bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of
Escherichia coli
Strains Causing Bacteriuria during Pregnancy: Selection for Strains That Do Not Express Type 1 Fimbriae
Authors: Graham J. C. , Leathart J. B. S. , Keegan S. J. , Pearson J. , Bint A. , Gally D. L. 
Published: July 2002

ABSTRACT

Escherichia coli
isolates from patients with bacteriuria of pregnancy were compared by PCR with isolates from patients with community-acquired cystitis for the presence of established virulence determinants. The strains from patients with bacteriuria of pregnancy were less likely to carry genes for P-family, S-family, and F1C adhesins, cytotoxic necrotizing factor 1, and aerobactin, but virtually all of the strains carried the genes for type 1 fimbriae. Standard mannose-sensitive agglutination of yeast cells showed that only 15 of 42 bacteriuria strains (36%) expressed type 1 fimbriae compared with 32 of 42 strains from community-acquired symptomatic infections (76%) (
P
< 0.01). This difference was confirmed by analysis of all isolates for an allele of the type 1 fimbrial regulatory region (
fim
switch), which negates type 1 fimbrial expression by preventing the
fim
switch from being inverted to the on phase. This allele,
fimS49
, was found in 8 of 47 bacteriuria strains from pregnant women (17.0%) compared with 2 of 60 strains isolated from patients with cystitis (3.3%) (
P
< 0.05). Determination of the phase switch orientation in vivo by analysis of freshly collected infected urine from patients with bacteriuria showed that the
fim
switch was detectable in the off orientation in 17 of 23 urine samples analyzed (74%). These data indicate that type 1 fimbriae are not necessary to maintain the majority of
E. coli
bacteriurias in pregnant women since there appears to be selection against their expression in this particular group. This is in contrast to the considered role of this adhesin in community-acquired symptomatic infections. The lack of type 1 fimbria expression is likely to contribute to the asymptomatic nature of bacteriuria in pregnant women, although approximately one-third of the bacteriuria isolates do possess key virulence determinants. If left untreated, this subset of isolates pose the greatest threat to the health of the mother and unborn child.
Transcription of Candidate Virulence Genes of
Haemophilus ducreyi
during Infection of Human Volunteers
Authors: Throm Robert E. , Spinola Stanley M. 
Published: July 2002

ABSTRACT

Haemophilus ducreyi
expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (
momp, ftpA, losB, lst, cdtC
, and
hhdB
) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with
H. ducreyi
were subjected to reverse transcription-PCR. Transcripts corresponding to
momp, ftpA, losB, lst, cdtB
, and
hhdA
were expressed in vivo. In addition, transcripts for other putative virulence determinants such as
ompA2, tdhA, lspA1
, and
lspA2
were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.
Major Histocompatibility Complex Class II-Independent Generation of Neutralizing Antibodies against T-Cell-DependentBorrelia burgdorferiAntigens Presented by Dendritic Cells: Regulation by NK and γδ T Cells
Authors: Mbow M. Lamine , Zeidner Nordin , Gilmore Robert D. , Dolan Marc , Piesman Joseph , Titus Richard G. 
Published: July 2002

ABSTRACTWe previously showed that adoptive transfer ofBorrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized withB. burgdorferi-pulsed MHC class II-deficient (MHC class II−/−) DCs mounted a humoral response against protective antigens, includingB. burgdorferiouter surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by γδ T cells. In contrast, anti-OspA Ab production required the presence of αβ T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II−/−DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as γδ and NK cells, can influence the generation of a protective humoral response againstB. burgdorferiantigens.
Potential Virulence Role of theLegionella pneumophila ptsPOrtholog
Authors: Higa Futoshi , Edelstein Paul H. 
Published: July 2002

ABSTRACTWe previously identified theLegionella pneumophila ptsP(phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties ofL. pneumophila ptsPand its complete sequence. TheL. pneumophila ptsPwas 2,295 bases in length. Its deduced amino acid sequence had high similarity withptsPorthologs ofPseudomonas aeruginosa, Azotobacter vinelandii, andEscherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of theL. pneumophila ptsPmutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. TheL. pneumophila ptsPmutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. TheL. pneumophila ptsPmutant expressed DotA and IcmX in apparently normal amounts, suggesting that theptsPmutation did not affectdotAandicmXregulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show thatL. pneumophila ptsPis required for full in vivo virulence ofL. pneumophila, most probably by affecting intracellular growth.
Dissociated Linkage of Cytokine-Inducing Activity and Cytotoxicity to Different Domains of Listeriolysin O from
Listeria monocytogenes
Authors: Kohda Chikara , Kawamura Ikuo , Baba Hisashi , Nomura Takamasa , Ito Yutaka , Kimoto Terumi , Watanabe Isao , Mitsuyama Masao 
Published: July 2002

ABSTRACT

Listeriolysin O (LLO), a cholesterol-binding cytolysin of
Listeria monocytogenes
, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-γ)-inducing activities. rLLO529 exhibited both IFN-γ-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-γ production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-γ-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-γ production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-γ-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.
Regulatory Role of Interleukin-10 in Experimental Group B Streptococcal Arthritis
Authors: Puliti Manuela , von Hunolstein Christina , Verwaerde Claudie , Bistoni Francesco , Orefici Graziella , Tissi Luciana 
Published: July 2002

ABSTRACTIntravenous inoculation of CD-1 mice with 107CFU of type IV group BStreptococcus(GBS) results in a high incidence of diffuse septic arthritis,associated with high levels of systemic and local production of interleukin-1β (IL-1β) and IL-6. In this study, the role of the anti-inflammatory cytokine IL-10 in the evolution of GBS systemic infection and arthritis was evaluated. IL-10 production was evident in sera and joints of GBS-infected mice. Neutralization of endogenous IL-10 by administration of anti-IL-10 antibodies (1 mg/mouse) at the time of infection resulted in worsening of articular lesions and 60% mortality associated with early sustained production of IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α). The effect of IL-10 supplementation was assessed by administering IL-10 (100, 200, or 400 ng/mouse) once a day for 5 days, starting 1 h after infection. Treatment with IL-10 had a beneficial effect on GBS arthritis, and there was a clear-cut dose dependence. The decrease in pathology was associated with a significant reduction in IL-6, IL-1β, and TNF-α production. Histological findings showed limited periarticular inflammation and a few-cell influx in the articular cavity of IL-10-treated mice, confirming clinical observations. In conclusion, this study provides further information concerning the role of IL-10 in regulating the immune response and inflammation and calls attention to the potential therapeutic use of IL-10 in GBS arthritis.
Transformation of
Rickettsia prowazekii
to Erythromycin Resistance Encoded by the
Escherichia coli ereB
Gene
Authors: Rachek Lyudmila I. , Hines Andria , Tucker Aimee M. , Winkler Herbert H. , Wood David O. 
Published: July 2002

ABSTRACT

Rickettsia prowazekii
, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an
R. prowazekii rpoB
chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in
R. prowazekii
of the
Escherichia coli ereB
gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in
R. prowazekii
. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.
HARO7
Encodes Chorismate Mutase of the Methylotrophic Yeast
Hansenula polymorpha
and Is Derepressed upon Methanol Utilization
Authors: Krappmann Sven , Pries Ralph , Gellissen Gerd , Hiller Mark , Braus Gerhard H. 
Published: July 2002

ABSTRACT

The
HARO7
gene of the methylotrophic, thermotolerant yeast
Hansenula polymorpha
was cloned by functional complementation.
HARO7
encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC
5.4.99.5
) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The
HARO7
gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (
k
cat
= 319.1 s
−1
,

n
H

= 1.56, [
S
]
0.5
= 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of
Saccharomyces cerevisiae
. Using the Cre-
loxP
recombination system, we constructed mutant strains carrying a disrupted
HARO7
gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb
HARO7
mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on
HARO7
expression.
Sequence Variations within PrfA DNA Binding Sites and Effects on
Listeria monocytogenes
Virulence Gene Expression
Authors: Williams James R. , Thayyullathil Chetna , Freitag Nancy E. 
Published: July 2002

ABSTRACT

Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to
Listeria monocytogenes
virulence gene expression. Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as
hly
and
mpl
. Other virulence genes, such as
actA
and
plcB
, may depend upon additional factors for full regulation of gene expression.
Accumulation of the PhaP Phasin of
Ralstonia eutropha
Is Dependent on Production of Polyhydroxybutyrate in Cells
Authors: York Gregory M. , Junker Björn H. , Stubbe JoAnne , Sinskey Anthony J. 
Published: July 2002

ABSTRACT

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of
Ralstonia eutropha. R. eutropha phaC
PHA synthase and
phaP
phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a
phaP
-
gfp
translational fusion in place of the wild-type alleles of
phaC
and
phaP
. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the
phaP
-
gfp
fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that
R. eutropha
has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.
Global Analysis of the General Stress Response of
Bacillus subtilis
Authors: Petersohn Anja , Brigulla Matthias , Haas Stefan , Hoheisel Jörg D. , Völker Uwe , Hecker Michael 
Published: July 2002

ABSTRACT

Gene arrays containing all currently known open reading frames of
Bacillus subtilis
were used to examine the general stress response of
Bacillus
. By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as ς
B
-dependent general stress genes. The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor ς
B
. At least 24 of these 125 ς
B
-dependent genes seemed to be subject to a second, ς
B
-independent stress induction mechanism. Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the ς
B
regulon indicates that even this approach has not yet elucidated the entire regulon. Most of the ς
B
-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their classification as ς
B
-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance. A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress. The first results of this program, showing the diminished salt resistance of
yjbC
and
yjbD
mutants compared to that of the wild type, are presented. Only a few new ς
B
-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase. In addition to analysis of the ς
B
-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a ς
B
-independent manner is presented. Perhaps the most interesting of the ς
B
-independent stress phenomena was the induction of the extracytoplasmic function sigma factor ς
W
and its entire regulon by salt shock.
Redox Signal Transduction by the ArcB Sensor Kinase of
Haemophilus influenzae
Lacking the PAS Domain
Authors: Georgellis Dimitris , Kwon Ohsuk , Lin Edmund C. C. , Wong Sandy M. , Akerley Brian J. 
Published: July 2002

ABSTRACT

The Arc (anoxic redox control) two-component signal transduction system of
Escherichia coli
, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the
arcA
and
arcB
genes of
Haemophilus influenzae
specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of
H. influenzae
mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in
E. coli
, indicating a high degree of functional conservation of the signal transduction system. The
H. influenzae
ArcB, however, lacks the PAS domain present in the region of
E. coli
ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of
H. influenzae
mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.
Biochemical Analysis of Interactions between Outer Membrane Proteins That Contribute to Starch Utilization by
Bacteroides thetaiotaomicron
Authors: Cho Kyu Hong , Salyers Abigail A. 
Published: July 2002

ABSTRACT

An early step in the utilization of starch by
Bacteroides thetaiotaomicron
is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of
B. thetaiotaomicron
that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (μ
max
, 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex.
Ability for Anaerobic Growth Is Not Sufficient for Development of the Petite Phenotype in
Saccharomyces kluyveri
Authors: Mo/ller Kasper , Olsson Lisbeth , Piškur Jure 
Published: July 2002

ABSTRACT

Saccharomyces cerevisiae
is a petite-phenotype-positive (“petite-positive”) yeast, which can successfully grow in the absence of oxygen. On the other hand,
Kluyveromyces lactis
as well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that
Saccharomyces kluyveri
can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view,
S. kluyveri
is apparently more closely related to
S. cerevisiae
than to
K. lactis
. These observations suggest that the progenitor of the modern
Saccharomyces
and
Kluyveromyces
yeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the
K. lactis
and
S. kluyveri-S. cerevisiae
lineages, the latter developed the ability to grow anaerobically. However, while the
S. kluyveri
lineage has remained petite negative, the lineage leading to the modern
Saccharomyces
sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.
Characterization of In Vitro Interactions between a Truncated TonB Protein fromEscherichia coliand the Outer Membrane Receptors FhuA and FepA
Authors: Moeck Gregory S. , Letellier Lucienne 
Published: July 2002

ABSTRACTHigh-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB structure and function, we expressed a recombinant form ofEscherichia coliTonB lacking the N-terminal cytoplasmic membrane anchor. This protein (H6-′TonB;Mr, 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation experiments indicated that the H6-′TonB preparation was almost monodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography. The frictional ratio (2.0) suggested that H6-′TonB adopts a highly asymmetrical form with an axial ratio of 15. H6-′TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified H6-′TonB was found to be stable and thus shows promise for high-resolution structural studies.