bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Lateral Transfers of Dissimilatory Sulfite Reductase Genes between Major Lineages of Sulfate-Reducing Prokaryotes
Authors: Klein Michael , Friedrich Michael , Roger Andrew J. , Hugenholtz Philip , Fishbain Susan , Abicht Heike , Blackall Linda L. , Stahl David A. , Wagner Michael
Published: July 2002

A large fragment of the dissimilatory sulfite reductase genes (
dsrAB
) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the
dsrAB
gene homologs of the sulfite reducer
Desulfitobacterium dehalogenans
was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several
Desulfotomaculum
species (low G+C gram-positive division) and two members of the genus
Thermodesulfobacterium
(a separate bacterial division) were monophyletic with δ-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that
dsrAB
genes from ancestors of as-yet-unrecognized sulfate reducers within the δ-
Proteobacteria
were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of
dsrAB
genes. Evidence for a
dsrAB
lateral gene transfer event also was found within the δ-
Proteobacteria,
affecting
Desulfobacula toluolica
. The root of the
dsr
tree was inferred to be within the
Thermodesulfovibrio
lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the
dsrAB
genes in
Archaeoglobus
species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of
dsrAB
genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.
SseBCD Proteins Are Secreted by the Type III Secretion System of
Salmonella
Pathogenicity Island 2 and Function as a Translocon
Authors: Nikolaus Thomas , Deiwick Jörg , Rappl Catherine , Freeman Jeremy A. , Schröder Werner , Miller Samuel I. , Hensel Michael
Published: July 2002

The type III secretion system encoded by
Salmonella
pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of
Salmonella enterica
. This system is induced by intracellular
Salmonella
and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg
2+
or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular
Salmonella
.
TspO as a Modulator of the Repressor/Antirepressor (PpsR/AppA) Regulatory System in
Rhodobacter sphaeroides
2.4.1
Authors: Zeng Xiaohua , Kaplan Samuel
Published: July 2002

The TspO outer membrane protein of
Rhodobacter sphaeroides
has been shown to be involved in controlling the transcription of a number of genes which encode enzymes involved in photopigment biosynthesis and the
puc
operon. The display of regulated genes appears identical to those genes encompassing the PpsR/AppA repressor/antirepressor regulon, although the effect of TspO is modest relative to that of PpsR/AppA. To directly address the hypothesis that TspO is effective through the PpsR/AppA system, we constructed mutant strains with mutations in both
tspO
and
appA
. In all cases, the phenotypes examined resembled those of the
appA
lesion by itself, leading us to conclude that TspO works through or modulates the PpsR/AppA system and acts upstream of the site of action of these regulatory proteins. In earlier publications, we had suggested that TspO is involved in the efflux of a certain intermediate(s) of the porphyrin biosynthesis pathway and that transcriptional regulation of target gene expression could be explained by the accumulation of a coactivator of AppA function. Although the data reported here do not precisely identify this coactivator, they lend support to this hypothesis. We discuss the importance of this form of gene control as the result of the recent extension of the TspO system to
Sinorhizobium meliloti
, as described by Davey and de Bruijn (M. E. Davey and F. J. de Bruijn, Appl. Environ. Microbiol. 66:5353–5359, 2000). It is therefore possible that this system constitutes a more widely, although not universally, demonstrated form of gene regulation.
Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in
Bacillus subtilis
Depends on Active Transcription
Authors: Weber Michael H. W. , Volkov Arsen V. , Fricke Ingo , Marahiel Mohamed A. , Graumann Peter L.
Published: July 2002

Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells
Bacillus subtilis
CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in
smc
mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal in
cspB cspC
and
cspB cspD
double-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that
cspB cspD
and
cspB cspC
double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.
DNA Microarray Analysis of the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Evidence for a New Type of Sulfur-Reducing Enzyme Complex
Authors: Schut Gerrit J. , Zhou Jizhong , Adams Michael W. W.
Published: July 2002

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon
Pyrococcus furiosus.
They were used to investigate the effects of elemental sulfur (S°) on the levels of gene expression in cells grown at 95°C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S° and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S°. Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S°-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S°-grown cells, and these are also thought be involved in S° reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S°-grown cells, a finding consistent with the fact that growth on peptides is a S°-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S°, although the reason for this is as yet unknown. Of the 20 S°-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from
P. furiosus,
but none of the products of the 34 S°-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.
The Initiation Codon Affects Ribosome Binding and Translational Efficiency in
Escherichia coli
of
c
I mRNA with or without the 5′ Untranslated Leader
Authors: O'Donnell Sean M. , Janssen Gary R.
Published: July 2002

Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless
c
I mRNA from bacteriophage λ. In a
c
I-
lacZ
translational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels. Addition of an untranslated
lac
leader and Shine-Dalgarno sequence to
c
I increased expression but still showed a dependence on an AUG for maximum expression.
c
I-
lacZ
mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5′ untranslated leader.
Control of
hemA
Expression in
Rhodobacter sphaeroides
2.4.1: Effect of a Transposon Insertion in the
hbdA
Gene
Authors: Fales Linda , Kryszak Luiza , Zeilstra-Ryalls Jill
Published: July 2002

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in
Rhodobacter sphaeroides
its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in
R. sphaeroides
2.4.1:
hemA
and
hemT
. In our investigations of
hemA
regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which
hemA
expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985–993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of
hbdA
, coding for
S
-(+)-β-hydroxybutyryl–coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the
hbdA
gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of
hemA
expression.
Regulation of Carbon and Electron Flow in
Clostridium butyricum
VPI 3266 Grown on Glucose-Glycerol Mixtures
Authors: Saint-Amans Sylvie , Girbal Laurence , Andrade Jose , Ahrens Kerstin , Soucaille Philippe
Published: July 2002

The metabolism of
Clostridium butyricum
was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for
Clostridium acetobutylicum
(L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of
C. butyricum
is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H
+
ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
Evolutionary Divergence of an Elongation Factor 3 from
Cryptococcus neoformans
Authors: Blakely Greg , Hekman James , Chakraburtty Kalpana , Williamson Peter R.
Published: July 2002

Elongation factor 3 (EF3) is considered a promising drug target for the control of fungal diseases because of its requirement for protein synthesis and survival of fungi and a lack of EF3 in the mammalian host. However, EF3 has been characterized only in ascomycete yeast. In order to understand the role of EF3 in a basidiomycete yeast, we cloned the gene encoding EF3 from
Cryptococcus neoformans
(
CnEF3
), an important fungal pathogen in immunocompromised patients, including those infected with human immunodeficiency virus.
CnEF3
was found to encode a 1,055-amino-acid protein and has 44% identity with EF3 from
Saccharomyces cerevisiae
(
YEF3
). Expressed CnEF3 exhibited ATPase activity that was only modestly stimulated by ribosomes from
S. cerevisiae
. In contrast, CnEF3 showed tight binding to cryptococcal ribosomes, as shown by an inability to be removed under conditions which successfully remove
Saccharomyces
EF3 from ribosomes (0.5 M KCl or 2 M LiCl).
CnEF3
also poorly complemented a
YEF3
defect in a diploid null mutant and two temperature-sensitive mutants which have been shown previously to be complemented well by
EF3
from other ascomycetes, such as
Candida albicans
. These data clearly identify the presence of a functioning
EF3
in the basidiomycete yeast
C. neoformans
, which demonstrates an evolutionary divergence from
EF3
of ascomycete yeast.
Development of a New Integration Site within the
Bacillus subtilis
Chromosome and Construction of Compatible Expression Cassettes
Authors: Härtl Barbara , Wehrl Wolfgang , Wiegert Thomas , Homuth Georg , Schumann Wolfgang
Published: July 2002

The
Bacillus subtilis lacA
gene, coding for β-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the
lacA
locus was able to control the expression of a transcriptional fusion at the
amyE
locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.
Mapping of the Rsd Contact Site on the Sigma 70 Subunit of
Escherichia coli
RNA Polymerase
Authors: Jishage Miki , Dasgupta Dipak , Ishihama Akira
Published: July 2002

Rsd (regulator of sigma D) is an anti-sigma factor for the
Escherichia coli
RNA polymerase ς
70
subunit. The contact site of Rsd on ς
70
was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-
p
-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted ς
70
and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter −35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and ς
70
contact transcription factors.
Utilization of
l
-Ascorbate by
Escherichia coli
K-12: Assignments of Functions to Products of the
yjf-sga
and
yia-sgb
Operons
Authors: Yew Wen Shan , Gerlt John A.
Published: July 2002

Escherichia coli
K-12 can ferment
l
-ascorbate. The operon encoding catabolic enzymes in the utilization of
l
-ascorbate (
ula
) has been identified; this operon of previously unknown function had been designated the
yif-sga
operon. Three enzymes in the pathway that produce
d
-xylulose 5-phosphate have been functionally characterized: 3-keto-
l
-gulonate 6-phosphate decarboxylase (UlaD),
l
-xylulose 5-phosphate 3-epimerase (UlaE), and
l
-ribulose 5-phosphate 4-epimerase (UlaF). Several products of the
yia-sgb
operon were also functionally characterized, although the substrate and physiological function of the operon remain unknown: 2,3-diketo-
l
-gulonate reductase (YiaK), 3-keto-
l
-gulonate kinase (LyxK), 3-keto-
l
-gulonate 6-phosphate decarboxylase (SgbH), and
l
-ribulose 5-phosphate 4-epimerase (SgbE).
Functional Interaction of Region 4 of the Extracytoplasmic Function Sigma Factor FecI with the Cytoplasmic Portion of the FecR Transmembrane Protein of the
Escherichia coli
Ferric Citrate Transport System
Authors: Mahren Susanne , Enz Sabine , Braun Volkmar
Published: July 2002

Transcriptional regulation of the ferric citrate transport genes of
Escherichia coli
is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the
fecABCDE
genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and
fecB-lacZ
transcription. The cloned region 4 of FecI interfered with
fecB-lacZ
transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of
Pseudomonas aeruginosa
was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the
P. aeruginosa
proteins and between a
P. aeruginosa
FecI homolog and
E. coli
FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.
Diversity of Tn4001Transposition Products: the Flanking IS256Elements Can Form Tandem Dimers and IS Circles
Authors: Prudhomme M. , Turlan C. , Claverys J.-P. , Chandler M.
Published: July 2002

We show that both flanking IS256elements carried by transposon Tn4001are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256belonging to one Tn4001copy attacks an IS256end carried by the second Tn4001copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis inEscherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such asStreptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.
Mutations in the GTPase Center of
Escherichia coli
23S rRNA Indicate Release Factor 2-Interactive Sites
Authors: Xu Wenbing , Pagel Frances T. , Murgola Emanuel J.
Published: July 2002

Mutations in the GTPase center of
Escherichia coli
23S rRNA were characterized in vivo as UGA-specific nonsense suppressors. Some site-directed mutations did not exhibit suppressor activity and were interspersed among suppressor mutations. Our results demonstrate the involvement of the two adjacent loops of this conserved rRNA structure in UGA-dependent translation termination and, taken with previous in vitro analyses and with consideration of the crystal structure of the GTPase center RNA, indicate that nucleotides 1067, 1093, 1094, and 1095 are sites of interaction with release factor 2.
An Extracellular Matrix-Associated Zinc Metalloprotease Is Required for Dilauroyl Phosphatidylethanolamine Chemotactic Excitation in
Myxococcus xanthus
Authors: Kearns Daniel B. , Bonner Pamela J. , Smith Daniel R. , Shimkets Lawrence J.
Published: July 2002

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium
Myxococcus xanthus
is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of
fibA
had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the
fibA
mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.
Force and Velocity of
Mycoplasma mobile
Gliding
Authors: Miyata Makoto , Ryu William S. , Berg Howard C.
Published: July 2002

The effects of temperature and force on the gliding speed of
Mycoplasma mobile
were examined. Gliding speed increased linearly as a function of temperature from 0.46 μm/s at 11.5°C to 4.0 μm/s at 36.5°C. A polystyrene bead was attached to the tail of
M. mobile
using a polyclonal antibody raised against whole
M. mobile
cells. Cells attached to beads glided at the same speed as cells without beads. When liquid flow was applied in a flow chamber, cells reoriented and moved upstream with reduced speeds. Forces generated by cells at various gliding speeds were calculated by multiplying their estimated frictional drag coefficients with their velocities relative to the liquid. The gliding speed decreased linearly with force. At zero speed, the force measurements extrapolated to 26 pN at 22.5 and 27.5°C. At zero force, the speed extrapolated to 2.3 and 3.3 μm/s at 22.5 and 27.5°C, respectively—the same speeds as those observed for free gliding cells. Cells attached to beads were also trapped by an optical tweezer, and the stall force was measured to be 26 to 28 pN (17.5 to 27.5°C). The gliding speed depended on temperature, but the maximum force did not, suggesting that the mechanism is composed of at least two steps, one that generates force and another that allows displacement. Other implications of these results are discussed.
Single-Tube Single-Enzyme Reverse Transcriptase PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pooled Bovine Serum
Authors: Weinstock Daniel , Bhudevi Bodreddigari , Castro Anthony E.
Published: July 2002

A reverse transcriptase PCR (RT-PCR) was developed for use as a diagnostic screening test for the detection of bovine viral diarrhea virus (BVDV) in pooled bovine serum samples. Individual serum samples from 60 dairy cattle herds located in Pennsylvania were evaluated by the microplate virus isolation method, and pooled sera were analyzed by RT-PCR. RT-PCR was sensitive and specific and detected a single viremic serum sample in up to 100 pooled serum samples. RT-PCR analysis of pooled sera provides a rapid and cost-effective method for the screening of cattle herds for the presence of animals persistently infected with BVDV.
Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes
Authors: Lindsley Mark D. , Hurst Steven F. , Iqbal Naureen J. , Morrison Christine J.
Published: July 2002

Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA fromHistoplasma capsulatum,Blastomyces dermatitidis,Coccidioides immitis,Paracoccidioides brasiliensis,Penicillium marneffei,Sporothrix schenckii,Cryptococcus neoformans, fiveCandidaspecies, andPneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA fromH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis, andP.marneffeibut not with DNA from nondimorphic fungi. Specific probes forH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis,P.marneffei,S.schenckii,C.neoformans, andP.cariniihybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for theB.dermititidisprobe used againstC.immitisDNA and for theH.capsulatumprobe used againstCandida albicansDNA. However, theC.immitisprobe did not cross-react withB.dermititidisDNA, nor did the dimorphic probe hybridize withC.albicansDNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.
Epidemiology and Susceptibility of 3,051Staphylococcus aureusIsolates from 25 University Hospitals Participating in the European SENTRY Study
Authors: Fluit A. C. , Wielders C. L. C. , Verhoef J. , Schmitz F.-J.
Published: July 2002

A total of 3,051 methicillin-susceptibleStaphylococcus aureus(MSSA) isolates and methicillin-resistantS. aureus(MRSA) isolates in Europe were compared. MRSA isolates constituted 25% of all isolates and were more prevalent in southern Europe. MRSA isolates appeared to be more prevalent in intensive care units than in outpatient departments. Only a small minority of MSSA isolates were multidrug resistant, whereas the majority of MRSA isolates were multidrug resistant.
Characterization ofNeospora caninumSurface Protein NcSRS2 Based on Baculovirus Expression System and Its Application for Serodiagnosis ofNeosporaInfection
Authors: Nishikawa Yoshifumi , Kousaka Yuko , Tragoolpua Khajornsak , Xuan Xuenan , Makala Levi , Fujisaki Kozo , Mikami Takeshi , Nagasawa Hideyuki
Published: July 2002

The baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have characterized theNeospora caninumsurface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis ofNeosporainfection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera againstToxoplasma gondii,Hammondia heydorni, orN. caninum. Anti-N. caninummouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera againstN. caninumfrom IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis ofN. caninuminfection.
First European Pediatric Case of Human Granulocytic Ehrlichiosis
Authors: Arnež Maja , Petrovec Miroslav , Lotrič-Furlan Stanka , Zupanc Tatjana Avsic , Strle Franc
Published: July 2002

Herein we report on the first confirmed pediatric case of acute human granulocytic ehrlichiosis in Europe. Presentation in this 11-year-old girl was comparable to clinical findings seen in adult European patients with human granulocytic ehrlichiosis; i.e., she had self-limited febrile illness with leukopenia, thrombocytopenia, and elevated serum C-reactive protein concentration. It is of interest that the patient not only had a fourfold change in antibody titer to
Ehrlichia phagocytophila
but also developed antibodies to
Ehrlichia chaffeensis
and that her PCR test result was positive on the third as well as on the 22nd day after the onset of illness, that is, 16 days after spontaneous defervescence.
Dogs as Sentinels for Human Lyme Borreliosis in The Netherlands
Authors: Goossens H. A. T. , van den Bogaard A. E. , Nohlmans M. K. E.
Published: July 2002

Serum samples from hunters (n= 440), their hunting dogs (n= 448), and hunters without dog ownership (n= 53) were collected in The Netherlands at hunting dog trials and were tested for antibodies againstBorrelia burgdorferiby a whole-cell enzyme-linked immunosorbent assay. Additionally, 75 healthy pet dogs were tested. The results of this study indicate that the seroprevalence among hunting dogs (18%) was of the same order as the seroprevalence among pet dogs (17%) and hunters (15%). The seropositivity of a hunting dog was not a significant indicator of increased risk of Lyme borreliosis for its owner. No significant rise in seroprevalence was found in dogs older than 24 months. This indicated that seropositivity after an infection withB. burgdorferiin dogs is rather short, approximately 1 year. In humans this is considerably longer but is also not lifelong. Therefore, the incidence ofB. burgdorferiinfections among dogs was greater than that among hunters, despite a similar prevalence of seropositivity among hunters and their hunting dogs. Because no positive correlation was observed between the seropositivity of a hunter and the seropositivity of the hunter's dog, direct transfer of ticks between dog and hunter does not seem important and owning a dog should not be considered a risk factor for Lyme borreliosis.
Comparison of Cultivation and PCR-Hybridization for Detection ofSalmonellain Porcine Fecal and Water Samples
Authors: Feder Ingrid , Nietfeld Jerome C. , Galland John , Yeary Teresa , Sargeant Jan M. , Oberst Richard , Tamplin Mark L. , Luchansky John B.
Published: July 2002

A total of 150 fecal and water samples from four swine farms were tested for the presence ofSalmonella entericausing different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37°C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viableSalmonellabacteria. PresumptiveSalmonellaisolates were biochemically and serologically confirmed. For the PCR detection ofSalmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to theinvAandinvEgenes were confirmed asSalmonellaspecific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for theSalmonella invAgene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone.Salmonellabacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement betweenSalmonelladetection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement betweenSalmonelladetection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, whileSalmonellabacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detectingSalmonellain swine feces or water samples from swine farms when using the medium combinations evaluated in this study.
Interaction betweenMycoplasma hyopneumoniaeand Swine Influenza Virus
Authors: Thacker Eileen L. , Thacker Brad J. , Janke Bruce H.
Published: July 2002

An experimental respiratory model was used to investigate the interaction betweenMycoplasma hyopneumoniaeand swine influenza virus (SIV) in the induction of pneumonia in susceptible swine. Previous studies demonstrated thatM. hyopneumoniae, which produces a chronic bronchopneumonia in swine, potentiates a viral pneumonia induced by the porcine reproductive and respiratory syndrome virus (PRRSV). In this study, pigs were inoculated withM. hyopneumoniae21 days prior to inoculation with SIV. Clinical disease as characterized by the severity of cough and fever was evaluated daily. Percentages of lung tissue with visual lesions and microscopic lesions were assessed upon necropsy at 3, 7, 14, and 21 days following SIV inoculation. Clinical observations revealed that pigs infected with both SIV andM. hyopneumoniaecoughed significantly more than pigs inoculated with a single agent. Macroscopic pneumonia on necropsy at days 3 and 7 was greatest in both SIV-infected groups, with minimal levels of pneumonia in theM. hyopneumoniae-only-infected pigs. At 14 days post-SIV inoculation, pneumonia was significantly more severe in pigs infected with both pathogens. However, by 21 days postinoculation, the level of pneumonia in the dual-infected pigs was similar to that of theM. hyopneumoniae-only-infected group, and the pneumonia in the pigs inoculated with only SIV was nearly resolved. Microscopically, there was no apparent increase in the severity of pneumonia in pigs infected with both agents compared to that of single-agent-challenged pigs. The results of this study found that while pigs infected with both agents exhibited more severe clinical disease, the relationship between the two pathogens lacked the profound potentiation found with dual infection withM. hyopneumoniaeand PRRSV. These findings demonstrate that the relationship between mycoplasmas and viruses varies with the individual agent.
Heminested Multiplex Reverse Transcription-PCR for Detection and Differentiation of Norwalk-Like Virus Genogroups 1 and 2 in Fecal Samples
Authors: Yuen Lilly K. W. , Catton Michael G. , Cox Bradley J. , Wright Peter J. , Marshall John A.
Published: July 2002

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 μl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.
Application of Real-Time Fluorescent PCR for Quantitative Assessment of
Neospora caninum
Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue
Authors: Müller Norbert , Vonlaufen Nathalie , Gianinazzi Christian , Leib Stephen L. , Hemphill Andrew
Published: July 2002

The previously described Nc5-specific PCR test for the diagnosis of
Neospora caninum
infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with
N. caninum
tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral
N. caninum
infection.
Comparison of Real-Time, Quantitative PCR with Molecular Beacons to Nested PCR and Culture Methods for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Fecal Samples
Authors: Fang Ying , Wu Wai-Hong , Pepper Jessica L. , Larsen Jill L. , Marras Salvatore A. E. , Nelson Eric. A. , Epperson William B. , Christopher-Hennings Jane
Published: July 2002

An automated PCR with fluorescent probes (molecular beacons) detectedMycobacterium aviumsubsp.paratuberculosisin bovine feces. When the PCR was compared with culture in testing 41 fecal samples, kappa scores of 0.94 to 0.96, a sensitivity of 93 to 96%, and a specificity of 92% were obtained. Results were quantitated by using a standard curve derived from a plasmid containing IS900. A minimum quantity of 1.7 × 10−4pg of DNA, correlating to 1 to 8 CFU, was detected.
Evaluation of Use of Epstein-Barr Viral Load in Patients after Allogeneic Stem Cell Transplantation To Diagnose and Monitor Posttransplant Lymphoproliferative Disease
Authors: Gärtner Barbara C. , Schäfer Hansjörg , Marggraff Katja , Eisele Günter , Schäfer Marco , Roemer Klaus , Laws Hans-Jürgen , Sester Martina , Sester Urban , Einsele Hermann , Mueller-Lantzsch Nikolaus
Published: July 2002

Epstein-Barr virus (EBV)-induced posttransplant lymphoproliferative disease (PTLD) continues to be a serious complication following transplantation. The aim of the present study was to evaluate the EBV load as a parameter for the prediction and monitoring of PTLD. The EBV load was analyzed by a quantitative competitive PCR with 417 whole-blood samples of 59 patients after allogeneic stem cell transplantation (SCT). The EBV load was positive for all 9 patients with PTLD and for 17 patients without PTLD. The viral loads of patients with manifest PTLD differed from the loads of those without PTLD (median loads, 1.4 × 10
6
versus 4 × 10
4
copies/μg of DNA;
P
< 0.0001). A threshold value of 10
5
copies/μg of DNA showed the best diagnostic efficacy (sensitivity, 87%; specificity, 91%). However, in patients with less than three major risk factors for PTLD, the positive predictive value of this threshold was rather low. One week prior to the manifestation of PTLD, the EBV load was as low in patients who developed PTLD as in patients without disease (median, 2.2 × 10
4
copies/μg of DNA;
P
was not significant). EBV DNA tested positive first at 20 to 71 days prior to the clinical manifestation of PTLD and occurred with the same delay after transplantation regardless of disease (median delay, 52 versus 63 days;
P
was not significant). EBV DNA was detected earlier in patients with primary infections than in those with reactivations (33 versus 79 days;
P
= 0.01), but the peak levels were similar in the two groups. EBV primary infection or EBV reactivation is frequent in patients after allogeneic SCT but results in PTLD only in a subgroup of patients. Although evaluation of the EBV load has limitations, the EBV load represents a valuable parameter to guide therapy.
Quantification of Feline Herpesvirus 1 DNA in Ocular Fluid Samples of Clinically Diseased Cats by Real-Time TaqMan PCR
Authors: Vögtlin A. , Fraefel C. , Albini S. , Leutenegger C. M. , Schraner E. , Spiess B. , Lutz H. , Ackermann M.
Published: July 2002

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R2= 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.
Unusual Outbreak of Clinical Mastitis in Dairy Sheep Caused by
Streptococcus equi
subsp.
zooepidemicus
Authors: Las Heras Alfonso , Vela Ana I. , Fernández Elena , Legaz Emilio , Domínguez Lucas , Fernández-Garayzábal Jose F.
Published: July 2002

This work describes an outbreak of clinical mastitis affecting 13 of 58 lactating ewes due to
Streptococcus equi
subsp.
zooepidemicus. S. equi
subsp.
zooepidemicus
was isolated in pure culture from all milk samples. All the clinical isolates had identical biochemical profiles and antimicrobial susceptibility patterns and also exhibited indistinguishable macrorestriction patterns by pulsed-field gel electrophoresis, indicating that all cases of mastitis were produced by a single strain.
Trends in Antifungal Susceptibility of
Candida
spp. Isolated from Pediatric and Adult Patients with Bloodstream Infections: SENTRY Antimicrobial Surveillance Program, 1997 to 2000
Authors: Pfaller M. A. , Diekema D. J. , Jones R. N. , Messer S. A. , Hollis R. J.
Published: July 2002

From 1 January 1997 through 31 December 2000, 2,047 bloodstream infections (BSIs) due to
Candida
spp. were reported from hospitals in the United States, Canada, Latin America, and Europe participating in the SENTRY Antifungal Surveillance Program. Among individuals in four age groups (≤1, 2 to 15, 16 to 64, and ≥65 years)
Candida albicans
was the most common species, causing 60, 55, 55, and 50% of infections, respectively.
C. glabrata
caused 17 to 23% of BSIs in those ages 16 to 64 and ≥65 years, whereas it caused only 3% of BSIs in the individuals in the two younger age groups (
P
< 0.001).
C. parapsilosis
(which caused 21 to 24% of BSIs) and
C. tropicalis
(which caused 7 to 10% of BSIs) were more common than
C. glabrata
in individuals ages ≤1 year and 2 to 15 years. Isolates of
Candida
spp. showed a trend of decreasing susceptibility to fluconazole, itraconazole, and amphotericin B with increasing patient age (
P
≤ 0.01). None of the
C. glabrata
isolates from individuals ≤1 year old were resistant to fluconazole, whereas they made up 5 to 9% of isolates from individuals ages 16 to 64 and ≥65 years. Isolates of
C. tropicalis
from patients ≤1 year old were more susceptible to flucytosine (MIC at which 90% of isolates are inhibited [MIC
90
], 0.5 μg/ml; 0% resistant isolates) than those from patients ≥65 years old (MIC
90
, 32 μg/ml; 11% resistant isolates). The investigational triazoles posaconazole, ravuconazole, and voriconazole were all highly active against all species of
Candida
from individuals in all age groups. These data demonstrate differences in the species distributions of pathogens and differences in antifungal resistance among isolates from individuals in the pediatric and adult age groups. Ongoing surveillance will enhance efforts to limit the extent of antifungal resistance in individuals in various age groups.