodrı́guez David , Olano Carlos , Braña Alfredo F. , Méndez Carmen , Salas José A.
Published: July 2002

Oleandomycin, a macrolide antibiotic produced by
Streptomyces antibioticus
, contains two sugars attached to the aglycon:
l
-oleandrose and
d
-desosamine.
oleY
codes for a methyltransferase involved in the biosynthesis of
l
-oleandrose. This gene was overexpressed in
Escherichia coli
to form inclusion bodies and in
Streptomyces lividans
, producing a soluble protein.
S. lividans
overexpressing
oleY
was used as a biotransformation host, and it converted the precursor
l
-olivosyl-erythronolide B into its 3-
O
-methylated derivative,
l
-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase:
l
-rhamnosyl- and
l
-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co
2+
. The
S. antibioticus
OleG2 glycosyltransferase is proposed to transfer
l
-olivose to the oleandolide aglycon, which is then converted into
l
-oleandrose by the OleY methyltransferase. This represents an alternative route for
l
-oleandrose biosynthesis from that in the avermectin producer
Streptomyces avermitilis
, in which
l
-oleandrose is transferred to the aglycon by a glycosyltransferase.
Disulfide Bond in
Pseudomonas aeruginosa
Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase
Authors: Liebeton Klaus , Zacharias Annette , Jaeger Karl-Erich
Published: July 2002

Pseudomonas aeruginosa
secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from
Erwinia carotovora
were expressed in
Escherichia coli
, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of
P. aeruginosa
. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.
Escherichia coli
Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation
Authors: Pichoff Sebastien , Lutkenhaus Joe
Published: July 2002

The
min
system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of
ftsZ
, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.
Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na
+
(Li
+
)/H
+
Antiporter Activity on
Escherichia coli
: Characterization of the Recovered Genes and the Corresponding Gene Products
Authors: Majernı́k Alan , Gottschalk Gerhard , Daniel Rolf
Published: July 2002

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na
+
(Li
+
)/H
+
antiporter activity on the antiporter-deficient
Escherichia coli
strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive
E. coli
clones were obtained during the initial screening of 1,480,000 recombinant
E. coli
strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li
+
-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na
+
/H
+
antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of
E. coli
K-12 containing
nhaA
,
nhaR
, and
gef
. This region is flanked by highly conserved insertion elements. The sequence identity with
E. coli
decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from
E. coli
. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of
E. coli
and enabled the antiporter-deficient
E. coli
mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na
+
/H
+
antiporter activity in everted membrane vesicles that were derived from the
E. coli
strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The

K
m

values of both antiporters for Na
+
were approximately 10-fold higher than the values for Li
+
.
Analysis of Early Promoters of the
Bacillus
Bacteriophage GA-1
Authors: Horcajadas José A. , Meijer Wilfried J. J. , Rojo Fernando , Salas Margarita
Published: July 2002

Bacteriophage GA-1, which infects
Bacillus
sp. strain G1R, is evolutionarily related to phage φ29, which infects
Bacillus subtilis
. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of φ29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.
Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed
oriT
Cleavage In Vivo
Authors: Karl Wolfgang , Bamberger Martina , Zechner Ellen L.
Published: July 2002

The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (
oriT
) in vivo was investigated. As expected, the cleavage reaction was not detected in
Escherichia coli
cells expressing
tral
and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at
oriT
in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1
oriT
constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations,
traY
was dispensable for mobilization in the presence of
traM
, but mobilization did not occur in the absence of both
traM
and
traY
. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.
The Hook Gene (
flgE
) Is Expressed from the
flgBCDEF
Operon in
Rhodobacter sphaeroides
: Study of an
flgE
Mutant
Authors: Ballado Teresa , Camarena Laura , González-Pedrajo Bertha , Silva-Herzog Eugenia , Dreyfus Georges
Published: July 2002

In this work we identified the
flgE
gene encoding the flagellar hook protein from
Rhodobacter sphaeroides
. Our results show that this gene is part of a flagellar cluster that includes the genes
flgB, flgC, flgD, flgE
, and
flgF
. Two different types of mutants in the
flgE
gene were isolated, and both showed a Fla
−
phenotype, indicating the functionality of this sequence. Complementation studies of these mutant strains suggest that
flgE
is included in a single transcriptional unit that starts in
flgB
and ends in
flgF
. In agreement with this possibility, a specific transcript of approximately 3.5 kb was identified by Northern blot. This mRNA is large enough to represent the complete
flgBCDEF
operon. FlgE showed a relatively high proline content; in particular, a region of 12 amino acids near the N terminus, in which four prolines were identified. Cells expressing a mutant FlgE protein lacking this region showed abnormal swimming behavior, and their hooks were curved. These results suggest that this region is involved in the characteristic quaternary structure of the hook of
R. sphaeroides
and also imply that a straight hook, or perhaps the rigidity associated with this feature, is important for an efficient swimming behavior in this bacterium.
The
secE
Gene of
Helicobacter pylori
Authors: Médigue Claudine , Wong Benjamin Chun-Yu , Lin Marie Chia-Mi , Bocs Stéphanie , Danchin Antoine
Published: July 2002

Despite extensive annotation by two independent teams, the
Helicobacter pylori
genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing
secE
component of the major secretion machinery of
Helicobacter pylori
.
Identification and Characterization of
pvuA
, a Gene Encoding the Ferric Vibrioferrin Receptor Protein in
Vibrio parahaemolyticus
Authors: Funahashi Tatsuya , Moriya Kaoru , Uemura Sachi , Miyoshi Shin-ichi , Shinoda Sumio , Narimatsu Shizuo , Yamamoto Shigeo
Published: July 2002

We previously reported that
Vibrio parahaemolyticus
expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of
V. parahaemolyticus
WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (
psuA
) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (
pvuA
) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by
pvuA
displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of
Morganella morganii
. Primer extension and Northern blot analyses indicated that
psuA
and
pvuA
constitute an operon which is transcribed from a Fur-repressed promoter upstream of
psuA
. The product of the
pvuA
gene and its function were confirmed by generating a
pvuA
-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream
psuA
gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.
TrwD, the Hexameric Traffic ATPase Encoded by Plasmid R388, Induces Membrane Destabilization and Hemifusion of Lipid Vesicles
Authors: Machón Cristina , Rivas Susana , Albert Armando , Goñi Félix M. , de la Cruz Fernando
Published: July 2002

TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases. It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus. In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium. It also induced extensive leakage of the vesicular aqueous contents. A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP. Structure prediction methods revealed a close similarity to
Helicobacter pylori
protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known. The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization. TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria.
Membrane Topology of theStreptococcus pneumoniaeFtsW Division Protein
Authors: Gérard Philippe , Vernet Thierry , Zapun André
Published: July 2002

The topology of FtsW fromStreptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm.
Population Genetic Structure of
Legionella pneumophila
Inferred from RNA Polymerase Gene (
rpoB
) and DotA Gene (
dotA
) Sequences
Authors: Ko Kwan Soo , Lee Hae Kyung , Park Mi-Yeoun , Park Man-Suk , Lee Keun-Hwa , Woo So-Yon , Yun Yeo-Jun , Kook Yoon-Hoh
Published: July 2002

The population structure of
Legionella pneumophila
was studied by using partial RNA polymerase gene (
rpoB
) and DotA gene (
dotA
) sequences. Trees inferred from
rpoB
sequences showed that two subspecies of
L. pneumophila
,
Legionella pneumophila
subsp.
pneumophila
and
Legionella pneumophila
subsp.
fraseri
, were clearly separated genetically. In both
rpoB
and
dotA
trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in
L. pneumophila
subsp.
pneumophila
and two of which (designated subgroups F-I and F-II) were identified in
L. pneumophila
subsp.
fraseri
. Although the relationships among subgroups were not identical, such subgrouping was congruent between the
rpoB
and
dotA
trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within
L. pneumophila
. Contrary to the phylogeny from
rpoB
and the taxonomic context, subgroups P-III and P-IV of
L. pneumophila
subsp.
pneumophila
proved to be closely related to those of
L. pneumophila
subsp.
fraseri
or showed a distinct clustering in the
dotA
tree. It can be inferred that
dotA
of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of
L. pneumophila
.
Rapid Identification of Laboratory Contamination with
Mycobacterium tuberculosis
Using Variable Number Tandem Repeat Analysis
Authors: Gascoyne-Binzi Deborah M. , Barlow Rachael E. L. , Frothingham Richard , Robinson Grant , Collyns Timothy A. , Gelletlie Ruth , Hawkey Peter M.
Published: July 2002

Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study of
Mycobacterium tuberculosis
isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.
Magnetic Immuno-PCR Assay with Inhibitor Removal for Direct Detection of
Helicobacter pylori
in Human Feces
Authors: Monteiro Lurdes , Gras Nathalie , Megraud Francis
Published: July 2002

A PCR protocol was developed to detect
Helicobacter pylori
in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47
H. pylori
-positive and 57
H. pylori
-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.
Genetic Diversity among
Mycobacterium bovis
Isolates: a Preliminary Study of Strains from Animal and Human Sources
Authors: Sales M. P. U. , Taylor G. M. , Hughes S. , Yates M. , Hewinson G. , Young D. B. , Shaw R. J.
Published: July 2002

Mycobacterium bovis
has the broadest host range of species in the
Mycobacterium tuberculosis
complex and is responsible for disease in humans and diverse animal species. We report on genotypic differences at multiple loci among 13 isolates derived from a range of human and animal infections. All isolates were classified as
M. bovis
by phenotypic analysis but could be subdivided into five distinct genotypes based on polymorphisms at the
pncA
and
oxyR
loci, the status of the RD5 deletion region, and the spoligotype pattern. These findings suggest the existence of a spectrum of strains with genotypic characteristics between those of
M. tuberculosis
and
M. bovis
.
Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity
Authors: Gomez Arley , Mve-Obiang Armand , Vray Bernard , Rudnicka Wieslawa , Shamputa Isdore C. , Portaels Françoise , Meyers Wayne M. , Fonteyne Pierre-Alain , Realini Laurence
Published: July 2002

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused byClostridium perfringensandListeria monocytogenes. Previous studies have reported multiple copies ofplcgenes homologous toPseudomonas aeruginosa plcHandplcNgenes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes ofMycobacterium tuberculosis, M. marinum, M. bovis, andM. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences ofM. kansasiiandM. marinumhomologous to the genes encoding phospholipase C fromM. tuberculosisandM. ulceranswere identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected inM. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis,andM. kansasiimycobacteria with phospholipase C activity, but not inM. fortuitum. No hemolytic activity was detected inM. smegmatis, M. gordonae, andM. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.
Specific Detection of
Pasteurella multocida
in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization
Authors: Mbuthia Paul Gichohi , Christensen Henrik , Boye Mette , Petersen Kamille Majken Dumong , Bisgaard Magne , Nyaga Phillip Njeru , Olsen John Elmerdahl
Published: July 2002

A
Pasteurella multocida
species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated
P. multocida
from other bacterial species of the families
Pasteurellaceae
and
Enterobacteriaceae
and also from divergent species of the order
Cytophagales
(except biovar 2 strains of
Pasteurella avium
and
Pasteurella canis,
which have high 16S rRNA similarity to
P. multocida
). The potential of the probe for specific identification and differentiation of
P. multocida
was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues
P. multocida
cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected
P. multocida
was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of
P. multocida
in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to
P. multocida
.
Reproducibility of
Bordetella pertussis
Genomic DNA Fragments Generated by
Xba
I Restriction and Resolved by Pulsed-Field Gel Electrophoresis
Authors: Hardwick Terri H. , Plikaytis Brian , Cassiday Pamela K. , Cage Gary , Peppler Mark S. , Shea Deborah , Boxrud David , Sanden Gary N.
Published: July 2002

The intra- and interlaboratory variabilities of the molecular size measurements of each DNA fragment contributing to three pulsed-field gel electrophoresis (PFGE) profiles were assessed, as were the reproducibilities of the entire PFGE profiles for three
Bordetella pertussis
strains. The major source of variability within a laboratory occurred between subcultures rather than within gels or between gels. Each PFGE profile was generated reproducibly and was objectively defined by the molecular sizes of its composite fragments. A strain or profile most suitable for use as an internal reference standard was identified.
Ca3 Fingerprinting of
Candida albicans
Isolates from Human Immunodeficiency Virus-Positive and Healthy Individuals Reveals a New Clade in South Africa
Authors: Blignaut Elaine , Pujol Claude , Lockhart Shawn , Joly Sophie , Soll David R.
Published: July 2002

To examine the question of strain specificity in oropharyngeal candidiasis associated with human immunodeficiency virus (HIV) infection, oral samples were collected from 1,196 HIV-positive black South Africans visiting three clinics and 249
Candida albicans
isolates were selected for DNA fingerprinting with the complex DNA fingerprinting probe Ca3. A total of 66
C. albicans
isolates from healthy black South Africans and 46 from healthy white South Africans were also DNA fingerprinted as controls. Using DENDRON software, a cluster analysis was performed and the identified groups were compared to a test set of isolates from the United States in which three genetic groups (I, II, and III) were previously identified by a variety of genetic fingerprinting methods. All of the characterized South African collections (three from HIV-positive black persons, two from healthy black persons, and one from healthy white persons) included group I, II, and III isolates. In addition, all South African collections included a fourth group (group SA) completely absent in the U.S. collection. The proportion of group SA isolates in HIV-positive and healthy black South Africans was 53% in both cases. The proportion in healthy white South Africans was 33%. In a comparison of HIV-positive patients with and without oropharyngeal symptoms of infection, the same proportions of group I, II, III, and SA isolates were obtained, indicating no shift to a particular group on infection. However, by virtue of its predominance as a commensal and in infections, group SA must be considered the most successful in South Africa. Why group SA isolates represent 53 and 33% of colonizing strains in black and white South Africans and are absent in the U.S. collection represents an interesting epidemiological question.
In Vitro Activity of Nystatin Compared with Those of Liposomal Nystatin, Amphotericin B, and Fluconazole against ClinicalCandidaIsolates
Authors: Arikan Sevtap , Ostrosky-Zeichner Luis , Lozano-Chiu Mario , Paetznick Victor , Gordon David , Wallace Tom , Rex John H.
Published: July 2002

We investigated the in vitro activity of nystatin and liposomal nystatin against 103Candidaisolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 μg/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and 1 μg/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against allCandidaspp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of 1 to 2 and 0.5 to 1 μg/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were ≥0.25 μg/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of ≥0.25 μg/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.
Multicenter Evaluation of a New Automated Fourth-Generation Human Immunodeficiency Virus Screening Assay with a Sensitive Antigen Detection Module and High Specificity
Authors: Weber Bernard , Gürtler Lutz , Thorstensson Rigmor , Michl Ulrike , Mühlbacher Annelies , Bürgisser Philippe , Villaescusa Roberto , Eiras Adolfo , Gabriel Christian , Stekel Herbert , Tanprasert Srivilai , Oota Sinenaart , Silvestre Maria-Jose , Marques Cristina , Ladeira Maria , Rabenau Holger , Berger Annemarie , Schmitt Urban , Melchior Walter
Published: July 2002

Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 antigen test, and HIV type 1 (HIV-1) RNA reverse transcriptase PCR (RT-PCR). A total of 94 seroconversion panels (
n
= 709 sera), samples from the acute phase of infection after seroconversion (
n
= 32), anti-HIV-1-positive specimens (
n
= 730) from patients in different stages of the disease, 462 subtyped samples from different geographical locations, anti-HIV-2-positive sera (
n
= 302), dilutions of cell culture supernatants (
n
= 62) from cells infected with different HIV-1 subtypes, selected performance panels from Boston Biomedica Inc., 7,579 unselected samples from blood donors, 303 unselected daily routine samples, 997 specimens from hospitalized patients, and potentially interfering samples (
n
= 1,222) were tested with Cobas Core HIV Combi EIA. The new assay showed a sensitivity comparable to that of the Abbott HIV-1 AG Monoclonal A for early detection of HIV infection in seroconversion panels. The mean time delay of Cobas Core HIV Combi EIA (last negative sample plus 1 day) in comparison to that for HIV-1 RT-PCR for 87 panels tested with both methods was 2.75 days. The diagnostic window was reduced with Cobas Core HIV Combi EIA by between 3.6 and 5.7 days from that for third-generation assays. The specificities of Cobas Core HIV Combi EIA in blood donors were 99.84 and 99.85% (after repeated testing). Overall, 30 repeatedly reactive false-positive results out of 10,031 HIV-negative samples were obtained with Cobas Core HIV Combi EIA. Our results show that a fourth-generation assay with improved specificity such as Cobas Core HIV Combi EIA is suitable for blood donor screening because of its low number of false positives and because it detects HIV p24 antigen with a sensitivity comparable to that of single-antigen assays.
Opposite Effects of Dextran Sulfate 500, the Polyene Antibiotic MS-8209, and Congo Red on Accumulation of the Protease-Resistant Isoform of PrP in the Spleens of Mice Inoculated Intraperitoneally with the Scrapie Agent
Authors: Beringue Vincent , Adjou Karim T. , Lamoury François , Maignien Thomas , Deslys Jean-Philippe , Race Richard , Dormont Dominique
Published: July 2002

The mode and the site of action of the major antiscrapie drugs have been studied by investigating their effects on the abnormal protease-resistant isoform of PrP (PrPres) and on its accumulation in mouse spleen. Day-by-day PrPres accumulation in the spleen and in other peripheral organs was first monitored to describe the early steps of scrapie pathogenesis. Three phases were identified: the detection of scrapie inoculum on the day of scrapie infection, a clearance phase, and then the peripheral accumulation of PrPres. In a second step, the effects of the polyene antibiotic MS-8209, the polyanion dextran sulfate 500 (DS500), and Congo red were assessed on these phases, after the drugs were coincubated with scrapie inoculum. Highly different mechanisms and sites of action were apparent. MS-8209 had a weak effect on the accumulation of PrPres in spleen, suggesting another site of intervention for this drug. DS500 delayed the beginning of the clearance phase but then blocked PrPres synthesis for a long period of time, probably because of its immunological effects on the spleen. Surprisingly, Congo red suppressed the clearance phase of scrapie inoculum and then increased transiently accumulation of PrPres in spleen. We showed in vitro that this effect was related to a direct enhancement of the protease resistance of PrPres by the drug.
Feline Leukemia Virus Envelope Sequences That Affect T-Cell Tropism and Syncytium Formation Are Not Part of Known Receptor-Binding Domains
Authors: Gwynn Samuel R. , Hankenson F. Claire , Lauring Adam S. , Rohn Jennifer L. , Overbaugh Julie
Published: July 2002

The envelope protein is a primary pathogenic determinant for T-cell-tropic feline leukemia virus (FeLV) variants, the best studied of which is the immunodeficiency-inducing virus, 61C. We have previously demonstrated that T-cell-tropic, cytopathic, and syncytium-inducing viruses evolve in cats infected with a relatively avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T variant, which encoded scattered single-amino-acid changes throughout the envelope as well as a 4-amino-acid insertion in the C-terminal half of the surface unit (SU) of envelope, was sufficient to confer the T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser, S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol. 72:2686–2696, 1998). In the present study, we examined the role of the 4-amino-acid insertion in determining viral replication and tropism of FeLV-81T. The 4-amino-acid insertion was found to be functionally equivalent to a 6-amino-acid insertion at an identical location in the 61C variant. However, viruses expressing a chimeric 61E/81T SU, containing the insertion together with the N terminus of 61E SU, were found to be replication defective and were impaired in the processing of the envelope precursor into the functional SU and transmembrane (TM) proteins. In approximately 10% of cultured feline T cells (3201) transfected with the 61E/81T envelope chimeras and maintained over time, replication-competent tissue culture-adapted variants were isolated. Compensatory mutations in the SU of the tissue culture-adapted viruses were identified at positions 7 and 375, and each was shown to restore envelope protein processing when combined with the C-terminal 81T insertion. Unexpectedly, these viruses displayed different phenotypes in feline T cells: the virus with a change from glutamine to proline at position 7 acquired a T-cell-tropic, cytopathic phenotype, whereas the virus with a change from valine to leucine at position 375 had slower replication kinetics and caused no cytopathic effects. Given the differences in the replication properties of these viruses, it is noteworthy that the insertion as well as the two single-amino-acid changes all occur outside of predicted FeLV receptor-binding domains.
DNA Sequence Motifs Which Direct Adeno-Associated Virus Site-Specific Integration in a Model System
Authors: Meneses Patricio , Berns Kenneth I. , Winocour Ernest
Published: July 2002

The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288–11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966–7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.
Identification of a Gammaherpesvirus Selective Chemokine Binding Protein That Inhibits Chemokine Action
Authors: van Berkel Victor , Barrett John , Tiffany H. Lee , Fremont Daved H. , Murphy Philip M. , McFadden Grant , Speck Samuel H. , Virgin Herbert W.
Published: July 2002

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of γHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1α (MIP-1α), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX3C chemokine fractalkine with high affinity (Kd= 1.6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (Kd> 1 μM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1α and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.
Type 2 Cytokines Predominate in the Human CD4+T-Lymphocyte Response to Epstein-Barr Virus Nuclear Antigen 1
Authors: Steigerwald-Mullen Patricia , Kurilla Michael G. , Braciale Thomas J.
Published: July 2002

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persistently infects 85% of the adult population worldwide. In this report, we examine the proliferative response and cytokine secretion profile of CD4+T lymphocytes from a panel of unrelated EBV-positive donors against two EBV latent antigens, EBNA1 and EBNA3C. Substantial proliferative responses by CD4+lymphocytes were demonstrated to both antigens in multiple, randomly selected donors. Surprisingly, we observed a striking and consistent difference in cytokine response to EBNA1 and EBNA3C. EBNA1-specific CD4+T lymphocytes from multiple unrelated donors preferentially produced type 2-like cytokines in response to antigenic stimulation, while the response to EBNA3C was a characteristic type 1 response. The implications of these findings for EBV persistence and the development of EBV-associated malignancies are discussed.
Measles Virus-Induced Immunosuppression In Vitro Is Independent of Complex Glycosylation of Viral Glycoproteins and of Hemifusion
Authors: Weidmann Armin , Fischer Christian , Ohgimoto Shinji , Rüth Claudia , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: July 2002

Expression of the measles virus (MV) F/H complex on the surface of viral particles, infected cells, or cells transfected to express these proteins (presenter cells [PC]) is necessary and sufficient to induce proliferative arrest in both human and rodent lymphoid cells (responder cells [RC]). This inhibition was found to occur independent of apoptosis and soluble mediators excluded by a pore size filter of 200 nm released from either PC or RC. We now show that reactive oxygen intermediates which might be released by RC or PC also do not contribute to MV-induced immunosuppression in vitro. Using an inhibitor of Golgi-resident mannosidases (deoxymannojirimycin), we found that complex glycosylation of the F and H proteins is not required for the induction of proliferative arrest of RC. As revealed by our previous studies, proteolytic cleavage of the MV F protein precursor into its F1 and F2 subunits, but not of F/H-mediated cellular fusion, was found to be required, since fusion-inhibitory peptides such as Z-d-Phe-l-Phe-Gly (Z-fFG) did not interfere with the induction of proliferative inhibition. We now show that Z-fFG inhibits cellular fusion at the stage of hemifusion by preventing lipid mixing of the outer membrane layer. These results provide strong evidence for a receptor-mediated signal elicited by the MV F/H complex which can be uncoupled from its fusogenic activity is required for the induction of proliferative arrest of human lymphocytes.
MM1, a Temperate Bacteriophage of the Type 23F Spanish/USA Multiresistant Epidemic Clone of
Streptococcus pneumoniae
: Structural Analysis of the Site-Specific Integration System
Authors: Gindreau Emmanuel , López Rubens , García Pedro
Published: July 2002

We have characterized a temperate phage (MM1) from a clinical isolate of the multiply antibiotic-resistant Spanish/American 23F
Streptococcus pneumoniae
clone (Spain
23F
-1 strain). The 40-kb double-stranded genome of MM1 has been isolated as a DNA-protein complex. The use of MM1 DNA as a probe revealed that the phage genome is integrated in the host chromosome. The host and phage attachment sites,
attB
and
attP
, respectively, have been determined. Nucleotide sequencing of the attachment sites identified a 15-bp core site (5′-TTATAATTCATCCGC-3′) that has not been found in any bacterial genome described so far. Sequence information revealed the presence of an integrase gene (
int
), which represents the first identification of an integrase in the pneumococcal system. A 1.5-kb DNA fragment embracing
attP
and the
int
gene contained all of the genetic information needed for stable integration of a nonreplicative plasmid into the
attB
site of a pneumococcal strain. This vector will facilitate the introduction of foreign genes into the pneumococcal chromosome. Interestingly, DNAs highly similar to that of MM1 have been detected in several clinical pneumococcal isolates of different capsular types, suggesting a widespread distribution of these phages in relevant pathogenic strains.
Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis
Authors: Huang Cheng , Kiyotani Katsuhiro , Fujii Yutaka , Fukuhara Noriko , Kato Atsushi , Nagai Yoshiyuki , Yoshida Tetsuya , Sakaguchi Takemasa
Published: July 2002

The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-
S
-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C
341
S and V-C
365
R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V
ΔC
, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.