bluuebunny/crossref_metadata_2025_split huggingface dataset:
Surface Loop Motion in FepA
Authors: Scott Daniel C. , Newton Salete M. C. , Klebba Phillip E. 
Published: October 2002

ABSTRACTUsing a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops ofEscherichia coliFepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanidem-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.
The Membrane-Bound α-Glucuronidase from
Pseudomonas cellulosa
Hydrolyzes 4-
O-
Methyl-
d
-Glucuronoxylooligosaccharides but Not 4-
O-
Methyl-
d
-Glucuronoxylan
Authors: Nagy Tibor , Emami Kaveh , Fontes Carlos M. G. A. , Ferreira Luis M. A. , Humphry David R. , Gilbert Harry J. 
Published: October 2002

ABSTRACT

The microbial degradation of xylan is a key biological process. Hardwood 4-
O-
methyl-
d
-glucuronoxylans are extensively decorated with 4-
O
-methyl-
d
-glucuronic acid, which is cleaved from the polysaccharides by α-glucuronidases. In this report we describe the primary structures of the α-glucuronidase from
Cellvibrio mixtus
(
C. mixtus
GlcA67A) and the α-glucuronidase from
Pseudomonas cellulosa
(
P. cellulosa
GlcA67A) and characterize
P. cellulosa
GlcA67A. The primary structures of
C. mixtus
GlcA67A and
P. cellulosa
GlcA67A, which are 76% identical, exhibit similarities with α-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad α-glucuronidase released 4-
O
-methyl-
d
-glucuronic acid from 4-
O
-methyl-
d
-glucuronoxylooligosaccharides but not from 4-
O-
methyl-
d
-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-
O-
methyl-
d
-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.
Filamentous Bacteriophages of Vibrios Are Integrated into the
dif
-Like Site of the Host Chromosome
Authors: Iida Tetsuya , Makino Kozo , Nasu Hatsumi , Yokoyama Katsushi , Tagomori Kenichi , Hattori Akiko , Okuno Toshihiro , Shinagawa Hideo , Honda Takeshi 
Published: October 2002

ABSTRACT

The
dif
site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (
Vibrio parahaemolyticus
) and CTXφ (
V. cholerae
), are integrated into the
dif
-like site of host chromosome.
Conserved Amplification of Chemotactic Responses through Chemoreceptor Interactions
Authors: Lamanna Allison C. , Gestwicki Jason E. , Strong Laura E. , Borchardt Sara L. , Owen Robert M. , Kiessling Laura L. 
Published: October 2002

ABSTRACT

Many bacteria concentrate their chemoreceptors at the cell poles. Chemoreceptor location is important in
Escherichia coli
, since chemosensory responses are sensitive to receptor proximity. It is not known, however, whether chemotaxis in other bacteria is similarly regulated. To investigate the importance of receptor-receptor interactions in other bacterial species, we synthesized saccharide-bearing multivalent ligands that are designed to cluster relevant chemoreceptors. As has been shown with
E. coli
, we demonstrate that the behaviors of
Bacillus subtilis
,
Spirochaete aurantia
, and
Vibrio furnissii
are sensitive to the valence of the chemoattractant. Moreover, in
B. subtilis
, chemotactic responses to serine were increased by pretreatment with saccharide-bearing multivalent ligands. This result indicates that, as in
E. coli
, signaling information is transferred among chemoreceptors in
B. subtilis
. These results suggest that interreceptor communication may be a general mechanism for modulating chemotactic responses in bacteria.
Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination
Authors: Arkov Alexey L. , Hedenstierna Klas O. F. , Murgola Emanuel J. 
Published: October 2002

ABSTRACT

Nucleotide 1093 in domain II of
Escherichia coli
23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.
barS1, a Gene for Biosynthesis of a γ-Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production inStreptomycesSpecies
Authors: Shikura Noriyasu , Yamamura Junji , Nihira Takuya 
Published: October 2002

ABSTRACTFromStreptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M1and S is tightly regulated by a low-molecular-weightStreptomyceshormone called virginiae butanolide (VB), which is a member of the γ-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression inEscherichia coliand by gene disruption inS. virginiae. ThebarS1gene (a 774-bp open reading frame encoding a 257-amino-acid protein [Mr, 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene,barA. The deduced BarS1 protein is weakly homologous to β-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In theΔbarS1mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by theΔbarS1mutant was fully recovered by the external addition of VB to the culture, which indicates that thebarS1gene is essential in the biosynthesis of the autoregulator VBs inS. virginiaeand that the failure of virginiamycin production was a result of the loss of VB production.
R391: a Conjugative Integrating Mosaic Comprised of Phage, Plasmid, and Transposon Elements
Authors: Böltner Dietmar , MacMahon Claire , Pembroke J. Tony , Strike Peter , Osborn A. Mark 
Published: October 2002

ABSTRACTThe conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South AfricanProvidencia rettgeristrain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element fromVibrio choleraeO139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences fromSalmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.
Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis
Authors: Lievano Fabio A. , Reynolds Meredith A. , Waring Alfred L. , Ackelsberg Joel , Bisgard Kristine M. , Sanden Gary N. , Guris Dalya , Golaz Anne , Bopp Dianna J. , Limberger Ronald J. , Smith Perry F. 
Published: October 2002

ABSTRACT

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for
Bordetella pertussis
was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for
B. pertussis
during both outbreaks, which confirmed that
B. pertussis
was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that
B. pertussis
may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for
B. pertussis
. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.
Pharmacoepidemiological Analysis of Provincial Differences between Consumption of Macrolides and Rates of Erythromycin Resistance among
Streptococcus pyogenes
Isolates in Spain
Authors: García-Rey C. , Aguilar L. , Baquero F. , Casal J. , Martín J. E. 
Published: October 2002

ABSTRACT

The M phenotype is by far the most common mechanism of erythromycin resistance among
Streptococcus pyogenes
isolates in Spain. A geographic analysis of the relationship between within-country differences in the prevalence of M-type resistance to erythromycin in
S. pyogenes
and the level of consumption of 14- and 15-membered macrolides within different provinces was carried out. From 1998 to 1999, a nationwide multicenter surveillance study yielded 2,039 consecutive pharyngeal isolates of
S. pyogenes
. Data on antibiotic consumption for the same period were gathered from IMS Health, and the corresponding daily defined doses per 1,000 inhabitants per day were calculated according to the Anatomic Therapeutic Classification index. Macrolide use was subdivided into dosages given three times a day (TID), twice a day (BID), or once a day (OD). Spearman nonparametric correlation coefficients (
R
) were calculated, and variables proving to be significantly associated (
P
< 0.1) were introduced into a linear regression model. The total consumption of macrolides presented a significant correlation with the prevalence of resistance (
R
= 0.527;
P
= 0.032). Neither TID nor BID macrolide consumption showed significant correlations. Only OD consumption had a significance below 0.1. These data are consistent with the hypothesis that only the total consumption of macrolides influences the local rates of M-type erythromycin resistance in
S. pyogenes,
and subgroups of macrolides seem to have an additive rather than a selective effect by contributing to increasing the final amount of macrolides used. Local variations in total consumption were associated only with BID consumption (
R
= 0.849;
P
= 0.004). The simple linear regression with total macrolide consumption showed a considerable determination coefficient (
R
2
= 0.678;
P
= 0.006). The model explains up to 68% of the measured variation and is clearly better as a predictor of the prevalence of resistance than the mere mean is. By solving the regression equation, the resultant value of 2.2 defined doses per 1,000 inhabitants per day fits with the existence of a critical threshold of selective pressure.
External Quality Assessment Program for Qualitative and Quantitative Detection of Hepatitis C Virus RNA in Diagnostic Virology
Authors: Schirm Jurjen , van Loon Anton M. , Valentine-Thon Elizabeth , Klapper Paul E. , Reid Jim , Cleator Graham M. 
Published: October 2002

ABSTRACT

To assess the performance of laboratories in detecting and quantifying hepatitis C virus (HCV) RNA levels in HCV-infected patients, we distributed two proficiency panels for qualitative and quantitative HCV RNA testing. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each panel consisted of two negative samples and six positive samples, with HCV RNA target levels from 200 to 500,000 copies/ml. Panel 1 had four samples with at least 50,000 copies/ml, and panel 2 had two samples with at least 50,000 copies/ml. Fifty-seven laboratories submitted 45 qualitative and 35 quantitative data sets on panel 1, and 81 laboratories submitted 75 qualitative and 48 quantitative data sets on panel 2. In both panels, about two-thirds of the qualitative data sets and >90% of the quantitative data sets were obtained with commercial assays. With each panel, two data sets gave one false-positive result, corresponding to false-positivity rates of 1.3% and 0.8% for panel 1 and panel 2, respectively. Samples containing at least 50,000 copies/ml were found positive in 97% and 99% of the cases with panel 1 and panel 2, respectively. In contrast, the positive samples containing ≤5,000 copies/ml were reported positive in only 71% and 77% of the cases with panel 1 and panel 2, respectively. Adequate or better scores on qualitative results (all results correct or only the low-positive samples missed) were obtained in 84% (panel 1) and 80% (panel 2) of the data sets. In the analysis of quantitative results, 60% (panel 1) and 73% (panel 2) of the data sets obtained an adequate or better score (≥80% of the positive results within the range of the geometric mean ± 0.5 log
10
). Our results indicate that considerable improvements in molecular detection and quantitation of HCV have been achieved, particularly through the use of commercial assays. However, the lowest detection levels of many assays are still too high, and further standardization is still needed. Finally, this study underlines the importance of proficiency panels for monitoring the quality of diagnostic laboratories.
Analysis of Sequences and Loci of
p44
Homologs Expressed by
Anaplasma phagocytophila
in Acutely Infected Patients
Authors: Lin Quan , Zhi Ning , Ohashi Norio , Horowitz Harold W. , Aguero-Rosenfeld Maria E. , Raffalli John , Wormser Gary P. , Rikihisa Yasuko 
Published: October 2002

ABSTRACT

Anaplasma phagocytophila
is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of
A. phagocytophila
are encoded by a
p44
multigene family. In the present study, expression profiles of
p44
genes in the blood of acutely infected patients in the year 2000 were characterized. A single
p44
gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different
p44
genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several
p44
genes were identical among the isolates from the three patients and a 1995
A. phagocytophila
isolate.
A. phagocytophila
was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995
A. phagocytophila
isolates with probes specific to the most dominant
p44
transcript in each patient showed that the
p44
loci in the
A. phagocytophila
genome were conserved. Analysis of the predicted amino acid sequences of 43 different
p44
genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of
p44
are not random but are restricted. Furthermore, several
p44
genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.
Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii
Authors: Larsen Hans Henrik , Kovacs Joseph A. , Stock Frida , Vestereng Vibeke H. , Lundgren Bettina , Fischer Steven H. , Gill Vee J. 
Published: October 2002

ABSTRACTA method for reliable quantification ofPneumocystis cariniiin research models ofP. cariniipneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detectingP. cariniif. sp.carinii, the subspecies ofP. cariniicommonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r= 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions ofP. cariniiorganisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r= 0.99). The assay was applied to a recently reported in vitro axenic cultivation system forP. cariniiand confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease inP. cariniiDNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay forP. cariniif. sp.cariniihas been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.
Large-Scale Screening of Nasal Swabs for
Bacillus anthracis
: Descriptive Summary and Discussion of the National Institutes of Health's Experience
Authors: Kiratisin Pattarachai , Fukuda Caroline D. , Wong Alexandra , Stock Frida , Preuss Jeanne C. , Ediger Laura , Brahmbhatt Trupti N. , Fischer Steven H. , Fedorko Daniel P. , Witebsky Frank G. , Gill Vee J. 
Published: October 2002

ABSTRACT

In October 2001, a letter containing a large number of anthrax spores was sent through the Brentwood post office in Washington, D.C., to a United States Senate office on Capitol Hill, resulting in contamination in both places. Several thousand people who worked at these sites were screened for spore exposure by collecting nasal swab samples. We describe here a screening protocol which we, as a level A laboratory, used on very short notice to process a large number of specimens (3,936 swabs) in order to report preliminary results as quickly as possible. Six isolates from our screening met preliminary criteria for
Bacillus anthracis
identification and were referred for definitive testing. Although none of the isolates was later confirmed to be
B. anthracis
, we studied these isolates further to define their biochemical characteristics and 16S rRNA sequences. Four of the six isolates were identified as
Bacillus megaterium
, one was identified as
Bacillus cereus
, and one was an unidentifiable
Bacillus
sp. Our results suggest that large-scale nasal-swab screening for potential exposure to anthrax spores, particularly if not done immediately postexposure, may not be very effective for detecting
B. anthracis
but may detect a number of
Bacillus
spp. that are phenotypically very similar to
B. anthracis
.
Presence of Integron-Associated Resistance in the Community Is Widespread and Contributes to Multidrug Resistance in the Hospital
Authors: Leverstein-van Hall M. A. , Paauw A. , Box A. T. A. , Blok H. E. M. , Verhoef J. , Fluit A. C. 
Published: October 2002

ABSTRACT

Integrons are strongly associated with the multidrug resistance seen in gram-negative bacilli in the hospital environment. No data, however, are available on their prevalence in the community. This study is the first to show that integrons are widespread in
Enterobacteriaceae
in the community and that integron-associated resistance genes in the community constitute a substantial reservoir for multidrug resistance in the hospital.
Hepatitis D Virus Genotypes in Intravenous Drug Users in Taiwan: Decreasing Prevalence and Lack of Correlation with Hepatitis B Virus Genotypes
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn 
Published: October 2002

ABSTRACTOf 368 hepatitis B virus (HBV)-infected intravenous drug users, 144 (39%) were positive for antibody to hepatitis D virus (anti-HDV). Anti-HDV-positive HBV carriers had a lower rate of positivity for HBV DNA than did anti-HDV-negative carriers (52 versus 73%, respectively). From 1986 to 1997, the average rate of decrease in the prevalence of HDV infection in this population was 4.7%/year.