bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of the Metalloprotease Toxin Produced by Enterotoxigenic
Bacteroides fragilis
Authors: Wu Shaoguang , Dreyfus Lawrence A. , Tzianabos Art O. , Hayashi Chika , Sears Cynthia L.
Published: July 2002

Enterotoxigenic
Bacteroides fragilis
(ETBF) strains produce a 20-kDa zinc metalloprotease toxin (BFT) associated with diarrheal disease of animals, young children, and adults. BFT stimulates secretion in intestinal loops in vivo and modifies epithelial cell morphology in vitro. The
B. fragilis
toxin (
bft
) gene from ETBF strain 86-5443-2-2 (piglet;
bft
-
2
) revealed significant nucleotide and predicted amino acid differences when compared to the
bft
gene from ETBF strain VPI 13784 (lamb;
bft
-
1
). This study compares BFT-1 and BFT-2, respectively, produced by ETBF strains VPI 13784 and 86-5443-2-2 purified using the Van Tassell method (38) and a modified purification scheme described herein. Multiple differences in the protein toxins produced by these ETBF strains were identified. First, purified BFT-1 eluted from a high-resolution anion-exchange column (Mono Q) at 0.22 ± 0.005 M NaC1 versus 0.18 ± 0.001 M NaC1 for BFT-2 (
P
< 0.001). Second, BFT-1 and BFT-2 exhibited different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase fast protein liquid chromatography. Third, each BFT reacted with greater specificity to homologous rather than heterologous antisera. Fourth, BFT-2 had modest, but consistently, greater biological activity than BFT-1 when tested on HT29/C1 cells (
P
≤ 0.01). Together, these data indicate that these ETBF strains produce two distinct isotypes of BFT, termed BFT-1 (VPI 13784 BFT) and BFT-2 (86-5443-2-2 BFT) to recognize the order in which the proteins were purified and genetic sequences identified. The modified purification scheme described in this report yields about two to three times more purified BFT protein than previous protocols and is less time consuming.
Localization within a Proline-Rich Antigen (Ag2/PRA) of Protective Antigenicity against Infection withCoccidioides immitisin Mice
Authors: Peng Tao , Shubitz Lisa , Simons Julie , Perrill Robert , Orsborn Kris I. , Galgiani John N.
Published: July 2002

Subunits of a proline-rich coccidioidal antigen (Ag2/PRA) ofCoccidioides immitiswere analyzed by comparison as vaccines in mice. The optimal dose of plasmid vaccine encoding full-length Ag2/PRA was determined to be between 10 and 100 μg. Mice vaccinated with plasmids encoding amino acids (aa) 1 to 106 were as protective as full-length Ag2/PRA (aa 1 to 194). The subunit from aa 27 to 106 was significantly but less protective. Plasmids encoding aa 90 to 151 or aa 90 to 194 were not protective. Analogous results were obtained with recombinant vaccines of the same amino acid sequences. In addition, mixtures of aa 90 to 194 with either aa 1 to 106 or aa 27 to 106 did not enhance protection compared to the active single-recombinant subunits alone. Humoral response of total immunoglobulin G (IgG) and subclasses IgG1 and IgG2a were detectable in subunit vaccinations but at significantly (100-fold) lower concentrations than after vaccination with plasmids encoding full-length Ag2/PRA. Since virtually all protection by vaccination with full-length Ag2/PRA can be accounted for in the first half of the protein (aa 1 to 106), this subunit could make a multicomponent vaccine more feasible by reducing the quantity of protein per dose and the possibility of an untoward reactions to a foreign protein.
Assessment of Virulence of Uropathogenic
Escherichia coli
Type 1 Fimbrial Mutants in Which the Invertible Element Is Phase-Locked On or Off
Authors: Gunther IV Nereus W. , Snyder Jennifer A. , Lockatell Virginia , Blomfield Ian , Johnson David E. , Mobley Harry L. T.
Published: July 2002

Type 1 fimbria is a proven virulence factor of uropathogenic
Escherichia coli
(UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as “on,” and the opposite orientation, in which no transcription occurs, is defined as “off.” During the course of a urinary tract infection, we have observed that the infecting
E. coli
population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (
P
≤ 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (
P
≤ 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (
P
= 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder.
Neisseria lactamica
Protects against Experimental Meningococcal Infection
Authors: Oliver Kerry J. , Reddin Karen M. , Bracegirdle Philippa , Hudson Michael J. , Borrow Ray , Feavers Ian M. , Robinson Andrew , Cartwright Keith , Gorringe Andrew R.
Published: July 2002

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal
Neisseria
spp., particularly with
N. lactamica
. We report here that immunization with
N. lactamica
killed whole cells, outer membrane vesicles, or outer membrane protein (OMP) pools and protected mice against lethal challenge by a number of diverse serogroup B and C meningococcal isolates in a model of bacteremic infection. Sera raised to
N. lactamica
killed whole cells, OMPs, or protein pools were found to cross-react with meningococcal isolates of a diverse range of genotypes and phenotypes. The results confirm the potential of
N. lactamica
to form the basis of a vaccine against meningococcal disease.
opdA
, a
Salmonella enterica
Serovar Typhimurium Gene Encoding a Protease, Is Part of an Operon Regulated by Heat Shock
Authors: Conlin Christopher A. , Miller Charles G.
Published: July 2002

The
opdA
(
prlC
) gene of
Salmonella enterica
serovar Typhimurium and
Escherichia coli
encodes the metalloprotease oligopeptidase A (OpdA). We report that
opdA
is cotranscribed with a downstream open reading frame,
yhiQ
. Transcription of this operon is induced after a temperature shift (30 to 42°C), and this induction depends on the heat shock sigma factor encoded by the
rpoH
(
htpR
) gene.
Arginine Catabolism in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Involves the Urea Cycle and Arginase Pathway
Authors: Quintero María José , Muro-Pastor Alicia María , Herrero Antonia , Flores Enrique
Published: July 2002

Cells of the unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 supplemented with micromolar concentrations of
l
-[
14
C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of
l
-[
14
C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [
14
C]ornithine preceded that of [
14
C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with
l
-[
14
C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Δ
1
pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an
argF
(ornithine carbamoyltransferase) insertional mutant was impaired in the production of [
14
C]citrulline from [
14
C]arginine; (ii) a
proC
(Δ
1
pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [
14
C]proline, [
14
C]glutamate, and [
14
C]glutamine from [
14
C]arginine or [
14
C]ornithine; and (iii) a
putA
(proline oxidase) insertional mutant did not produce [
14
C]glutamate from
l
-[
14
C]arginine,
l
-[
14
C]ornithine, or
l
-[
14
C]proline. Mutation of two open reading frames (
sll0228
and
sll1077
) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of
argD
(
N
-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Δ
1
pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [
14
C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-
l
-arginyl-poly(
l
-aspartic acid)], a reserve material unique to cyanobacteria.
Penicillin Binding Protein 5 Affects Cell Diameter, Contour, and Morphology of
Escherichia coli
Authors: Nelson David E. , Young Kevin D.
Published: July 2002

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of
Escherichia coli
, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of
E. coli
mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in Δ
dacA
mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.
Purification and Properties of Ornithine Racemase from
Clostridium sticklandii
Authors: Chen Hao-Ping , Lin Chin-Fen , Lee Ya-Jung , Tsay San-San , Wu Shih-Hsiung
Published: July 2002

Ornithine racemase has been purified to homogeneity from
Clostridium sticklandii
, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an
M
r
of 92,000, with a

K
m

for
l
-ornithine of 0.77 ± 0.05 mM and a

k
cat

of 980 ± 20 s
−1
.
Autophagy and the cvt Pathway Both Depend on
AUT9
Authors: Lang Thomas , Reiche Steffen , Straub Michael , Bredschneider Monika , Thumm Michael
Published: July 2002

In growing cells of the yeast
Saccharomyces cerevisiae
, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen for
aut
−
mutants and isolated
aut9-1
cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid
aut9-1
mutant cells with a genomic library, in this study we identified and characterized the
AUT9
gene, which is allelic with
CVT7. aut9
-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.
Roles of Aconitase in Growth, Metabolism, and Morphological Differentiation of
Streptomyces coelicolor
Authors: Viollier P. H. , Nguyen K. T. , Minas W. , Folcher M. , Dale G. E. , Thompson C. J.
Published: July 2002

The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184–3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in
Streptomyces coelicolor
development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned
acoA
gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an
acoA
disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an
acoA citA
mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription of
acoA
was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.
Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria
Authors: Karlin Samuel , Mrázek Jan , Campbell Allan , Kaiser Dale
Published: July 2002

Predicted highly expressed (PHX) genes are characterized for the completely sequenced genomes of the four fast-growing bacteria
Escherichia coli,
Haemophilus influenzae, Vibrio cholerae,
and
Bacillus subtilis
. Our approach to ascertaining gene expression levels relates to codon usage differences among certain gene classes: the collection of all genes (average gene), the ensemble of ribosomal protein genes, major translation/transcription processing factors, and genes for polypeptides of chaperone/degradation complexes. A gene is predicted highly expressed (PHX) if its codon frequencies are close to those of the ribosomal proteins, major translation/transcription processing factor, and chaperone/degradation standards but strongly deviant from the average gene codon frequencies. PHX genes identified by their codon usage frequencies among prokaryotic genomes commonly include those for ribosomal proteins, major transcription/translation processing factors (several occurring in multiple copies), and major chaperone/degradation proteins. Also PHX genes generally include those encoding enzymes of essential energy metabolism pathways of glycolysis, pyruvate oxidation, and respiration (aerobic and anaerobic), genes of fatty acid biosynthesis, and the principal genes of amino acid and nucleotide biosyntheses. Gene classes generally not PHX include most repair protein genes, virtually all vitamin biosynthesis genes, genes of two-component sensor systems, most regulatory genes, and most genes expressed in stationary phase or during starvation. Members of the set of PHX aminoacyl-tRNA synthetase genes contrast sharply between genomes. There are also subtle differences among the PHX energy metabolism genes between
E. coli
and
B. subtilis
, particularly with respect to genes of the tricarboxylic acid cycle. The good agreement of PHX genes of
E. coli
and
B. subtilis
with high protein abundances, as assessed by two-dimensional gel determination, is verified. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are also examined. The spatial distribution of PHX genes within each genome reveals clusters and significantly long regions without PHX genes.
Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate
Authors: Miyazaki Junichi , Kobashi Nobuyuki , Nishiyama Makoto , Yamane Hisakazu
Published: July 2002

Our previous studies revealed that lysine is synthesized through α-aminoadipate in an extremely thermophilic bacterium,
Thermus thermophilus
HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from α-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an
argD
homolog of
T. thermophilus
HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the
argD
-like gene, now termed
lysJ
, in
T. thermophilus
HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The
lysJ
gene was cloned into a plasmid and overexpressed in
Escherichia coli
, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the ɛ-amino group of
N
2
-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When
N
2
-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the δ-amino group of
N
2
-acetylornithine to 2-oxoglutarate 16 times more efficiently than when
N
2
-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in
T. thermophilus
HB27 is functionally and evolutionarily related to arginine biosynthesis.
Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in
Bacillus subtilis
168
Authors: Tsuge Kenji , Itaya Mitsuhiro
Published: July 2002

Transformation of
Bacillus subtilis
by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic
B. subtilis
recombinational repair pathway. The vector, pGETS103, a derivative of the θ-type replicating plasmid pTB19 of thermophilic
Bacillus
, had the full length of
Escherichia coli
plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of
B. subtilis
. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.
Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein
Authors: Umeyama Takashi , Horinouchi Sueharu
Published: July 2002

A protein serine/threonine kinase, AfsK, and its target protein AfsR globally control physiological and morphological differentiation in the bacterial genus
Streptomyces
. A protein (KbpA) of 252 amino acids encoded by an open reading frame in a region upstream of
afsK
in
Streptomyces coelicolor
A3(2) was identified as an AfsK-interacting protein. The interaction site of AfsK was in the N-terminal portion containing the kinase catalytic domain. KbpA bound a nonphosphorylated form of AfsK and inhibited its autophosphorylation at serine and threonine residues. KbpA in the reaction mixture containing AfsK and AfsR also inhibited the phosphorylation of AfsR by AfsK, presumably because KbpA inhibited the conversion from the inactive, nonphosphorylated form of AfsK to the active, phosphorylated form.
kbpA
was transcribed throughout growth, and the transcription was enhanced when production of actinorhodin had already started. KbpA thus appeared to play an inhibitory role in a negative feedback system in the AfsK-AfsR regulatory pathway. Consistent with these in vitro observations,
kbpA
served as a repressor for actinorhodin production in
S. coelicolor
A3(2); disruption of
kbpA
greatly enhanced actinorhodin production, and overexpression of
kbpA
reduced the production.
Regulation of
Escherichia coli
RelA Requires Oligomerization of the C-Terminal Domain
Authors: Gropp Michal , Strausz Yael , Gross Miriam , Glaser Gad
Published: July 2002

The
E. coli
RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in
relA
+
cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length
relA
gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.
Characterization of the Promoter of
PRS1
in
Saccharomyces cerevisiae
Identifies Three Regions Potentially Involved in Control of Expression
Authors: Hernando Yolanda , Carter Andrew T. , Sickinger Stefan , Schweizer Michael
Published: July 2002

The transcription initiation site of the
Saccharomyces cerevisiae PRS1
gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted
PRS1
promoter linked to
lacZ
and integrated at the
ura3
locus defined three DNA regions involved in the control of
PRS1
expression. Gel shift analysis confirmed the data.
Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria
Authors: Smits Theo H. M. , Balada Stefanie B. , Witholt Bernard , van Beilen Jan B.
Published: July 2002

We have cloned homologs of the
Pseudomonas putida
GPo1 alkane hydroxylase from
Pseudomonas aeruginosa
PAO1,
Pseudomonas fluorescens
CHA0,
Alcanivorax borkumensis
AP1,
Mycobacterium tuberculosis
H37Rv, and
Prauserella rugosa
NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the
Pseudomonas
alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts,
Escherichia coli
GEc137 and
P. putida
GPo12, were equipped with pGEc47ΔB, which encodes all proteins necessary for growth on medium-chain-length alkanes (C
6
to C
12
), except a functional alkane hydroxylase. The third host was an
alkB
knockout derivative of
P. fluorescens
CHA0, which is no longer able to grow on C
12
to C
16
alkanes. All alkane hydroxylase homologs, except the
Acinetobacter
sp. ADP1 AlkM, allowed at least one of the three hosts to grow on
n
-alkanes.
Ralstonia paucula
(Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other
Ralstonia
Species (
R. eutropha
,
R. pickettii
,
R. gilardii
, and
R. solanacearum
)
Authors: Moissenet Didier , Bidet Philippe , Garbarg-Chenon Antoine , Arlet Guillaume , Vu-Thien Hoang
Published: July 2002

Ralstonia paucula
(formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate
R. paucula
isolates. Eighteen strains belonging to other
Ralstonia
species (one
R. eutropha
strain, six
R. pickettii
strains, three
R. solanacearum
strains, and eight
R. gilardii
strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of
R. paucula
contains the tRNA
Ile
and tRNA
Ala
genes, which are identical to genes described for
R. pickettii
and
R. solanacearum
.
Determining Confidence Intervals When Measuring Genetic Diversity and the Discriminatory Abilities of Typing Methods for Microorganisms
Authors: Grundmann Hajo , Hori Satoshi , Tanner Gregor
Published: July 2002

We describe here a method for determining confidence intervals for a commonly used index of diversity. This approach facilitates the comparison of the genetic population structure of microorganisms isolated from different environments and improves the objective assessment of the discriminatory power of typing techniques.
Application of the Sherlock Mycobacteria Identification System Using High-Performance Liquid Chromatography in a Clinical Laboratory
Authors: Kellogg James A. , Bankert David A. , Withers Gisela S. , Sweimler William , Kiehn Timothy E. , Pfyffer Gaby E.
Published: July 2002

There is a growing need for a more accurate, rapid, and cost-effective alternative to conventional tests for identification of clinical isolates of
Mycobacterium
species. Therefore, the ability of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.) using computerized software and a Hewlett-Packard series 1100 high-performance liquid chromatograph to identify mycobacteria was compared to identification using phenotypic characteristics, biochemical tests, probes (Gen-Probe, Inc.), gas-liquid chromatography, and, when necessary, PCR-restriction enzyme analysis of the 65-kDa heat shock protein gene and 16S rRNA gene sequencing. Culture, harvesting, saponification, extraction, derivatization, and chromatography were performed following MIDI's instructions. Of 370 isolates and stock cultures tested, 327 (88%) were given species names by the SMIS. SMIS software correctly identified 279 of the isolates (75% of the total number of isolates and 85% of the named isolates). The overall predictive value of accuracy (correct calls divided by total calls of a species) for SMIS species identification was 85%, ranging from only 27% (3 of 11) for
M. asiaticum
to 100% for species or groups including
M. malmoense
(8 of 8),
M. nonchromogenicum
(11 of 11), and the
M. chelonae-abscessus
complex (21 of 21). By determining relative peak height ratios (RPHRs) and relative retention times (RRTs) of selected mycolic acid peaks, as well as phenotypic properties, all 48 SMIS-misidentified isolates and 39 (91%) of the 43 unidentified isolates could be correctly identified. Material and labor costs per isolate were $10.94 for SMIS, $26.58 for probes, and $42.31 for biochemical identification. The SMIS, combined with knowledge of RPHRs, RRTs, and phenotypic characteristics, offers a rapid, reasonably accurate, cost-effective alternative to more traditional methods of mycobacterial species identification.
Nucleic Acid Sequence-Based Amplification of
Aspergillus
RNA in Blood Samples
Authors: Loeffler Juergen , Hebart Holger , Cox Philipp , Flues Nicole , Schumacher Ulrike , Einsele Hermann
Published: July 2002

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of
Aspergillus
RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the
Aspergillus
18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant
Aspergillus
species, including
Aspergillus fumigatus
. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of
Aspergillus
RNA with potential for routine diagnosis, including the possibility to test the viability of cells.
Specificities and Functions of the
recA
and
pps1
Intein Genes of
Mycobacterium tuberculosis
and Application for Diagnosis of Tuberculosis
Authors: Saves Isabelle , Lewis Lee-Ann , Westrelin Fabrice , Warren Robin , Daffé Mamadou , Masson Jean-Michel
Published: July 2002

The worldwide recrudescence of tuberculosis and the widespread appearance of antibiotic resistance have strengthened the need for rapid and specific diagnostic tools. The prevailing microbiological identification of
Mycobacterium tuberculosis
, the causative agent of tuberculosis, which implies the use of in vitro cultures and acid-fast staining microscopy, is time-consuming. Detection of
M. tuberculosis
directly in clinical samples through PCR amplification of mycobacterium-specific genes, designed to shorten diagnostic delay, demonstrated reliability and high sensitivity. However, the quality of the diagnosis depends on the specificity of the target sequence for
M. tuberculosis
complex strains. In the present study, we demonstrated the specificity of
recA
and
pps1
inteins for this complex and thus the feasibility of using intein-coding sequences as a new target for PCR diagnosis. Indeed, the
recA
and
pps1
genes of 36 clinical isolates of
M. tuberculosis
and 10 field strains of
M. bovis
were found to be interrupted by an intein sequence at the RecA-a and Pps1-b sites, respectively, while a large number of nontuberculous mycobacterial species failed to demonstrate these insertions. Besides, the MtuPps1, which was cloned and expressed in
Escherichia coli
, was shown to possess an endonuclease activity. The intein cleaves the 40-bp sequence spanning the intein insertion site Pps1-b in the inteinless
pps1
gene. In addition to the PCR amplification of
recA
and
pps1
intein genes as a tool for diagnosis, the specific endonuclease activity could represent a new molecular approach to identify
M. tuberculosis
.
Real-Time LightCycler PCR for Detection and Discrimination of
Bordetella pertussis
and
Bordetella parapertussis
Authors: Kösters Katrin , Reischl Udo , Schmetz Johanna , Riffelmann Marion , Wirsing von König Carl Heinz
Published: July 2002

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of
Bordetella pertussis
and
Bordetella parapertussis
in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for
B
.
pertussis
and
B
.
parapertussis
, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for
B
.
pertussis
and/or
B
.
parapertussis
. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (
P
= 0.08) to 2.3-fold (simplex PCR) (
P
= 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.
Maternal SDF1 3′A Polymorphism Is Associated with Increased Perinatal Human Immunodeficiency Virus Type 1 Transmission
Authors: John Grace C. , Rousseau Christine , Dong Tao , Rowland-Jones Sarah , Nduati Ruth , Mbori-Ngacha Dorothy , Rostron Tim , Kreiss Joan K. , Richardson Barbra A. , Overbaugh Julie
Published: July 2002

Genetic polymorphisms in chemokine and chemokine receptor genes influence susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression, but little is known regarding the association between these allelic variations and the ability of the host to transmit virus. In this study, we show that the maternal heterozygous SDF1 genotype (SDF1 3′A/wt) is associated with perinatal transmission of HIV-1 (risk ratio [RR], 1.8; 95% confidence interval [CI], 1.0 to 3.3) and particularly postnatal breastmilk transmission (RR, 3.1; 95% CI, 1.1 to 8.6). In contrast, the infant SDF1 genotype had no effect on mother-to-infant transmission. These data suggest that SDF1, which is a ligand for the T-tropic HIV-1 coreceptor CXCR4, may affect the ability of a mother to transmit the virus to her infant. This suggests that a genetic polymorphism in a gene encoding a chemokine receptor ligand may be associated with increased infectivity of the index case and highlights the importance of considering transmission as well as clinical outcome in designing chemokine-based therapies for HIV-1.
Immune Responses and Viral Replication in Long-Term Inapparent Carrier Ponies Inoculated with Equine Infectious Anemia Virus
Authors: Hammond Scott A. , Li Feng , McKeon Brian M. , Cook Sheila J. , Issel Charles J. , Montelaro Ronald C.
Published: July 2002

Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627–9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840–3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10
3
-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.
Internalization of Adenovirus by Alveolar Macrophages Initiates Early Proinflammatory Signaling during Acute Respiratory Tract Infection
Authors: Zsengellér Zsuzsanna , Otake Kazuhisa , Hossain Shaikh-Abu , Berclaz Pierre-Yves , Trapnell Bruce C.
Published: July 2002

Adenovirus is a common respiratory pathogen which causes a broad range of distinct clinical syndromes and has recently received attention for its potential for in vivo gene delivery. Although adenovirus respiratory tract infection (ARTI) results in dose-dependent, local inflammation, the pathogenesis of this remains unclear. We hypothesized that alveolar macrophages (AMφ) rapidly internalize adenovirus following in vivo pulmonary administration and then initiate inflammatory signaling within the lung. To evaluate the role of AMφ in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AMφ and correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF-
α
], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and MIP-1α) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-PCR]) and protein (by enzyme-linked immunosorbent assay) and by identification of cells expressing TNF-α and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labeled with the fluorescent dye (Cy3), was rapidly and widely distributed on epithelial surfaces of airways and alveoli and was very rapidly (∼1 min) localized within AMφ. At 30 min after infection AMφ but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-α and IL-6, thus identifying AMφ as the cell source of initial cytokine signaling. IL-6, TNF-α, MIP-2, and MIP-1α levels progressively increased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h (
P
< 0.05). To begin to define the molecular mechanism(s) by which adenovirus initiates the inflammatory signaling in macrophages, TNF-α expression from adenovirus-infected RAW264.7 macrophages was evaluated in vitro. TNF-α expression was readily detected in adenovirus-infected RAW cell supernatant with kinetics similar to AMφ during in vivo infection. Blockage of virus uptake at specific cellular sites, including internalization (by wortmannin), endosome acidification and/or lysis (by chloroquine) or by Ca
2+
chelation (by BAPTA) completely blocked TNF-α expression. In conclusion, results showed that during ARTI, (i) AMφ rapidly internalized adenovirus, (ii) expression of inflammatory mediators was initiated within AMφ and not airway epithelial or other cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidification. These results have implications for our understanding of the role of the AMφ in the initiation of inflammation following adenovirus infection and adenovirus-mediated gene transfer to the lung.
Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon
Authors: Wang Xiuyan , Li Ming , Zheng Hongyong , Muster Thomas , Palese Peter , Beg Amer A. , Garcı́a-Sastre Adolfo
Published: July 2002

The alpha/beta interferon (IFN-α/β) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-α/β-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-α/β genes, including IFN-β. It is known that transactivation of the IFN-β promoter depends on NF-κB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-κB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-κB pathway during delNS1 virus infection prevented the transactivation of the IFN-β promoter, demonstrating a functional link between NF-κB activation and IFN-α/β synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-κB pathway and of IFN-β synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-κB pathway may thus play a key role in the pathogenesis of influenza A virus.
Lymphotoxin-α-Deficient Mice Can Clear a Productive Infection with Murine Gammaherpesvirus 68 but Fail To Develop Splenomegaly or Lymphocytosis
Authors: Lee Bong Joo , Santee Sybil , Von Gesjen Sigrid , Ware Carl F. , Sarawar Sally R.
Published: July 2002

Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- α-deficient (LTα−/−) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTα−/−mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTα−/−and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTα−/−mice. Furthermore, LTα−/−mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTα−/−mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.
Modulation of Antigen-Specific Humoral Responses in Rhesus Macaques by Using Cytokine cDNAs as DNA Vaccine Adjuvants
Authors: Kim Jong J. , Yang Joo-Sung , VanCott Thomas C. , Lee Daniel J. , Manson Kelledy H. , Wyand Michael S. , Boyer Jean D. , Ugen Kenneth E. , Weiner David B.
Published: July 2002

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.
Dominant-Negative Inhibition of Prion Formation Diminished by Deletion Mutagenesis of the Prion Protein
Authors: Zulianello Laurence , Kaneko Kiyotoshi , Scott Michael , Erpel Susanne , Han Dong , Cohen Fred E. , Prusiner Stanley B.
Published: July 2002

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPCact as dominant-negative, inhibitors of PrPScformation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069–10074, 1997). Trafficking of substituted PrPCto caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPCare responsible for dominant-negative inhibition of PrPScformation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPCto PrPSc. We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPCmolecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPCto an auxiliary molecule that participates in PrPScformation remains to be established.