bluuebunny/crossref_metadata_2025 huggingface dataset:
HreP, an In Vivo
-
Expressed Protease of
Yersinia enterocolitica
, Is a New Member of the Family of Subtilisin/Kexin-Like Proteases
Authors: Heusipp Gerhard , Young Glenn M. , Miller Virginia L.
Published: July 2002

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for
Yersinia enterocolitica
. Previously, we identified a gene,
hreP
, which showed significant similarity to proteases in a screen for chromosomal genes of
Y. enterocolitica
that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like
inv
, the gene encoding the invasin protein of
Y. enterocolitica
,
hreP
is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in
Escherichia coli, Salmonella,
and
Yersinia pestis
than in
Y. enterocolitica.
Analysis of the distribution of
hreP
between different
Yersinia
isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of
Anabaena variabilis
, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His
6
tag fusion protein in
E. coli
revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.
Analysis of Promoter Recognition In Vivo Directed by ς
F
of
Bacillus subtilis
by Using Random-Sequence Oligonucleotides
Authors: Amaya Edward , Khvorova Anastasia , Piggot Patrick J.
Published: July 2002

Formation of spores from vegetative bacteria by
Bacillus subtilis
is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase ς factors. Of these, ς
F
is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing ς
F
(E-ς
F
), and only two genes of known function are exclusively under the control of E-ς
F
,
spoIIR
and
spoIIQ
. In order to investigate the features of promoters that are recognized by E-ς
F
, we studied the effects of randomizing sequences for the −10 and −35 regions of the promoter for
spoIIQ
. The randomized promoter regions were cloned in front of a promoterless copy of
lacZ
in a vector designed for insertion by double crossover of single copies of the promoter-
lacZ
fusions into the
amyE
region of the
B. subtilis
chromosome. This system made it possible to test for transcription of
lacZ
by E-ς
F
in vivo. The results indicate a weak ς
F
-specific −10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated ς factors, as well as to ς
A
. There was a rather stronger −35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated ς factors. The looseness of the ς
F
promoter requirement contrasts with the strict requirement for ς
A
-directed promoters of
B. subtilis
. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-ς
F
in vivo.
EfficientvirGene Induction inAgrobacterium tumefaciensRequiresvirA,virG,andvirBox from the Same Ti Plasmid
Authors: Krishnamohan Atmakuri , Balaji V. , Veluthambi K.
Published: July 2002

Thevirgenes of octopine, nopaline, andl,l-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopalinevircomponents. The induction of an octopinevirEA6::lacZfusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopinevirGA6in a nopaline strain with pSM358 did not completely restorevirEA6induction. However, addition of the octopinevirAA6to the above strain increasedvirEA6induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopalinevirEC58::catfusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) andl,l-succinamopine (A281) strains. Supplementation of nopalinevirAC58andvirGC58in an octopine strain (A348) harboring pUCD1553 increased induction levels ofvirEC58::catfusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine andl,l-succinamopine VirG proteins induce the octopinevirEA6more efficiently than they do the nopalinevirEC58. Conversely, the nopaline VirG protein induces the nopalinevirEC58more efficiently than it does the octopinevirEA6. The ability of Bo542virGto bring about supervirulence in tobacco is observed for an octopinevirhelper (LBA4404) but not for a nopalinevirhelper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG andvirboxes of different Ti plasmids. Efficientvirgene induction in octopine and nopaline strains requiresvirA,virG,andvirboxes from the respective Ti plasmids.
Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-
cis,cis
-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A
Authors: Kaulmann Ursula , Kaschabek Stefan R. , Schlömann Michael
Published: July 2002

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-
cis,cis
-muconate to
cis
-dienelactone (
cis
-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-
cis,cis
-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of
cis
-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of
Pseudomonas putida
muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as
cis,cis
-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-
cis,cis
-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-
cis,cis
-muconate. Interestingly,
cis
-dienelactone and 2-chloro-
cis
-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.
Regulation and Mechanism of Action of the Small Heat Shock Protein from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Laksanalamai Pongpan , Maeder Dennis L. , Robb Frank T.
Published: July 2002

The small heat shock protein (sHSP) from the hyperthermophile
Pyrococcus furiosus
was specifically induced at the level of transcription by heat shock at 105°C. The gene encoding this protein was cloned and overexpressed in
Escherichia coli
. The recombinant sHSP prevented the majority of
E. coli
proteins from aggregating in vitro for up to 40 min at 105°C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56°C. Survivability of
E. coli
overexpressing the sHSP was enhanced approximately sixfold during exposure to 50°C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.
GerN, an Antiporter Homologue Important in Germination of
Bacillus cereus
Endospores
Authors: Thackray Penny D. , Behravan Javad , Southworth Thomas W. , Moir Anne
Published: July 2002

A homologue of the
grmA
spore germination gene of
Bacillus megaterium
and of a NaH-antiporter gene (
napA
) of
Enterococcus hirae
has been identified in
Bacillus cereus
569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this
B. cereus
gene, named
gerN
, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to
l
-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 μM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 μM
l
-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of
l
-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.
YkdA and YvtA, HtrA-Like Serine Proteases in
Bacillus subtilis
, Engage in Negative Autoregulation and Reciprocal Cross-Regulation of
ykdA
and
yvtA
Gene Expression
Authors: Noone David , Howell Alistair , Collery Ross , Devine Kevin M.
Published: July 2002

HtrA-type serine proteases participate in folding and degradation of aberrant proteins and in processing and maturation of native proteins. Mutation of the corresponding genes often confers a pleiotropic phenotype that can include temperature sensitivity, sensitivity to osmotic and oxidative stress, and attenuated virulence. There are three HtrA-type serine proteases, YkdA, YvtA, and YycK, encoded in the
Bacillus subtilis
genome. In this report we show that YkdA and YvtA display many similarities: their expression profiles during the growth cycle in wild-type and mutant backgrounds are very alike, with expression being directed by very similar promoters. Both are induced by temperature upshift and by heterologous amylases at the transition phase of the growth cycle. These characteristics are quite different for YycK, suggesting that it has a cellular function distinct from that of the other two proteases or that it performs the same function but under different conditions. We also show that inactivation of either
ykdA
or
yvtA
results in compensating overexpression of the other gene, especially during stress conditions, with a concomitant increase in resistance to heat and hydrogen peroxide stresses. Mutation of both
ykdA
and
yvtA
leads to growth defects and to thermosensitivity. The fact that their expression increases dramatically at the transition phase of the growth cycle under certain conditions suggests that the YkdA and YvtA proteases may function in the processing, maturation, or secretion of extracellular enzymes in
B. subtilis
.
Bacillus subtilis
Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture
Authors: Dauner Michael , Storni Tazio , Sauer Uwe
Published: July 2002

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing
Bacillus subtilis
. Based on [U-
13
C
6
]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited
B. subtilis
is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited
B. subtilis
, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.
NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of
Ralstonia eutropha
Strain Bo on Tetrahydrofurfuryl Alcohol
Authors: Schräder Thomas , Zarnt Grit , Andreesen Jan R.
Published: July 2002

Different aldehyde dehydrogenases (AlDHs) were formed during growth of
Ralstonia eutropha
Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4.
Sugar Transport through Maltoporin of
Escherichia coli
: Role of the Greasy Slide
Authors: Van Gelder Patrick , Dumas Fabrice , Bartoldus Ingrid , Saint Nathalie , Prilipov Alexei , Winterhalter Mathias , Wang Yanfei , Philippsen Ansgar , Rosenbusch Jürg P. , Schirmer Tilman
Published: July 2002

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.
Glycerol-3-Phosphate-Induced Catabolite Repression inEscherichia coli
Authors: Eppler Tanja , Postma Pieter , Schütz Alexandra , Völker Uwe , Boos Winfried
Published: July 2002

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction inmalTexpression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlcphosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlcdid not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlcis controlled by G3P and other phosphorylated sugars such asd-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), andd-tagatose-1,6-bisphosphate aldolase.
Acinetobacter baumannii
at a Tertiary-Care Teaching Hospital in Jerusalem, Israel
Authors: Simhon A. , Rahav G. , Shazberg G. , Block C. , Bercovier H. , Shapiro M.
Published: July 2002

In a retrospective 10-year analysis of 3,536 patient-unique isolates,
Acinetobacter baumannii
imipenem susceptibility declined from 98.1 (1990) to 64.1% (2000), and ciprofloxacin susceptibility decreased from 50.5 to 13.1%. Imipenem median zone diameters decreased from 27.7 (1997) to 18.8 mm (2000). No outbreaks were detected. Two clusters were identified for 41 strains genotyped by pulsed-field gel electrophoresis, but imipenem resistance was not clonal.
Analysis of Bacterial Vaginosis-Related Amines in Vaginal Fluid by Gas Chromatography and Mass Spectrometry
Authors: Wolrath Helen , Forsum Urban , Larsson P. G. , Borén Hans
Published: July 2002

The presence of various amines in vaginal fluid from women with malodorous vaginal discharge has been reported before. The investigations have used several techniques to identify the amines. However, an optimized quantification, together with a sensitive analysis method in connection with a diagnostic procedure for vaginal discharge, including the syndrome of bacterial vaginosis, as defined by the accepted “gold standard,” has not been done before. We now report a sensitive gas chromatographic and mass spectrometric method for identifying the amines isobutylamine, phenethylamine, putrescine, cadaverine, and tyramine in vaginal fluid. We used weighted samples of vaginal fluid to obtain a correct quantification. In addition, a proper diagnosis was obtained using Gram-stained smears of the vaginal fluid that were Nugent scored according to the method of Nugent et al. (R. P. Nugent et al., J. Clin. Microbiol., 29:297–301, 1991). We found that putrescine, cadaverine, and tyramine occurred in high concentrations in vaginal fluid from 24 women with Nugent scores between 7 and 10. These amines either were not found or were found only in very low concentrations in vaginal fluid from women with Nugent scores of 0 to 3. There is a strong correlation between bacterial vaginosis and the presence of putrescine, cadaverine, and tyramine in high concentrations in vaginal fluid.
PCR-Based Detection and Identification of
Burkholderia
cepacia
Complex Pathogens in Sputum from Cystic Fibrosis Patients
Authors: McDowell Andrew , Mahenthiralingam Eshwar , Moore John E. , Dunbar Kerstin E. A. , Webb A. Kevin , Dodd Mary E. , Martin S. Lorraine , Millar B. Cherie , Scott Christopher J. , Crowe Mary , Elborn J. Stuart
Published: July 2002

PCR amplification of the
recA
gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of
Burkholderia
cepacia
complex genomovars directly from sputum. Successful amplification of the
B
.
cepacia
complex
recA
gene from cystic fibrosis (CF) patient sputum samples containing
B
.
cepacia
genomovar I,
Burkholderia
multivorans
,
B
.
cepacia
genomovar III,
Burkholderia
stabilis
, and
Burkholderia
vietnamiensis
was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that
recA
-based PCR could reliably detect
B
.
cepacia
complex organisms to concentrations of 10
6
CFU g of sputum
−1
. To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for
B
.
cepacia
complex infection by selective culturing and
recA
-based PCR. Selective culturing identified 19 samples with presumptive
B
.
cepacia
complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by
recA
-based PCR, while 2 samples were negative. The isolates cultured from both
recA
-negative sputum samples were subsequently identified as
Burkholderia
gladioli
. RFLP analysis of the
recA
amplicons revealed 2 patients (12%) infected with
B
.
multivorans
, 11 patients (65%) infected with
B
.
cepacia
genomovar III-A, and 4 patients (23%) infected with
B
.
cepacia
genomovar III-B. These results demonstrate the potential of
recA
-based PCR-RFLP analysis for the rapid detection and identification of
B
.
cepacia
complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by
recA
-based analysis of the isolate.
Identification of
Pandoraea
Species by 16S Ribosomal DNA-Based PCR Assays
Authors: Coenye Tom , Liu Lixia , Vandamme Peter , LiPuma John J.
Published: July 2002

The recently described genus
Pandoraea
contains five named species (
Pandoraea apista
,
Pandoraea pulmonicola
,
Pandoraea pnomenusa
,
Pandoraea sputorum
, and
Pandoraea norimbergensis
) and four unnamed genomospecies.
Pandoraea
spp. have mainly been recovered from the respiratory tracts of cystic fibrosis (CF) patients. Accurate genus- and species-level identification by routine clinical microbiology methods is difficult, and differentiation from
Burkholderia cepacia
complex organisms may be especially problematic. This can have important consequences for the management of CF patients. On the basis of 16S ribosomal DNA sequences, PCR assays for the identification of
Pandoraea
spp. were developed. A first PCR assay was developed for the identification of
Pandoraea
isolates to the genus level. PCR assays for the identification of
P. apista
and
P. pulmonicola
as a group,
P. pnomenusa
,
P. sputorum
, and
P. norimbergensis
were also developed. All five assays were evaluated with a panel of 123 bacterial isolates that included 69
Pandoraea
sp. strains, 24
B. cepacia
complex strains, 6
Burkholderia gladioli
strains, 9
Ralstonia
sp. strains, 5
Alcaligenes xylosoxidans
strains, 5
Stenotrophomonas maltophilia
strains, and 5
Pseudomonas aeruginosa
strains. The use of these PCR assays facilitates the identification of
Pandoraea
spp. and avoids the misidentification of a
Pandoraea
sp. as a
B. cepacia
complex isolate.
Zoonotic Potential of
Enterocytozoon bieneusi
Authors: Dengjel Bianca , Zahler Monika , Hermanns Walter , Heinritzi Karl , Spillmann Thomas , Thomschke Angelika , Löscher Thomas , Gothe Rainer , Rinder Heinz
Published: July 2002

The reservoirs and the modes of transmission of the most frequent microsporidial species in humans,
Enterocytozoon bieneusi
, are still unknown. We have examined fecal samples of 26 humans and 350 animals from 37 species to find 18 samples containing this parasite from humans, cats, pigs, cattle, and a llama. Genotypic characterization of the internal transcribed spacer of the rRNA gene resulted in 14 different genotypes, 6 of them previously undescribed. Phylogenetic analysis revealed the lack of a transmission barrier between
E. bieneusi
from humans and animals (cats, pigs, and cattle). Thus,
E. bieneusi
appears to be a zoonotic pathogen.
In Vitro Fungicidal Activities of Voriconazole, Itraconazole, and Amphotericin B against Opportunistic Moniliaceous and Dematiaceous Fungi
Authors: Espinel-Ingroff Ana
Published: July 2002

The NCCLS proposed standard M38-P describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds); however, standard conditions are not available for testing their fungicidal activities (minimum fungicidal or lethal concentrations [MFCs]). This study evaluated the in vitro fungistatic and fungicidal activities of voriconazole, itraconazole, and amphotericin B against 260 common and emerging molds (174
Aspergillus
sp. isolates [five species], 23
Fusarium
sp. isolates [three species], 6
Paecilomyces lilacinus
isolates, 6
Rhizopus arrhizus
isolates, 23
Scedosporium
sp. isolates, 23 dematiaceous fungi, and 5
Trichoderma longibrachiatum
isolates). MICs were determined by following the NCCLS M38-P broth microdilution method. MFCs were the lowest drug dilutions that resulted in fewer than three colonies. Voriconazole showed similar or better fungicidal activity (MFC at which 90% of isolates tested are killed [MFC
90
], 1 to 2 μg/ml) than the reference agents for
Aspergillus
spp. with the exception of
Aspergillus terreus
(MFC
90
of voriconazole and amphotericin B, >8 μg/ml). The voriconazole geometric mean (G mean) MFC for
Scedosporium apiospermum
was lower (2.52 μg/ml) than those of the other two agents (5.75 to 7.5 μg/ml). In contrast, amphotericin B and itraconazole G mean MFCs for
R. arrhizus
were 2.1 to 2.2 μg/ml, but that for voriconazole was >8 μg/ml. Little or no fungicidal activity was shown for
Fusarium
spp. (2 to >8 μg/ml) and
Scedosporium prolificans
(>8 μg/ml) by the three agents, but voriconazole had some activity against
P. lilacinus
and
T. longibrachiatum
(G mean MFCs, 1.8 and 4 μg/ml, respectively). The fungicidal activity of the three agents was similar (G mean MFC, 1.83 to 2.36 μg/ml) for the dematiaceous fungi with the exception of the azole MFCs (>8 μg/ml) for some
Bipolaris spicifera
and
Dactylaria constricta
var.
gallopava
. These data extend and corroborate the available fungicidal results for the three agents. The role of the MFC as a predictor of clinical outcome needs to be established in clinical trials by following standardized testing conditions for determination of these in vitro values.
Genotypic Diversity of Mutans Streptococci in Brazilian Nursery Children Suggests Horizontal Transmission
Authors: Mattos-Graner Renata O. , Li Yihong , Caufield Page W. , Duncan Margaret , Smith Daniel J.
Published: July 2002

Streptococcus mutans
strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five
S. mutans
isolates, 29% carried two or more genotypes. The presence of matching genotypes of
S. mutans
among children attending one nursery suggests horizontal transmission.
Accurate, Noninvasive Detection of
Helicobacter pylori
DNA from Stool Samples: Potential Usefulness for Monitoring Treatment
Authors: Shuber Anthony P. , Ascaño Jennifer J. , Boynton Kevin A. , Mitchell Anastasia , Frierson Henry F. , El-Rifai Wa’el , Powell Steven M.
Published: July 2002

A novel DNA assay demonstrating sensitive and accurate detection of
Helicobacter pylori
from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of
H. pylori
DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify
H. pylori
DNA.
Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay
Authors: Kolk Daniel P. , Dockter Janel , Linnen Jeff , Ho-Sing-Loy Marcy , Gillotte-Taylor Kristin , McDonough Sherrol H. , Mimms Larry , Giachetti Cristina
Published: July 2002

While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.
Kaposi's Sarcoma Associated with Previous Human Herpesvirus 8 Infection in Heart Transplant Recipients
Authors: Emond Jean-Philippe , Marcelin Anne-Geneviève , Dorent Richard , Milliancourt Catherine , Dupin Nicolas , Frances Camille , Agut Henri , Gandjbakhch Iradj , Calvez Vincent
Published: July 2002

The aim of this study was to evaluate the seroprevalence of human herpesvirus 8 (HHV-8) in a group of 150 patients awaiting heart transplants and to detect HHV-8 seroconversion after transplantation. Four patients were HHV-8 seropositive before transplantation, and one of them developed Kaposi's sarcoma. One patient converted to seropositive HHV-8 status after transplantation.
Herpes Simplex Virus Type 1 Glycoprotein E Domains Involved in Virus Spread and Disease
Authors: Saldanha Charles E. , Lubinski John , Martin Claudia , Nagashunmugam Thandavarayan , Wang Liyang , van der Keyl Harjeet , Tal-Singer Ruth , Friedman Harvey M.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.
Nondeletional T-Cell Receptor Transgenic Mice: Model for the CD4+T-Cell Repertoire in Chronic Hepatitis B Virus Infection
Authors: Chen M. , Sällberg M. , Thung S. N. , Hughes J. , Jones J. , Milich D. R.
Published: July 2002

Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR α- and β-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency (∼67%) of CD4+T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4+T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4+T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4+T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4+T-cell repertoire present in chronically infected HBV patients.
Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking
Authors: Brister J. Rodney , Muzyczka Nicholas
Published: July 2002

The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (
trs
). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE′), and the
trs
. Previously, we determined the sequences at the
trs
required for Rep-mediated cleavage and demonstrated that the
trs
endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at the
trs
. In the second step, Rep transesterification activity cleaves the
trs
. Here we investigate the contribution of the RBE and RBE′ during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation during
trs
nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE′ and directing nicking to the
trs
. Accordingly, alterations in the polarity or position of the RBE relative to the
trs
greatly inhibit Rep nicking. Substitutions within the RBE′ also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE′ during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and
trs
cleavage steps of the endonuclease reaction. On the other hand, RBE′ contacts seem to be required primarily for TR unwinding and formation of the
trs
stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE′, and the
trs
.
Moving the Glycoprotein Gene of Vesicular Stomatitis Virus to Promoter-Proximal Positions Accelerates and Enhances the Protective Immune Response
Authors: Flanagan E. Brian , Ball L. Andrew , Wertz Gail W.
Published: July 2002

Vesicular stomatitis virus(VSV) is the prototype of theRhabdoviridaeand contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3′-N-P-M-G-L-5′, and transcription is obligatorily sequential from the single 3′ promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3′-G-N-P-M-L-5′ (G1N2), 3′-P-M-G-N-L-5′ (G3N4), and 3′-G-P-M-N-L-5′ (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.
Chimeric Yellow Fever/Dengue Virus as a Candidate Dengue Vaccine: Quantitation of the Dengue Virus-Specific CD8 T-Cell Response
Authors: van der Most Robbert G. , Murali-Krishna Kaja , Ahmed Rafi , Strauss James H.
Published: July 2002

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.
3′-End Formation of Baculovirus Late RNAs
Authors: Jin Jianping , Guarino Linda A.
Published: July 2002

Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mechanism of 3′-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3′-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3′ ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism.
The Ubiquity and Impressive Genomic Diversity of Human Skin Papillomaviruses Suggest a Commensalic Nature of These Viruses
Authors: Antonsson Annika , Forslund Ola , Ekberg Henrik , Sterner Gunnar , Hansson Bengt Göran
Published: July 2002

Human papillomaviruses (HPV) are epitheliotropic viruses, with some types suggested to be associated with skin cancer. In this study, swab samples collected from five different sites on the skin of renal transplant recipients, dialysis patients, and age- and sex-matched healthy controls were analyzed for HPV DNA by a newly designed PCR test. Most individuals were found to have asymptomatic HPV infections; more specifically, 94% of the renal transplant patients, 82% of the dialysis patients, and 80% of the healthy controls were positive for HPV DNA. The multiplicity of the HPVs detected was astounding: 20 previously described and 30 putatively new types were identified by cloning and sequencing of 33 samples from 13 individuals. These results demonstrate that normal human skin harbors an array of papillomaviruses, most of them previously unknown.
The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3
Authors: Nilsson Nina , Malmborg Ann-Christin , Borrebaeck Carl A. K.
Published: July 2002

The filamentous bacteriophage infects
Escherichia coli
by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression.
High Frequency of Virus-Specific Interleukin-2-Producing CD4+T Cells and Th1 Dominance during Lymphocytic Choriomeningitis Virus Infection
Authors: Varga Steven M. , Welsh Raymond M.
Published: July 2002

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4+T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4+T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8−/−mice, more than 20% of the CD4+T cells secreted IFN-γ after viral peptide stimulation, whereas less than 1% of the CD4+T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4+T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.
Vaccinia Virus A30L Protein Is Required for Association of Viral Membranes with Dense Viroplasm To Form Immature Virions
Authors: Szajner Patricia , Weisberg Andrea S. , Wolffe Elizabeth J. , Moss Bernard
Published: July 2002

The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.
Examining Human T-Lymphotropic Virus Type 1 Infection and Replication by Cell-Free Infection with Recombinant Virus Vectors
Authors: Derse David , Hill Shawn A. , Lloyd Patricia A. , Chung Hye-kyung , Morse Barry A.
Published: July 2002

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37°C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.