bluuebunny/crossref_metadata_2025 huggingface dataset:
Beryllium, an Adjuvant That Promotes Gamma Interferon Production
Authors: Lee Julia Y. , Atochina Olga , King Benjamin , Taylor Leslie , Elloso Merle , Scott Phillip , Rossman Milton D.
Published: July 2002

Beryllium is associated with a human pulmonary granulomatosis characterized by an accumulation of CD4+T cells in the lungs and a heightened specific lymphocyte proliferative response to beryllium (Be) with gamma interferon (IFN-γ) release (i.e., a T helper 1 [Th1] response). While an animal model of Be sensitization is not currently available, Be has exhibited adjuvant effects in animals. The effects of Be on BALB/c mice immunized with soluble leishmanial antigens (SLA) were investigated to determine if Be had adjuvant activity for IFN-γ production, an indicator of the Th1 response. In this strain ofLeishmania-susceptible BALB/c mice, a Th2 response is normally observed after in vivo SLA sensitization and in vitro restimulation with SLA. If interleukin-12 (IL-12) is given during in vivo sensitization with SLA, markedly increased IFN-γ production and decreased IL-4 production are detected. We show here that when beryllium sulfate (BeSO4) was added during in vivo sensitization of BALB/c mice with SLA and IL-12, significantly increased IFN-γ production and decreased IL-4 production from lymph node and spleen cells were detected upon in vitro SLA restimulation. No specific responses were observed to Be alone. Lymph node and spleen cells from all mice proliferated strongly and comparably upon in vitro restimulation with SLA and with SLA plus Be; no differences were noted among groups of mice that received different immunization regimens. In vivo, when Be was added to SLA and IL-12 for sensitization of BALB/c mice, more effective control ofLeishmaniainfection was achieved. This finding has implications for understanding not only the development of granulomatous reactions but also the potential for developing Be as a vaccine adjuvant.
Macrophage Inflammatory Protein 1α/CCL3 Is Required for Clearance of an AcuteKlebsiella pneumoniaePulmonary Infection
Authors: Lindell Dennis M. , Standiford Theodore J. , Mancuso Peter , Leshen Zachary J. , Huffnagle Gary B.
Published: July 2002

The objective of these studies was to determine the role of macrophage inflammatory protein 1α/CCL3 in pulmonary host defense duringKlebsiella pneumoniaeinfection. Following intratracheal inoculation, 7-day survival of CCL3−/−mice was less than 10%, compared to 60% for CCL3+/+mice. Survival of CCR5−/−mice was equivalent to that of controls, indicating that the enhanced susceptibility of CCL3−/−mice toK. pneumoniaeis mediated via another CCL3 receptor, presumably CCR1. At day 3, CFU burden in the lungs of CCL3−/−mice was 800-fold higher than in CCL3+/+mice, demonstrating that CCL3 is critical for control of bacterial growth in the lung. Surprisingly, CCL3−/−mice had no differences in the recruitment of monocytes/macrophages and even showed enhanced neutrophil recruitment at days 1, 2, and 3 postinfection, compared to CCL3+/+mice. Therefore, the defect in clearance was not due to insufficient recruitment of leukocytes. No significant differences in cytokine levels of monocyte chemoattractant protein 1 (MCP-1), interleukin 12, gamma interferon, or tumor necrosis factor alpha in lung lavages were found between CCL3+/+and CCL3−/−mice. CCL3−/−alveolar macrophages were found to have significantly lower phagocytic activity towardK. pneumoniaethan CCL3+/+alveolar macrophages. These findings demonstrate that CCL3 production is critical for activation of alveolar macrophages to control the pulmonary growth of the gram-negative bacteriumK. pneumoniae.
Comparison of Genomic Structures and Antigenic Reactivities of Orthologous 29-Kilodalton Outer Membrane Proteins of
Helicobacter pylori
Authors: Sumie Akihiro , Yamashiro Tetsu , Nakashima Kazutoshi , Nasu Masaru , Watanabe Makoto , Nishizono Akira
Published: July 2002

We purified a 29-kDa
Helicobacter pylori
outer membrane protein (Omp29 protein) and cloned the gene encoding the protein from
H. pylori
strain ATCC 43504. The Omp29 gene corresponded to the reported JHP73 and the HP78–79 genes of
H. pylori
strains. A corresponding nucleotide fragment was detected in all 150 tested
H. pylori
clinical isolates by PCR or Southern blotting. The amplified Omp29-corresponding fragments were categorized into a ca. 770-bp-long group and a larger-fragment group. Sequence analysis indicated that the larger fragments were likely synthesized from the 770-bp fragments by insertion of an irrelevant fragment via 17-bp-long repeat sequences. Immunoblot analysis implies that the ca. 770-bp fragment is responsible for the protein homologous to Omp29, whereas the larger fragments are not responsible for those proteins or encoding antigenically distinct proteins. We postulate that the
H. pylori
outer membrane protein Omp29 can alter its antigenicity through gene modifications mediated by nucleotide transfer.
Interactions ofNeisseria gonorrhoeaewith Mature Human Macrophage Opacity Proteins Influence Production of Proinflammatory Cytokines
Authors: Makepeace Benjamin L. , Watt Peter J. , Heckels John E. , Christodoulides Myron
Published: July 2002

The pathological features of ascending gonococcal infection suggest that proinflammatory mediators secreted by tissue-resident macrophages are important components of the host response. Challenge of fully differentiated, mature macrophages with variants ofNeisseria gonorrhoeaestrain P9 or purified bacterial surface components (pili, lipooligosaccharide, and outer membrane vesicles) induced the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha, growth-related protein α, macrophage inflammatory protein 1α (MIP-1α), and RANTES cytokines but had no effect on IL-8 production. No secretion of IL-1β, epithelial-derived neutrophil attractant 78, granulocyte-macrophage colony-stimulating factor, IL-10, or IL-12 cytokines was observed. Notably, the P9-Opabprotein, in comparison to P9-Opaa, increased the association of gonococci with macrophages and elevated the secretion of cytokines. Thus, variation in Opa protein expression by the gonococcus may be a determining factor in the severity of pelvic inflammatory disease.
Porphyromonas gingivalis
Gingipains and Adhesion to Epithelial Cells
Authors: Chen Tsute , Nakayama Koji , Belliveau Lynn , Duncan Margaret J.
Published: July 2002

Porphyromonas gingivalis
is one of the principal organisms associated with adult periodontitis. Bacterial surface proteins such as fimbriae and gingipain hemagglutinin domains have been implicated as adhesins that actuate colonization of epithelium lining the gingival sulcus. We investigated the genetics of
P. gingivalis
adhesion to monolayers of epithelial cells using wild-type and gingipain mutant strains. These experiments suggested that arginine-specific gingipain (Rgp) catalytic activity modulated adhesion. From the data obtained with
rgp
mutants, we constructed a working hypothesis predicting that attachment and detachment of
P. gingivalis
to epithelial cells were mediated by gingipain adhesin and Rgp catalytic domains, respectively. A membrane-based epithelial cell binding assay, used to locate adhesins in extracellular fractions of wild-type and mutant strains, recognized gingipain peptides as adhesins rather than fimbriae. We developed a capture assay that demonstrated the binding of gingipain adhesin peptides to oral epithelial cells. The adherence of fimbrillin to epithelial cells was detected after heat denaturation of cell fractions. The prediction that Rgp catalytic activities mediated detachment was substantiated when the high level of attachment of an
rgp
mutant was reduced in the presence of wild-type cell fractions that contained gingipain catalytic activities.
Immunogenicity and Protective Efficacy of aPlasmodium yoeliiHsp60 DNA Vaccine in BALB/c Mice
Authors: Sanchez Gloria I. , Sedegah Martha , Rogers William O. , Jones Trevor R. , Sacci John , Witney Adam , Carucci Daniel J. , Kumar Nirbhay , Hoffman Stephen L.
Published: July 2002

The gene encoding the 60-kDa heat shock protein ofPlasmodium yoelii(PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 μg of PyHsp60 DNA vaccine alone or in combination with 30 μg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognizedP. yoeliisporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50P. yoeliisporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test;P= 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 × 103irradiatedP. yoeliisporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.
Mutation of the Gene Encoding Cytotoxic Necrotizing Factor Type 1 (cnf1) Attenuates the Virulence of UropathogenicEscherichia coli
Authors: Rippere-Lampe Karen E. , O'Brien Alison D. , Conran Richard , Lockman Hank A.
Published: July 2002

Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenicEscherichia coli(UPEC). While both epidemiological studies that link CNF1 production byE. coliwith urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced by three different CNF1-producingE. colistrains with isogenic CNF1-deficient derivatives. The data from single-strain challenge experiments with C3H/HeOuJ mice indicated a trend toward higher counts of the wild-type strains in the urine and bladders of these animals up to 3 days after challenge in two of three strain pairs. Furthermore, this difference was statistically significant at day 2 of infection with one strain pair, C189 and C189cnf1. To control for the animal-to-animal variability inherent in this model, we infected C3H/HeOuJ mice with a mixture of CNF1-positive and -negative isogenic derivatives of CP9. The CNF1-positive strain was recovered in higher numbers than the CNF1-negative strain in the urine, bladders, and kidneys of the mice up to 9 days postinfection. These striking coinfection findings, taken with the trends observed in single-strain infections, led us to conclude that CNF1-negative strains were generally attenuated compared to the wild type in the C3H/HeOuJ mouse model of UTI. Furthermore, histopathological examination of bladder specimens from mice infected with CNF1-positive strains consistently showed deeper, more extensive inflammation than in those infected with the isogenic mutants. Lastly, we found that CNF1-positive strain CP9 was better able to resist killing by fresh human neutrophils than were CP9cnf1bacteria. From these data in aggregate, we propose that CNF1 production increases the capacity of UPEC strains to resist killing by neutrophils, which in turn permits these bacteria to gain access to deeper tissue and persist better in the lower urinary tract.
In Vivo Clearance of an Intracellular Bacterium,Francisella tularensisLVS, Is Dependent on the p40 Subunit of Interleukin-12 (IL-12) but Not on IL-12 p70
Authors: Elkins Karen L. , Cooper Allison , Colombini Susan M. , Cowley Siobhán C. , Kieffer Tara L.
Published: July 2002

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection withFrancisella tularensisLVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-γ), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4+and CD8+T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-γ than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-γ production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.
Comparative and Genetic Analyses of the Putative
Vibrio cholerae
Lipopolysaccharide Core Oligosaccharide Biosynthesis (
wav
) Gene Cluster
Authors: Nesper Jutta , Kraiß Anita , Schild Stefan , Blaβ Julia , Klose Karl E. , Bockemühl Jochen , Reidl Joachim
Published: July 2002

We identified five different putative
wav
gene cluster types, which are responsible for the synthesis of the core oligosaccharide (OS) region of
Vibrio cholerae
lipopolysaccharide. Preliminary evidence that the genes encoded by this cluster are involved in core OS biosynthesis came from analysis of the recently released O1 El Tor
V. cholerae
genome sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of O1 El Tor mutant strains defective in three genes (
waaF
,
waaL
, and
wavB
). Investigations of 38 different
V. cholerae
strains by Southern blotting, PCR, and sequencing analyses showed that the O1 El Tor
wav
gene cluster type is prevalent among clinical isolates of different serogroups associated with cholera and environmental O1 strains. In contrast, we found differences in the
wav
gene contents of 19 unrelated non-O1, non-O139 environmental and human isolates not associated with cholera. These strains contained four new
wav
gene cluster types that differ from each other in distinct gene loci, providing evidence for horizontal transfer of
wav
genes and for limited structural diversity of the core OS among
V. cholerae
isolates. Our results show genetic diversity in the core OS biosynthesis gene cluster and predominance of the type 1
wav
gene locus in strains associated with clinical cholera, suggesting that a specific core OS structure could contribute to
V. cholerae
virulence.
ideR
, an Essential Gene in
Mycobacterium tuberculosis
: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response
Authors: Rodriguez G. Marcela , Voskuil Martin I. , Gold Benjamin , Schoolnik Gary K. , Smith Issar
Published: July 2002

The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that
ideR
is an essential gene in
Mycobacterium tuberculosis. ideR
cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare
ideR
mutant was obtained in which the lethal effects of
ideR
inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from
ideR
inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type,
ideR
mutant, and
ideR
-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.
Mycobacterium tuberculosisCDC1551 Is Resistant to Reactive Nitrogen and Oxygen Intermediates In Vitro
Authors: Firmani Marcia A. , Riley Lee W.
Published: July 2002

Resistance to reactive oxygen intermediates and reactive nitrogen intermediates in vitro of a clinical isolate ofMycobacterium tuberculosis(CDC1551) that caused a large outbreak of tuberculosis was compared to that ofM. tuberculosisstrains CB3.3, H37Rv, H37Ra, Erdman, RJ2E, C.C. 13, and C.C. 22 as well asM. bovisstrains Ravenel and BCG. CDC1551 and CB3.3 were significantly more resistant to both hydrogen peroxide (H2O2) and acidified sodium nitrite than were the other strains tested. This biological phenotype may serve as an in vitro marker for clinical strains ofM. tuberculosislikely to cause a large outbreak of tuberculosis.
Positioning of Region 4 of the
Escherichia coli
RNA Polymerase ς
70
Subunit by a Transcription Activator
Authors: Bown Jonathan A. , Kolb Annie , Meares Claude F. , Ishihama Akira , Minchin Stephen D. , Busby Stephen J. W.
Published: July 2002

A DNA cleavage reagent, specifically tethered to residue 581 of the
Escherichia coli
RNA polymerase ς
70
subunit, has been used to investigate the location of ς
70
region 4 in different complexes at the
galp
1
promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the
galp
1
promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of ς
70
region 4.
Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo
Authors: Chernyaeva Natalya S. , Murgola Emanuel J.
Published: July 2002

We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I of
Escherichia coli
23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop in
Bacteria
,
Archaea
, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.
High-Frequency Flp Recombinase-Mediated Inversions of the
oriC
-Containing Region of the
Pseudomonas aeruginosa
Genome
Authors: Barekzi Nazir , Beinlich Kerry , Hoang Tung T. , Pham Xuan-Quynh , Karkhoff-Schweizer RoxAnn , Schweizer Herbert P.
Published: July 2002

The genomes of the two clonally derived
Pseudomonas aeruginosa
prototypic strains PAO1 and DSM-1707 differ by the presence of a 2.19-Mb inversion including
oriC
. Integration of two Flp recombinase target sites near the
rrn
operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including
oriC
, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the
oriC
-containing region of the
P. aeruginosa
chromosome can readily undergo and tolerate large inversions.
Single-Molecule Imaging of Interaction between Dextran and Glucosyltransferase from
Streptococcus sobrinus
Authors: Kaseda Kuniyoshi , Yokota Hiroaki , Ishii Yoshiharu , Yanagida Toshio , Inoue Tetsuyoshi , Fukui Kazuhiro , Kodama Takao
Published: July 2002

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of
Streptococcus sobrinus
. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constrants estimated for TMR-labeled GTF at pH 6.8 and 25°C in the absence and presence of sucrose were 9.2 and 13.3 s
−1
, respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (≥25 s
−1
) under comparable conditions.
Methylotrophic
Methylobacterium
Bacteria Nodulate and Fix Nitrogen in Symbiosis with Legumes
Authors: Sy Abdoulaye , Giraud Eric , Jourand Philippe , Garcia Nelly , Willems Anne , de Lajudie Philippe , Prin Yves , Neyra Marc , Gillis Monique , Boivin-Masson Catherine , Dreyfus Bernard
Published: July 2002

Rhizobia described so far belong to three distinct phylogenetic branches within the α-2 subclass of
Proteobacteria
. Here we report the discovery of a fourth rhizobial branch involving bacteria of the
Methylobacterium
genus. Rhizobia isolated from
Crotalaria
legumes were assigned to a new species, “
Methylobacterium nodulans
,” within the
Methylobacterium
genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of
Methylobacterium
spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the
mxaF
gene, encoding the α subunit of the methanol dehydrogenase, and the
nodA
gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and
nodA
amplification assays showed that “
M. nodulans
” is the only nodulating
Methylobacterium
sp. identified so far. Phylogenetic sequence analysis showed that “
M. nodulans
” NodA is closely related to
Bradyrhizobium
NodA, suggesting that this gene was acquired by horizontal gene transfer.
Indole Can Act as an Extracellular Signal in
Escherichia coli
Authors: Wang Dandan , Ding Xuedong , Rather Philip N.
Published: July 2002

Previous work has shown that
lacZ
fusions to the
cysK, astD, tnaB
, and
gabT
genes in
Escherichia coli
are activated by self-produced extracellular signals. Using a combination of ethyl acetate extraction, reversed-phase C
18
chromatography, and thin-layer chromatography, we have purified an extracellular activating signal from
E. coli
supernatants. Mass spectrometry revealed a molecule with an
m/z
peak of 117, consistent with indole. Nuclear magnetic resonance analysis of the purified
E. coli
factor and synthetic indole revealed identical profiles. Using synthetic indole, a dose-dependent activation was observed with
lacZ
fusions to the
gabT, astD
, and
tnaB
genes. However,
cysK
::
lacZ
and several control fusions were not significantly activated by indole. Conditioned medium prepared from a
tnaA
(tryptophanase) mutant, deficient in indole production, supported 26 to 41% lower activation of the
gabT
and
astD
fusions. The residual level of activation may be due to a second activating signal. Activation of the
tnaB
::
lacZ
fusion was reduced by greater than 70% in conditioned medium from a
tnaA
mutant.
Loss of
Pseudomonas aeruginosa
PhpA Aminopeptidase Activity Results in Increased
algD
Transcription
Authors: Woolwine Samuel C. , Sprinkle April B. , Wozniak Daniel J.
Published: July 2002

Inactivation of
Pseudomonas aeruginosa phpA
, encoding a putative leucine aminopeptidase, results in increased transcription of
algD
. The homologous protein in
Escherichia coli
, PepA, is multifunctional, possessing independent aminopeptidase and DNA-binding activities. Here we provide in vitro evidence that PhpA is an aminopeptidase and show that this activity is the relevant property with regard to
algD
expression. This regulation occurred at the previously mapped
algD
transcription initiation site and was not due to activation of an alternative promoter.
Role of DnaB Helicase in UV-Induced Illegitimate Recombination in
Escherichia coli
Authors: Hanada Katsuhiro , Yamashita Teruhito , Shobuike Yuko , Ikeda Hideo
Published: July 2002

To study the involvement of DNA replication in UV-induced illegitimate recombination, we examined the effect of temperature-sensitive
dnaB
mutations on illegitimate recombination and found that the frequency of illegitimate recombination was reduced by an elongation-deficient mutation,
dnaB14
, but not by an initiation-deficient mutation,
dnaB252
. This result indicates that DNA replication is required for UV-induced illegitimate recombination. In addition, the
dnaB14
mutation also affected spontaneous or UV-induced illegitimate recombination enhanced by the
recQ
mutation. Nucleotide sequence analyses of the recombination junctions showed that DnaB-mediated illegitimate recombination is short homology dependent. Previously, Michel et al. (B. Michel, S. Ehrlich, and M. Uzest, EMBO J. 16:430–438, 1997) showed that thermal treatment of the temperature-sensitive
dnaB8
mutant induces double-stranded breaks, implying that induction of illegitimate recombination occurs. To explain the discrepancy between the observations, we propose a model for DnaB function, in which the
dnaB
mutations may exhibit two types of responses, early and late responses, for double-stranded break formation. In the early response, replication forks stall at damaged DNA, resulting in the formation of double-stranded breaks, and the
dnaB14
mutation reduces the double-stranded breaks shortly after temperature shift-up. On the other hand, in the late response, the arrested replication forks mediated by the
dnaB8
mutation may induce double-stranded breaks after prolonged incubation.
The
Saccharomyces
cerevisiae
Isw2p-Itc1p Complex Represses
INO1
Expression and Maintains Cell Morphology
Authors: Sugiyama Minetaka , Nikawa Jun-Ichi
Published: July 2002

In the yeast
Saccharomyces
cerevisiae
,
IRE1
encodes a bifunctional protein with transmembrane kinase and endoribonuclease activities.
HAC1
encodes a transcription factor which has a basic leucine zipper domain. Both gene products play a crucial role in the unfolded protein response. Mutants in which one of these genes is defective also show the inositol-auxotrophic (Ino
−
) phenotype, but the reason for this has not been clear. To investigate the mechanism underlying the Ino
−
phenotype, we screened a multicopy suppressor gene which can suppress the Ino
−
phenotype of the Δ
hac1
strain. We obtained a truncated form of the
ITC1
gene that has a defect in its 3′ region. Although the truncated form of
ITC1
clearly suppressed the Ino
−
phenotype of the Δ
hac1
strain, the full-length
ITC1
had a moderate effect. The gene products of
ITC1
and
ISW2
are known to constitute a chromatin-remodeling complex (T. Tsukiyama, J. Palmer, C. C. Landel, J. Shiloach, and C. Wu, Genes Dev. 13:686–697, 1999). Surprisingly, the deletion of either
ITC1
or
ISW2
in the Δ
hac1
strain circumvented the inositol requirement and caused derepression of
INO1
even under repression conditions, i.e., in inositol-containing medium. These data indicate that the Isw2p-Itc1p complex usually represses
INO1
expression and that overexpression of the truncated form of
ITC1
functions in a dominant negative manner in
INO1
repression. It is conceivable that the repressor function of this complex is regulated by the C-terminal region of Itc1p.
Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of
Staphylococcus aureus
with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins
Authors: Hussain Muzaffar , Becker Karsten , von Eiff Christof , Schrenzel Jacques , Peters Georg , Herrmann Mathias
Published: July 2002

The ability to attach to host ligands is a well-established pathogenic factor in invasive
Staphylococcus aureus
disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (
srtA
) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene
emp
(1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR,
emp
was demonstrated in all 240
S. aureus
isolates of a defined clinical strain collection as well as in 6
S. aureus
laboratory strains, whereas it is lacking in all 10
S. epidermidis
strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of
S. carnosus
. rEmp expressed in
Escherichia coli
interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a
K

d

of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted
S. aureus
molecules that interact with an extended spectrum of host ligands and thereby contribute to
S. aureus
pathogenicity.
Gene Transfer to the Desiccation-Tolerant Cyanobacterium
Chroococcidiopsis
Authors: Billi Daniela , Friedmann E. Imre , Helm Richard F. , Potts Malcolm
Published: July 2002

The coccoid cyanobacterium
Chroococcidiopsis
dominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation of
Chroococcidiopsis
. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of
Nostoc
sp. strain PCC 7524, were transferred to different isolates of
Chroococcidiopsis
via conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than
Nostoc
and
Anabaena
. Following conjugation, both plasmids replicated in
Chroococcidiopsis
sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P

psb
A

-
luxAB
on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into
Chroococcidiopsis
sp. strain 029 were approximately 10
−2
and 10
−4
transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10
−4
electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain
Chroococcidiopsis thermalis
PCC 7203. The high efficiency of conjugative transfer of
Chroococcidiopsis
sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.
Pyridoxal 5-Phosphate Inhibition of Substrate Selectivity Mutants of UhpT, the Sugar 6-Phosphate Carrier of
Escherichia coli
Authors: Hall Jason A. , Maloney Peter C.
Published: July 2002

In the sugar phosphate transporter UhpT, gain-of-function derivatives that prefer phosphoenolpyruvate (PEP) as substrate have an uncompensated lysine residue on transmembrane segment 11. We show here that these variants are also highly susceptible to substrate-protectable inhibition by covalent modification of lysine with pyridoxal 5-phosphate. The chemical requirements of this interaction provide evidence that the gain-of-function phenotype results from the pairing of the uncompensated lysines in these mutants with the anionic carboxyl group of PEP.
Control of Butanol Formation in
Clostridium acetobutylicum
by Transcriptional Activation
Authors: Thormann Kai , Feustel Lothar , Lorenz Karin , Nakotte Stephan , Dürre Peter
Published: July 2002

The
sol
operon of
Clostridium acetobutylicum
is the essential transcription unit for formation of the solvents butanol and acetone. The recent proposal that transcriptional regulation of this operon is controlled by the repressor Orf5/SolR (R. V. Nair, E. M. Green, D. E. Watson, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 181:319-330, 1999) was found to be incorrect. Instead, regulation depends on activation, most probably by the multivalent transcription factor Spo0A. The operon is transcribed from a single promoter. A second signal identified in primer extension studies results from mRNA processing and can be observed only in the natural host, not in a heterologous host. The first structural gene in the operon (
adhE
, encoding a bifunctional butyraldehyde/butanol dehydrogenase) is translated into two different proteins, the mature AdhE enzyme and the separate butanol dehydrogenase domain. The promoter of the
sol
operon is preceded by three imperfect repeats and a putative Spo0A-binding motif, which partially overlaps with repeat 3 (R3). Reporter gene analysis performed with the
lacZ
gene of
Thermoanaerobacterium thermosulfurigenes
and targeted mutations of the regulatory region revealed that the putative Spo0A-binding motif, R3, and R1 are essential for control. The data obtained also indicate that an additional activator protein is involved.
Correlation between Mutations in the Interferon Sensitivity-Determining Region of NS5A Protein and Viral Load of Hepatitis C Virus Subtypes 1b, 1c, and 2a
Authors: Lusida Maria I. , Nagano-Fujii Motoko , Nidom Chairul A. , Soetjipto  , Handajani Retno , Fujita Tsunenori , Oka Kiyomasa , Hotta Hak
Published: July 2002

In the present study, we analyzed the possible relationship between interferon (IFN) sensitivity-determining region (ISDR) sequence variation of various hepatitis C virus (HCV) subtypes and serum HCV titers in Indonesian patients without IFN treatment. The viremia titers (mean ± standard deviation) of HCV subtype 1b (HCV-1b) isolates with low (three or fewer) and high (four or more) numbers of ISDR mutations were 5.4 ± 0.6 and 4.2 ± 0.9 log
10
RNA copies/ml, respectively, with the difference between the two groups being statistically significant (
P
< 0.01). Similarly, the viremia titers of HCV-1c isolates with low and high numbers of ISDR mutations were 5.3 ± 0.6 and <3.0 ± 0.0 log
10
RNA copies/ml, respectively, with the difference between the two groups being statistically significant (
P
< 0.01). Also, the virus titers of HCV-2a isolates with low and high numbers of ISDR mutations were 4.3 ± 0.7 and 3.5 ± 0.4 log
10
RNA copies/ml, respectively, with the difference between the two groups being statistically significant (
P
< 0.01). Thus, our results demonstrated that virus load in Indonesian patients infected with HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of mutations in the ISDR sequence, implying the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia.
Recurrent
Achromobacter piechaudii
Bacteremia in a Patient with Hematological Malignancy
Authors: Kay Stephen E. , Clark Robert A. , White Karen L. , Peel Margaret M.
Published: July 2002

We describe a recurrent bacteremia caused by
Achromobacter
(formerly
Alcaligenes
)
piechaudii
in association with an intravenous catheter in an immunocompromised 73-year-old man. This is the first reported case of bacteremia due to
A. piechaudii
.
Detection of Rifampin-Resistant
Mycobacterium tuberculosis
in Sputa by Nested PCR-Linked Single-Strand Conformation Polymorphism and DNA Sequencing
Authors: Kim Bum-Joon , Lee Keun-Hwa , Park Bo-Na , Kim Seo-Jeong , Park Eun-Mi , Park Young-Gil , Bai Gil-Han , Kim Sang-Jae , Kook Yoon-Hoh
Published: July 2002

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of
rpoB
DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of
Mycobacterium tuberculosis
. However, due to the nonspecific amplification of
rpoB
DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the
rpoB
DNA of
M. tuberculosis
on the basis of
rpoB
DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect
M. tuberculosis
and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.
Genetic and Phenotypic Features of
Streptococcus pyogenes
Strains Isolated in Brazil That Harbor New
emm
Sequences
Authors: Teixeira Lúcia M. , Barros Rosana R. , Castro Angela Christina D. , Peralta José Mauro , Da Glória S. Carvalho Maria , Talkington Deborah F. , Vivoni Adriana M. , Facklam Richard R. , Beall Bernard
Published: July 2002

In the present study, 37 group A
Streptococcus
(GAS) strains belonging to 13 new
emm
sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated
st204
,
st211
,
st213
,
st809
,
st833
,
st854
,
st2904
,
st2911
,
st2917
,
st2926
,
st3757
,
st3765
, and
st6735
. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the
speB
gene, and 94.6% produced detectable SpeB. Most strains belonging to a given
emm
type had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some
emm
types. Unlike the other isolates, all type
st213
isolates were
speA
positive and produced SpeA. Strains belonging to
st3765
were T6 and opacity factor (OF) negative. Individual isolates within OF-positive
emm
types were associated with unique
sof
gene sequence types, while OF-negative isolates were
sof
negative by PCR. This report provides information on new
emm
sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.
Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle
Authors: Agol Vadim I. , Belov George A. , Bienz Kurt , Egger Denise , Kolesnikova Marina S. , Romanova Lyudmila I. , Sladkova Larissa V. , Tolskaya Elena A.
Published: July 2002

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.
Efficient Lytic Infection of Human Arterial Endothelial Cells by Human Cytomegalovirus Strains
Authors: Kahl M. , Siegel-Axel D. , Stenglein S. , G.  , Jahn  , Sinzger C.
Published: July 2002

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661–5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.
The Explosive Human Immunodeficiency Virus Type 1 Epidemic among Injecting Drug Users of Kathmandu, Nepal, Is Caused by a Subtype C Virus of Restricted Genetic Diversity
Authors: Oelrichs Robert B. , Shrestha Iswar L. , Anderson David A. , Deacon Nicholas J.
Published: July 2002

An explosive epidemic of human immunodeficiency virus type 1 (HIV-1) has been documented among the injecting drug user population of Kathmandu, Nepal, whose seropositivity rate has risen from 0 to 40% between 1995 and 1997. By using Catrimox to preserve whole-blood RNA at ambient temperature for transportation, HIV-1 envelope V3–V4 sequences were obtained from 36 patients in this group. Analysis of the sequences indicated a homogenous epidemic of subtype C virus, with at least two independent introductions of the virus into the population. Viral diversity was restricted within two transmission subclusters, with the majority of variation occurring in V4. Calculation of the synonymous-to-nonsynonymous mutation ratio (Ks:Ka) across this region showed that significant evolutionary pressure had been experienced during the rapid horizontal spread of the virus in this population, most strongly directed to the region between V3 and V4.
Incorporation of Lysyl-tRNA Synthetase into Human Immunodeficiency Virus Type 1
Authors: Cen Shan , Khorchid Ahmad , Javanbakht Hassan , Gabor Juliana , Stello Timothy , Shiba Kiyotaka , Musier-Forsyth Karin , Kleiman Lawrence
Published: July 2002

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA
Lys
isoacceptors are selectively incorporated into virions and tRNA

3

Lys

is used as the primer for reverse transcription. We show herein that the tRNA
Lys
-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55

gag

alone will package LysRS into Pr55

gag

particles, independently of tRNA
Lys
. With the additional presence of the viral precursor protein Pr160

gag-pol

, tRNA
Lys
and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent
M
r
of 70,000, viral LysRS associated with tRNA
Lys
packaging is shorter, with an apparent
M
r
of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA

3

Lys

.