bluuebunny/crossref_metadata_2025 huggingface dataset:
Brachyspira
(
Serpulina
)
hyodysenteriae gyrB
Mutants and Interstrain Transfer of Coumermycin A
1
Resistance
Authors: Stanton Thaddeus B. , Matson Eric G. , Humphrey Samuel B.
Published: July 2002

To further develop genetic techniques for the enteropathogen
Brachyspira hyodysenteriae
, the
gyrB
gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes.
B. hyodysenteriae
coumermycin A
1
-resistant (Cn
r
) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A
1
/ml. The coumermycin A
1
MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cn
r
strains had single nucleotide changes in their
gyrB
genes, corresponding to GyrB amino acid changes of Gly
78
to Ser (two strains), Gly
78
to Cys, and Thr
166
to Ala. When Cn
r
strain 435A (Gly
78
to Ser) and Cm
r
Km
r
strain SH (Δ
flaA1
::
cat
Δ
nox
::
kan
) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A
1
, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn
r
Km
r
Cm
r
strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn
r
Km
r
Cm
r
cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a
gyrB
mutation is a useful selection marker for monitoring gene exchange between
B. hyodysenteriae
cells. Gene transfer readily occurs between
B. hyodysenteriae
cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.
Synthesis and Fermentation Properties of Novel Galacto-Oligosaccharides by β-Galactosidases from
Bifidobacterium
Species
Authors: Rabiu Bodun A. , Jay Andrew J. , Gibson Glenn R. , Rastall Robert A.
Published: July 2002

β-Galactosidase enzymes were extracted from pure cultures of
Bifidobacterium angulatum, B. bifidum
BB-12,
B. adolescentis
ANB-7,
B. infantis
DSM-20088, and
B. pseudolongum
DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase in growth rate, compared to Oligomate 55, with products derived from
B. angulatum, B. bifidum, B. infantis
, and
B. pseudolongum
. However
B. adolescentis
had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the
B. bifidum
BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.
Quantitative Detection of
Escherichia coli
O157 in Surface Waters by Using Immunomagnetic Electrochemiluminescence
Authors: Shelton Daniel R. , Karns Jeffrey S.
Published: July 2002

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

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

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

The 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

Much 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

In 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

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

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

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

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

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

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

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.
The Sulfur-Regulated Arylsulfatase Gene Cluster of
Pseudomonas aeruginosa
, a New Member of the
cys
Regulon
Authors: Hummerjohann Jörg , Laudenbach Sascha , Rétey Julia , Leisinger Thomas , Kertesz Michael A.
Published: July 2002

A gene cluster upstream of the arylsulfatase gene (
atsA
) in
Pseudomonas aeruginosa
was characterized and found to encode a putative ABC-type transporter, AtsRBC. Mutants with insertions in the
atsR
or
atsB
gene were unable to grow with hexyl-, octyl-, or nitrocatecholsulfate, although they grew normally with other sulfur sources, such as sulfate, methionine, and aliphatic sulfonates. AtsRBC therefore constitutes a general sulfate ester transport system, and desulfurization of aromatic and medium-chain-length aliphatic sulfate esters occurs in the cytoplasm. Expression of the
atsR
and
atsBCA
genes was repressed during growth with sulfate, cysteine, or thiocyanate. No expression of these genes was observed in the
cysB
mutant PAO-CB, and the
ats
genes therefore constitute an extension of the
cys
regulon in this species.
Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in
Synechocystis
sp. Strain PCC 6803
Authors: Ermakova-Gerdes Svetlana , Yu Zhenbao , Vermaas Wim
Published: July 2002

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium
Synechocystis
sp. strain PCC 6803, we randomly mutagenized a region of
psbDI
(coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a
Synechocystis
sp. strain PCC 6803 mutant that lacks both
psbD
genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q
A
-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in
Synechocystis
sp. strain PCC 6803 was developed to facilitate genetic analysis of
psbDI
mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in
Synechocystis
sp. strain PCC 6803 than was generally assumed.
Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium
Acidithiobacillus caldus
and Analysis of the Plasmid Replicon
Authors: Gardner Murray N. , Deane Shelly M. , Rawlings Douglas E.
Published: July 2002

A moderately thermophilic (45 to 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic
Acidithiobacillus caldus
strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the
A. caldus
cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in
Escherichia coli
. Autonomous replication was also demonstrated in
Pseudomonas putida
and
Agrobacterium tumefaciens
LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14
oriV
region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both
E. coli,
and
A. caldus
. The
rep
gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's
oriV
regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in
trans
. Two smaller open reading frames were found between the
repB
and
repA
genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.
Identification of the Outer Membrane Porin of
Thermus thermophilus
HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance
Authors: Maier Elke , Polleichtner Georg , Boeck Birgit , Schinzel Reinhard , Benz Roland
Published: July 2002

The outer membrane of the thermophilic bacterium
Thermus thermophilus
was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.
Twin-Arginine Translocation Pathway in
Streptomyces lividans
Authors: Schaerlaekens Kristien , Schierová Michaela , Lammertyn Elke , Geukens Nick , Anné Jozef , Van Mellaert Lieve
Published: July 2002

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in
Streptomyces lividans
, a gram-positive organism with a high secretion capacity. The presence of one
tatC
and two
hcf106
homologs in the
S. lividans
genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the
S. lividans
secretome. To demonstrate its functionality, a
tatC
deletion mutant was constructed. This mutation impaired the translocation of the
Streptomyces antibioticus
tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in
Escherichia coli
, could be translocated in wild-type
S. lividans
but not in the
tatC
mutant. In contrast, the secretion of the Sec-dependent
S. lividans
subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in
Streptomyces
spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the
E. coli
TorA signal peptide can direct Tat-dependent transport in
S. lividans
.
Alternative Translation Initiation Produces a Short Form of a Spore Coat Protein in
Bacillus subtilis
Authors: Ozin Amanda J. , Costa Teresa , Henriques Adriano O. , Moran Charles P.
Published: July 2002

During endospore formation in
Bacillus subtilis,
over two dozen polypeptides are localized to the developing spore and coordinately assembled into a thick multilayered structure called the spore coat. Assembly of the coat is initiated by the expression of morphogenetic proteins SpoIVA, CotE, and SpoVID. These morphogenetic proteins appear to guide the assembly of other proteins into the spore coat. For example, SpoVID forms a complex with the SafA protein, which is incorporated into the coat during the early stages of development. At least two forms of SafA are found in the mature spore coat: a full-length form and a shorter form (SafA-C
30
) that begins with a methionine encoded by codon 164 of
safA
. In this study, we present evidence that the expression of SafA-C
30
arises from translation initiation at codon 164. We found only a single transcript driving expression of SafA. A stop codon engineered just upstream of a predicted ribosome-binding site near codon M164 abolished formation of full-length SafA, but not SafA-C
30
. The same effect was observed with an alanine substitution at codon 1 of SafA. Accumulation of SafA-C
30
was blocked by substitution of an alanine codon at codon 164, but not by a substitution at a nearby methionine at codon 161. We found that overproduction of SafA-C
30
interfered with the activation of late mother cell-specific transcription and caused a strong sporulation block.
Alcohol-Induced Delay of Viability Loss in Stationary-Phase Cultures of
Escherichia coli
Authors: Vulić Marin , Kolter Roberto
Published: July 2002

During prolonged incubation in stationary phase
Escherichia coli
undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain
n
-alcohols within a specific time window in stationary phase also prevents viability loss, in an
rpoS
-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of σ
s
such as cell density signals and sensing the nutrient content of the medium.
Definition of the Mycobacterial SOS Box and Use To Identify LexA-Regulated Genes inMycobacterium tuberculosis
Authors: Davis Elaine O. , Dullaghan Edith M. , Rand Lucinda
Published: July 2002

The bases of the mycobacterial SOS box important for LexA binding were determined by replacing each base with every other and examining the effect on the induction of a reporter gene following DNA damage. This analysis revealed that the SOS box was longer than originally thought by 2 bp in each half of the palindromic site. A search of theMycobacterium tuberculosisgenome sequence with the new consensus, TCGAAC(N)4GTTCGA, identified 4 sites which were perfect matches and 12 sites with a single mismatch which were predicted to bind LexA. Genes which could potentially be regulated by these SOS boxes were ascertained from their positions relative to the sites. Examination of expression data for these genes following DNA damage identified 12 new genes which are most likely regulated by LexA as well as the knownM. tuberculosisDNA damage-inducible genesrecA,lexA, andruvC. Of these 12 genes, only 2 have a predicted function:dnaE2, a component of DNA polymerase III, andlinB, which is similar to 1,3,4,6-tetrachloro-1,4-cylcohexadiene hydrolase. Curiously, of the remaining 10 genes predicted to be LexA regulated, 7 are members of theM. tuberculosis13E12 repeat family, which has some of the characteristics of mobile elements.
Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of
Mycobacterium tuberculosis
in Mexico
Authors: Banaiee N. , Bobadilla-del-Valle M. , Bardarov S. , Riska P. F. , Small P. M. , Ponce-de-Leon A. , Jacobs W. R. , Hatfull G. F. , Sifuentes-Osornio J.
Published: July 2002

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of
Mycobacterium tuberculosis
was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of
p
-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) was used for selective identification of
M. tuberculosis
complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.
Rapid Real-Time PCR for Determination of Penicillin Susceptibility in Pneumococcal Meningitis, Including Culture-Negative Cases
Authors: Kearns Angela M. , Graham Clive , Burdess Doreen , Heatherington Jane , Freeman Roger
Published: July 2002

A novel real-time PCR-hybridization assay was developed for the rapid (<1 h) detection of penicillin susceptibility inStreptococcus pneumoniae. When applied to 24 pneumococcal DNA-positive cerebrospinal fluid extracts, penicillin-sensitiveS. pneumoniaewas detected in all instances. Real-time PCR proved more sensitive than culture, microscopy, or antigen detection and provided susceptibility data even in culture-negative cases.
Comparison of the ESP and BACTEC Systems for Testing Susceptibilities of
Mycobacterium tuberculosis
Complex Isolates to Pyrazinamide
Authors: LaBombardi Vincent J.
Published: July 2002

Fifty isolates of
Mycobacterium tuberculosis
complex were tested for susceptibility to pyrazinamide (PZA) by the ESP (Trek Diagnostic Systems, Westlake, Ohio) and BACTEC (BD Biosciences, Sparks, Md.) test systems. Initial results showed concordance for 48 of the isolates. On retest, the two discordant isolates were resolved in favor of the ESP system. The ESP Myco susceptibility test system generates rapid, reliable PZA test results.
Generation and Characterization of a Hepatitis C Virus NS3 Protease-Dependent Bovine Viral Diarrhea Virus
Authors: Lai Vicky C. H. , Zhong Weidong , Skelton Angela , Ingravallo Paul , Vassilev Venteislav , Donis Ruben O. , Hong Zhi , Lau Johnson Y. N.
Published: July 2002

Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P
H/B
(wild-type HCV NS3 protease) and P
H/B(S139A)
(mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P
H/B
clone yielded viable viruses, whereas the mutant clone, P
H/B(S139A)
, failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV−Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV−Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.
Innate Immune Response of the Human Host to Exposure with Herpes Simplex Virus Type 1: In Vitro Control of the Virus Infection by Enhanced Natural Killer Activity via Interleukin-15 Induction
Authors: Ahmad Ali , Sharif-Askari Ehsan , Fawaz Lama , Menezes José
Published: July 2002

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-γ], tumor necrosis factor alpha, or IFN-α). Anti-CD122 antibodies which block signaling through IL-2 receptor β chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.