bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Detection of
Escherichia coli
O157 in Surface Waters by Using Immunomagnetic Electrochemiluminescence
Authors: Shelton Daniel R. , Karns Jeffrey S. 
Published: July 2002

ABSTRACT

A protocol for the quantitative detection of
Escherichia coli
O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10
1
to 10
5
cells ml
−1
in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured
E. coli
O157 cells in the presence of ca. 10
8
cells of a non-O157 strain of
E. coli
ml
−1
. Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10
2
to 10
5
E. coli
O157 cells ml of concentrated water
−1
. To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000
E. coli
O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for
E. coli
O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25
E. coli
O157 cells ml of raw water
−1
, 25 cells 100 ml of 100-fold concentrated water
−1
, or 1 to 2 viable cells liter
−1
with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.
Rapid Immunoassays for Detection of UV-Induced Cyclobutane Pyrimidine Dimers in Whole Bacterial Cells
Authors: Peccia Jordan , Hernandez Mark 
Published: July 2002

ABSTRACT

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active
Mycobacterium parafortuitum
and
Serratia marcescens
cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled (
125
I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in
M. parafortuitum
and
S. marcescens
cells as well as photoenzymatic repair responses in
S. marcescens
cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.
Susceptibility of
Vibrio cholerae
O139 to Antibody-Dependent, Complement-Mediated Bacteriolysis
Authors: Attridge Stephen R. , Qadri Firdausi , Albert M. John , Manning Paul A. 
Published: July 2002

ABSTRACT

Volunteer studies with
Vibrio cholerae
O1 have shown that the best correlate of a vaccine's protective efficacy is its propensity to elicit serum bactericidal responses in its recipients. Attempts to detect such responses following infection with
V. cholerae
O139, however, have met with varying success. Using a tube-based assay which involves viable counting, we now report that strains of serogroup O139 can appear to be sensitive or resistant to a fixed concentration of complement in the presence of antibody, depending on assay conditions. Susceptibility to lysis is critically dependent on the availability of complement, but with O139 indicator strains this is not simply determined by the concentration of serum added to the reaction mix. The nature of the assay diluent and the concentration of indicator bacteria can also dramatically affect bactericidal end points, whereas such variables have minimal significance with O1 indicator bacteria. Although some laboratories use unencapsulated mutant strains to seek evidence of seroconversion following exposure to
V. cholerae
O139, this is not necessary, and our findings question the significance of capsule expression as a determinant of complement sensitivity when antibody is present. The medium used for growth of the indicator strain and the particular strain used appeared to be unimportant. Each of seven O139 isolates tested was found to be lysed by antibody and complement in our standard assay system, which allowed the detection of significant serum bactericidal responses in 9 of 11 cases of O139 disease.
Identification of Murine B-Cell and T-Cell Epitopes ofEscherichia coliOuter Membrane Protein F with Synthetic Polypeptides
Authors: Williams Kristina M. , Bigley Elmer C. , Raybourne Richard B. 
Published: July 2002

ABSTRACTThe major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. BecauseEscherichia coliOmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants ofE. coliOmpF may have broader application.
Mycobacterium tuberculosisChaperonin 60.1 Is a More Potent Cytokine Stimulator than Chaperonin 60.2 (Hsp 65) and Contains a CD14-Binding Domain
Authors: Lewthwaite Jo C. , Coates Anthony R. M. , Tormay Peter , Singh Mahavir , Mascagni Paolo , Poole Stephen , Roberts Michael , Sharp Lindsay , Henderson Brian 
Published: July 2002

ABSTRACTMuch attention has focused on theMycobacterium tuberculosismolecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However,M.tuberculosisis one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed bothM.tuberculosisproteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2. This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins. TheM.tuberculosisCpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies. The cytokine-inducing activity of both chaperonins is extremely resistant to heat. Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.
Immunological Basis for Reactivation of Tuberculosis in Mice
Authors: Turner J. , Gonzalez-Juarrero M. , Saunders B. M. , Brooks J. V. , Marietta P. , Ellis D. L. , Frank A. A. , Cooper A. M. , Orme I. M. 
Published: July 2002

ABSTRACTIn this study different inbred strains of mice appeared to control and contain a low dose aerosol infection withMycobacterium tuberculosisin a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection withM. tuberculosismay reflect an inability to localize antigen-specific lymphocytes within the lung.
Fimbria-Dependent Activation of Cell Adhesion Molecule Expression in
Porphyromonas gingivalis
-Infected Endothelial Cells
Authors: Khlgatian Mary , Nassar Hamdy , Chou Hsin-Hua , Gibson Frank C. , Genco Caroline Attardo 
Published: July 2002

ABSTRACT

Porphyromonas gingivalis
is an oral pathogen that has recently been associated with chronic inflammatory diseases such as atherosclerosis. The strength of the epidemiological associations of
P. gingivalis
with atherosclerosis can be increased by the demonstration that
P. gingivalis
can initiate and sustain growth in human vascular cells. We previously established that
P. gingivalis
can invade aortic, heart, and human umbilical vein endothelial cells (HUVEC), that fimbriae are required for invasion of endothelial cells, and that fimbrillin peptides can induce the expression of the chemokines interleukin 8 and monocyte chemotactic protein. In this study, we examined the expression of surface-associated cell adhesion molecules on endothelial cells in response to
P. gingivalis
infection by fluorescence-activated cell sorting FACS analysis and confocal microscopy. Coculture of HUVEC with
P. gingivalis
strain 381 or A7436 resulted in the induction in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P- and E-selectins, which was maximal at 48 h postinfection. In contrast, we did not observe induction of ICAM-1, VCAM-1, or P- or E-selectin expression in HUVEC cultured with the noninvasive
P. gingivalis fimA
mutant DPG3 or when
P. gingivalis
was incubated with fimbrillin peptide-specific anti-sera prior to the addition to HUVEC. Furthermore, the addition of a peptide corresponding to the N-terminal domain of fimbrillin to HUVEC resulted in an increase in ICAM-1, VCAM-1, and P- and E-selectins, which was maximal at 48 h and similar to that observed for live
P. gingivalis
. Treatment of
P. gingivalis
-infected HUVEC with cytochalsin D, which prevented
P. gingivalis
invasion, also resulted in the inhibition of ICAM-1, VCAM-1, or P- and E-selectin expression. Taken together, these results indicate that active
P. gingivalis
invasion of HUVEC mediated via the major fimbriae stimulates surface-associated cell adhesion molecule expression. Stimulation of adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by
P. gingivalis
may play a role in the pathogenesis of systemic inflammatory diseases associated with this microorganism, including atherosclerosis.
Identification of a Receptor-Binding Domain of
Bordetella
Dermonecrotic Toxin
Authors: Matsuzawa Takeshi , Kashimoto Takashige , Katahira Jun , Horiguchi Yasuhiko 
Published: July 2002

ABSTRACT

Bordetella
dermonecrotic toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating or polyaminating Gln63 of the small GTPase Rho. DNT is an A-B toxin which is composed of an N-terminal receptor-binding (B) domain and a C-terminal enzymatically active (A) domain. In this study, to analyze the functional and structural organization of DNT, we prepared 10 clones of hybridoma producing anti-DNT monoclonal antibodies. One of these antibodies, 2B3, neutralized the effects of DNT on target cells when mixed with the toxin. When microinjected into cells, however, 2B3 did not inhibit the intoxication by DNT. Western blot analysis revealed that 2B3 recognized the N-terminal region of DNT. To delineate the DNT-binding domain, we examined a series of truncated DNT mutants for the ability to competitively inhibit the intoxication of cells by the full-length DNT and found that a fragment consisting of the N-terminal 54 amino acids (DNT
1-54
) was the smallest inhibitory fragment. The radioiodinated DNT
1-54
actually bound to target cells, which was inhibited by 2B3. These results suggest that the N-terminal 54 amino acids of DNT are responsible for the binding to target cells. DNT
1-54
bound to none of the DNT-resistant cells, implying the presence of a cell surface receptor specific to DNT-sensitive cells.
The
Brucella abortus
CcrM DNA Methyltransferase Is Essential for Viability, and Its Overexpression Attenuates Intracellular Replication in Murine Macrophages
Authors: Robertson Gregory T. , Reisenauer Ann , Wright Rachel , Jensen Rasmus B. , Jensen Allen , Shapiro Lucille , Roop R. Martin 
Published: July 2002

ABSTRACT

The CcrM DNA methyltransferase of the α-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in
Caulobacter crescentus
and
Rhizobium meliloti
. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in
Brucella abortus
. Further, increased copy number of the
ccrM
gene results in striking changes in
B. abortus
morphology, DNA replication, and growth in murine macrophages. We generated strains that carry
ccrM
either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in
B. abortus
and increased
ccrM
copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.
Pheromone-Regulated Expression of Sex Pheromone Plasmid pAD1-Encoded Aggregation Substance Depends on at Least Six Upstream Genes and a
cis
-Acting, Orientation-Dependent Factor
Authors: Muscholl-Silberhorn Albrecht B. 
Published: July 2002

ABSTRACT

Conjugative transfer of
Enterococcus faecalis
-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an α-amylase reporter gene,
amyL
, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated α-amylase activity, a total of at least six genes,
traB
,
traC
,
traA
,
traE1
,
orfY
, and
orf1
, are required: TraB efficiently represses
asa1
(by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of
traC
); only
traC
can relieve
traB
-mediated repression in a pheromone-dependent manner. In addition to
traB
,
traA
is required but not sufficient for negative control. Mutational inactivation of
traE1
,
orfY
, or
orf1
, respectively, results in a total loss of α-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering
traA
, P
0
, and
traE1
without disrupting any gene or control element switches off
amyL
or
asa1
expression, indicating the involvement of a
cis
-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in
trans
, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a
trans
-acting positive regulator is discussed.
A New Circadian Class 2 Gene,
opcA
, Whose Product Is Important for Reductant Production at Night in
Synechococcus elongatus
PCC 7942
Authors: Min Hongtao , Golden Susan S. 
Published: July 2002

ABSTRACT

Gene expression in the cyanobacterium
Synechococcus elongatus
PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to
S. elongatus
promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which
purF
is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn
5
mutants for those in which
purF
phasing is altered revealed a mutant that carries an insertion in the
opcA
gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the
purF
promoter is not altered by
opcA
inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in
S. elongatus
and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The
opcA
gene, expected to be cotranscribed with
fbp
and
zwf
, was shown to have its own class 2 promoter, whereas the
fbp
promoter was determined to be in class 1. Thus,
opcA
expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases.
Characterization of a Thermostable DNA Glycosylase Specific for U/G and T/G Mismatches from the Hyperthermophilic Archaeon
Pyrobaculum aerophilum
Authors: Yang Hanjing , Fitz-Gibbon Sorel , Marcotte Edward M. , Tai Jennifer H. , Hyman Elizabeth C. , Miller Jeffrey H. 
Published: July 2002

ABSTRACT

U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeon
Methanobacterium thermoautotrophicum
, a thermophile with an optimal growth temperature of 65°C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeon
Pyrobaculum aerophilum
, whose optimal growth temperature is 100°C. The open reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in
Escherichia coli
and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon,
Aeropyrum pernix
. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.
Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of
Lactococcus lactis
Authors: Picon Antonia , Kunji Edmund R. S. , Lanfermeijer Frank C. , Konings Wil N. , Poolman Bert 
Published: July 2002

ABSTRACT

The kinetic properties of wild-type and mutant oligopeptide binding proteins of
Lactococcus lactis
were determined. To observe the properties of the mutant proteins in vivo, the
oppA
gene was deleted from the chromosome of
L. lactis
to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the
oppA
gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from
Salmonella enterica
serovar Typhimurium and
L. lactis
, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar

K
m

for transport, whereas the
V
max
was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.