bluuebunny/crossref_metadata_2025_split huggingface dataset:
Degradation of Aromatics and Chloroaromatics byPseudomonassp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase
Authors: Göbel Markus , Kassel-Cati Kerstin , Schmidt Eberhard , Reineke Walter 
Published: July 2002

ABSTRACT3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb fromPseudomonassp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termedcatF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp betweencatIandcatJand of 4 bp betweencatJandcatF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.
Yersinia enterocolitica
Type III Secretion: Mutational Analysis of the
yopQ
Secretion Signal
Authors: Ramamurthi Kumaran S. , Schneewind Olaf 
Published: July 2002

ABSTRACT

Pathogenic
Yersinia
spp. secrete Yop proteins via the type III pathway.
yopQ
codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of
yopQ
mRNA sequence do not abolish type III transport, suggesting a model in which
yopQ
mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the
yopE
signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of
yopE
abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of
yopE
codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of
yopQ
act as a minimal secretion signal. Further truncation of
yopQ
, either at codon 10 or at codon 2, abolished secretion. Replacement of
yopQ
AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with
npt
did not permit Npt secretion, suggesting that the translation of
yopQ
codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of
yopQ
codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of
yopQ
codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at
yopQ
positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of
yopQ
codons 1 to 15. An mRNA mutant of
yopQ
codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of
yopQ
mRNA.
Yop Fusions to Tightly Folded Protein Domains and Their Effects on
Yersinia enterocolitica
Type III Secretion
Authors: Lee Vincent T. , Schneewind Olaf 
Published: July 2002

ABSTRACT

Yersinia enterocolitica
organisms secrete Yop proteins via the type III pathway. Translational fusion of
yop
genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.
ClpXP Protease Regulates the Signal Peptide Cleavage of Secretory Preproteins inBacillus subtiliswith a Mechanism Distinct from That of the Ecs ABC Transporter
Authors: Pummi Tiina , Leskelä Soile , Wahlström Eva , Gerth Ulf , Tjalsma Harold , Hecker Michael , Sarvas Matti , Kontinen Vesa P. 
Published: July 2002

ABSTRACTIdentification and characterization of a suppressor mutation,sup-15, which partially restored secretion in the protein secretion-deficientBacillus subtilis ecsA26mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ α-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of thesipS,sipT,sipV, andlspsignal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation ofecsmutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of thesipgenes, pointing to a different mechanism of secretion regulation.
Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora
Authors: Vangnai Alisa S. , Arp Daniel J. , Sayavedra-Soto Luis A. 
Published: July 2002

ABSTRACTThe involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization inPseudomonas butanovora(ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption ofbdhorbohaffected adversely, but did not eliminate, butane utilization byP. butanovora. TheP. butanovoramutant with both genesbohandbdhinactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of theP. butanovorastrain withbdhdisrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD+-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway ofP. butanovora.
Perfringolysin O Expression in
Clostridium perfringens
Is Independent of the Upstream
pfoR
Gene
Authors: Awad Milena M. , Rood Julian I. 
Published: July 2002

ABSTRACT

The pathogenesis of
Clostridium perfringens
-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with
Escherichia coli
suggested that the perfringolysin O structural gene,
pfoA
, was positively regulated by the product of the upstream
pfoR
gene. In an attempt to confirm this hypothesis in
C
.
perfringens
, a
pfoR-pfoA
deletion mutant was complemented with isogenic

pfoA
+

shuttle plasmids that varied only in their ability to encode an intact
pfoR
gene. No difference in the ability to produce perfringolysin O was observed for
C
.
perfringens
strains carrying these plasmids. In addition, chromosomal
pfoR
mutants were constructed by homologous recombination in
C
.
perfringens
. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of
pfoR
, it was concluded that the
pfoR
gene product is unlikely to have a role in the regulation of
pfoA
expression in
C
.
perfringens
.
Differential Recognition of Surface Proteins inStreptococcus pyogenesby Two Sortase Gene Homologs
Authors: Barnett Timothy C. , Scott June R. 
Published: July 2002

ABSTRACTThe interaction ofStreptococcus pyogenes(group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous toStaphylococcus aureussortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GASsrtAmutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-typesrtAgene was provided intrans. The surface localization of T6, however, was unaffected by thesrtAmutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designatedsrtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented bysrtBintrans. AnsrtAprobe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, whilesrtBhybridized with DNA from only a few GAS strains. We conclude thatsrtAandsrtBencode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.
Simple and Rational Approach to the Identification of
Mycobacterium tuberculosis
,
Mycobacterium avium
Complex Species, and Other Commonly Isolated Mycobacteria
Authors: Wong Derek A. , Yip Peter C. W. , Cheung Danny T. L. , Kam Kai Man 
Published: July 2002

ABSTRACT

A novel PCR-restriction fragment length polymorphism analysis of the
hsp65
gene was developed. The restriction patterns for
Mycobacterium tuberculosis
and
Mycobacterium avium
complex (MAC) species were designed to be highly distinct, and the overall number of restriction patterns was designed to be limited. Four hundred specimens (17 reference strains and 383 clinical isolates) were tested, of which 98 were
M. tuberculosis
and 132 were MAC species. The assay was virtually 100% sensitive and specific for
M. tuberculosis
and MAC species. Moreover, it gave highly concordant results for other mycobacterial species other than
M. terrae
complex species. This assay can be completed in one day and is user-friendly and robust. Therefore, it is highly suitable for large-scale use in a clinical laboratory.
Performance of Candida ID, a New Chromogenic Medium for Presumptive Identification of
Candida
Species, in Comparison to CHROMagar Candida
Authors: Willinger Birgit , Hillowoth Cornelia , Selitsch Brigitte , Manafi Mammad 
Published: July 2002

ABSTRACT

Candida ID agar allows identification of
Candida albicans
and differentiation of other
Candida
species. In comparison with CHROMagar Candida, we evaluated the performance of this medium directly from 596 clinical specimens. In particular, detection of
C. albicans
after 24 h of incubation was easier on Candida ID (sensitivity, 96.8%) than on CHROMagar (sensitivity, 49.6%).
Rapid and Accurate Determination of Genotypic Clarithromycin Resistance in Cultured
Helicobacter pylori
by Fluorescent In Situ Hybridization
Authors: Rüssmann Holger , Adler Kristin , Haas Rainer , Gebert Bettina , Koletzko Sibylle , Heesemann Jürgen 
Published: July 2002

ABSTRACT

Phenotypic susceptibility testing for clarithromycin by E-test and disk diffusion of 109 cultured
Helicobacter pylori
isolates was compared with the genotypic susceptibility determination by fluorescent in situ hybridization (FISH). No discrepancies were found between these three methods. However, FISH has the advantage of providing results after 3 h.
Purification and Characterization of PCR-Inhibitory Components in Blood Cells
Authors: Al-Soud Waleed Abu , Rådström Peter 
Published: July 2002

ABSTRACTIn a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jönsson, and P. Rådström. J. Clin. Microbiol. 38:345–350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 μl containing 10 different thermostable DNA polymerases and 1 ng ofListeria monocytogenesDNA as template DNA, AmpliTaqGold,Pwo, andUltmawere inhibited in the presence of ≤1.3 μg of hemoglobin and ≤25 ng of lactoferrin, whilerTthandTliwere found to resist inhibition of at least 100 μg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis ofrTthusing the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3′ end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 μM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaqGold to amplify DNA in the presence of 20 instead of 1 μg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaqGold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.
Normalized Quantification by Real-Time PCR of Epstein-Barr Virus Load in Patients at Risk for Posttransplant Lymphoproliferative Disorders
Authors: Jabs Wolfram J. , Hennig Holger , Kittel Michael , Pethig Klaus , Smets Françoise , Bucsky Peter , Kirchner Holger , Wagner Hans J. 
Published: July 2002

ABSTRACT
The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.
Analysis of
Clostridium difficile
Isolates from Nosocomial Outbreaks at Three Hospitals in Diverse Areas of Japan
Authors: Kato Haru , Kato Naoki , Watanabe Kunitomo , Yamamoto Toshinobu , Suzuki Kanzo , Ishigo Shiomi , Kunihiro Seiko , Nakamura Isao , Killgore George E. , Nakamura Shinichi 
Published: July 2002

ABSTRACT

Clostridium difficile
isolates recovered from patients with
C. difficile
-associated diarrhea (CDAD) at three hospitals located in diverse areas of Japan were analyzed by three typing systems, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), and Western immunoblotting. At the three hospitals examined, a single PCR ribotype strain (type smz) was predominant and accounted for 22 (65%) of 34, 18 (64%) of 28, and 11 (44%) of 25 isolates, respectively. All of the 51 isolates that represented PCR ribotype smz were nontypeable by PFGE because of DNA degradation. Since the type smz strain did not react with any of the antisera against 10 different serogroups (A, B, C, D, F, G, H, I, K, and X), we prepared a new antiserum against a type smz isolate. All 51 type smz isolates presented identical banding patterns, reacting with the newly prepared antiserum (designated subserogroup JP-0 of serogroup JP). These results were compared with those of a strain from a hospital outbreak that occurred in New York, which has been identified as type J9 by restriction enzyme analysis and type 01/A by arbitrarily primed PCR but was nontypeable by PFGE because of DNA degradation. This strain was reported to be epidemic at multiple hospitals in the United States. The J9 strain represented a PCR ribotype pattern different from that of a type smz strain and was typed as subserogroup G-1 of serogroup G by immunoblot analysis. A single outbreak type causing nosocomial CDAD in Japan was found to be different from the strain causing multiple outbreaks in the United States, even though the outbreak strains from the two countries were nontypeable by PFGE because of DNA degradation.
Diagnosis of Bacteriuria by Detection of Volatile Organic Compounds in Urine Using an Automated Headspace Analyzer with Multiple Conducting Polymer Sensors
Authors: Aathithan S. , Plant J. C. , Chaudry A. N. , French G. L. 
Published: July 2002

ABSTRACTThe Osmetech Microbial Analyzer (OMA) is an automated headspace analyzer fitted with a novel detector system consisting of an array of polymer sensors, each of which responds to different volatile organic compounds. The system can be used for screening clinical urine specimens for significant bacteriuria by sampling urine headspace and subjecting the output of the multiple-detector response to principal component analysis. The OMA readily distinguished artificially infected urine samples from sterile controls. The OMA was then used to analyze 534 unselected clinical urine specimens, of which 21.5% had significant bacteriuria (containing >105CFU of bacteria/ml). The sensitivity and specificity of the OMA compared with conventional culture were 83.5 and 87.6%, respectively. The OMA is a promising automated system for the rapid routine screening of urine specimens, and further clinical trials are in progress.
Population-Based Survey of Antimicrobial Susceptibility and Serotype Distribution of
Streptococcus pneumoniae
from Meningitis Patients in Salvador, Brazil
Authors: Reis Joice Neves , Cordeiro Soraia Machado , Coppola Steven J. , Salgado Kátia , Carvalho Maria G. S. , Teixeira Lúcia M. , Thompson Terry A. , Facklam Richard R. , Reis Mitermayer G. , Ko Albert I. 
Published: July 2002

ABSTRACT
Penicillin-nonsusceptible strains were isolated from 15% of 303 individuals with pneumococcal meningitis identified during a 4-year surveillance study in Salvador, Brazil. The estimated rate of coverage of the seven-valent conjugate vaccine was 74% among patients <5 years of age and 94% among those infected with nonsusceptible isolates, indicating that the use of conjugate vaccines may be an approach to the control of emerging penicillin resistance in Brazil.
Diagnosis of Respiratory Syncytial Virus Infection: Comparison of Reverse Transcription-PCR to Viral Culture and Serology in Adults with Respiratory Illness
Authors: Falsey Ann R. , Formica Maria A. , Walsh Edward E. 
Published: July 2002

ABSTRACT
Diagnosis of respiratory syncytial virus (RSV) during acute infection in adults is difficult because of the poor sensitivity of viral culture and antigen detection. A recently developed single-tube nested reverse transcription-PCR (RT-PCR) was compared to viral culture and serology by enzyme immunoassay for the diagnosis of RSV in adults with respiratory illness. Nasal swab samples were collected during respiratory illnesses from five groups of subjects: healthy young adults, healthy elderly adults, adults with chronic heart and lung disease, nursing home residents, and adults admitted to the hospital during the winter with cardiopulmonary illnesses. Of 1,112 samples for which all three tests were available, 117 were positive by at least one method and 995 were negative by all methods. One hundred ten were considered true positives because culture and/or serology was positive. Of these, 80 (73%) were PCR positive compared to 43 (39%) that were culture positive. Seven PCR results were considered false positives due to negative culture and serology. The overall RT-PCR sensitivity was 73%, and specificity was 99%. These data indicate that RT-PCR is an excellent method for the diagnosis of acute RSV infection in adults.