bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic
Bradyrhizobium
sp. Strain HW13 Isolated from a Pristine Environment
Authors: Kitagawa Wataru , Takami Sachiko , Miyauchi Keisuke , Masai Eiji , Kamagata Yoichi , Tiedje James M. , Fukuda Masao 
Published: July 2002

ABSTRACT

The
tfd
genes of
Ralstonia eutropha
JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes,
cadRABKC
, was cloned and characterized from
Bradyrhizobium
sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The
cadR
gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The
cadABC
genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of
Burkholderia cepacia
AC1100 and with a putative ferredoxin, ThcC, of
Rhodococcus erythropolis
NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene,
tfdA
, of
R. eutropha
JMP134. The
cadK
gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134.
Sinorhizobium meliloti
Rm1021 cells containing
cadRABKC
transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the
cadRABC
genes was essential for 2,4-D conversion in strain Rm1021 but that
cadK
was not. Five 2,4-D degraders, including
Bradyrhizobium
and
Sphingomonas
strains, were found to have
cadA
gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13
cadABC
.
Glyoxylate Regeneration Pathway in the Methylotroph
Methylobacterium extorquens
AM1
Authors: Korotkova Natalia , Chistoserdova Ludmila , Kuksa Vladimir , Lidstrom Mary E. 
Published: July 2002

ABSTRACT

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of
14
C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and β-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.
Enterococcus faecalis
Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis
Authors: Hedl Matija , Sutherlin Autumn , Wilding E. Imogen , Mazzulla Marie , McDevitt Damien , Lane Pamela , Burgner John W. , Lehnbeuter Kevin R. , Stauffacher Cynthia V. , Gwynn Michael N. , Rodwell Victor W. 
Published: July 2002

ABSTRACT

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame,
mvaE
, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the
mvaE
gene product is a single polypeptide in
Enterococcus faecalis
,
Enterococcus faecium
, and
Enterococcus hirae
. The
mvaE
gene was cloned from
E. faecalis
and was expressed with an N-terminal His tag in
Escherichia coli
. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa
mvaE
gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, Δ

H
a

and

K
m

values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar

K
i

for a statin drug confirmed that
E. faecalis
HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The
mvaE
gene product represents the first example of an HMG-CoA reductase fused to another enzyme.
Gene Transfer betweenSalmonella entericaSerovar Typhimurium inside Epithelial Cells
Authors: Ferguson Gayle C. , Heinemann Jack A. , Kennedy Martin A. 
Published: July 2002

ABSTRACTVirulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here thatSalmonella entericaserovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmidtragenes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.
TetR Family Member PsrA Directly Binds the
Pseudomonas rpoS
and
psrA
Promoters
Authors: Kojic Milan , Aguilar Claudio , Venturi Vittorio 
Published: July 2002

ABSTRACT

We have previously described a
Pseudomonas
gene,
psrA
, which enhances transcription of the
rpoS
sigma factor gene at stationary phase. We present molecular data which demonstrate that in
Pseudomonas putida
PsrA binds specifically to the
rpoS
and
psrA
promoters in DNA regions having similar palindromic sequences, C/GAAAC N
2-4
GTTTG/C, where N is any nucleotide. The position of the initiation of transcription was determined for both promoters, and PsrA binds from positions −59 to −35 in the
rpoS
promoter and from −18 to +20 in the
psrA
promoter with respect to the +1 transcription site. Expression studies with a
psrA-lacZ
transcriptional fusion in wild-type and
psrA
::Tn
5
knockout mutants revealed that
psrA
was under additional control in response to growth phase. A model for the role of PsrA in the regulation of
rpoS
and
psrA
is presented.
Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR
Authors: van Elden L. J. R. , Nijhuis M. , Schipper P. , Schuurman R. , van Loon A. M. 
Published: July 2002

ABSTRACT
Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.
Evaluation of Two Commercial Enzyme Immunoassays, Testing Immunoglobulin G (IgG) and IgA Responses, for Diagnosis of
Helicobacter pylori
Infection in Children
Authors: Kindermann Angelika , Konstantopoulos Nikolaos , Lehn Norbert , Demmelmair Hans , Koletzko Sibylle 
Published: July 2002

ABSTRACT

Serological testing to diagnose
Helicobacter pylori
infection in children is still controversial, although commonly used in clinical practice. We compared the immunoglobulin G (IgG) and IgA results of two commercially available enzyme immunoassays (EIAs) (Pyloriset IgG and IgA and Enzygnost II IgG and IgA) for 175 children with abdominal symptoms divided into three age groups (0 to ≤6 years,
n
= 47; >6 to ≤12 years,
n
= 77; >12 years,
n
= 51). A child was considered
H. pylori
infected if at least two of three tests (histology, rapid urease test,
13
C-urea breath test) or culture were positive and noninfected if all results were concordantly negative. Of 175 children, 93 (53%) were
H. pylori
negative and 82 were
H. pylori
positive. With the recommended cutoff values, the overall specificity was excellent for all four EIAs (95.7 to 97.8%) regardless of age. Sensitivity varied markedly between tests and was 92.7, 70.7, 47.5, and 24.4% for Enzygnost II IgG, Pyloriset IgG, Enzygnost II IgA, and Pyloriset IgA, respectively. Sensitivity was low in the youngest age group (25 to 33.3%), except for Enzygnost II IgG (91.6%). Receiver-operating curve analyses revealed that lower cutoff values would improve the accuracy of all of the tests except Enzygnost II IgG. Measurement of specific IgA, in addition to IgG, antibodies hardly improved the sensitivity. The specificity of commercial serological tests is high in children when the cutoff values obtained from adults are used. In contrast, sensitivity is variable, with a strong age dependence in some, but not all, tests. We speculate that young children may have a different immune response to
H. pylori
, with preferable responses to certain antigens, as well as lower titers than adults. The Pyloriset test may fail to recognize these specific antibodies.
Effect of Endocervical Specimen Adequacy on Ligase Chain Reaction Detection of
Chlamydia trachomatis
Authors: Loeffelholz Mike J. , Jirsa Sandra J. , Teske Rebecca K. , Woods Juliet N. 
Published: July 2002

ABSTRACT

Several studies have demonstrated that the sensitivity of a commercially available PCR test for the detection of
Chlamydia trachomatis
(Roche Diagnostics) is affected by the cellular quality of the endocervical swab specimens. The cellular adequacies of 1,633 female endocervical swab specimens were assessed and compared with the results of
C. trachomatis
detection obtained by ligase chain reaction (LCR; Abbott Laboratories). Specimen adequacy studies and LCR were performed with samples from the same swab, after demonstration of the stability of human epithelial cells in LCR transport medium. Prior to heat treatment of the swab specimen, an aliquot was removed and cytocentrifuged onto a slide. Cell spots were stained and examined at ×400 magnification for endocervical (columnar epithelial or metaplastic) cells and erythrocytes. The overall rate of positivity of the LCR was 6.5% (106 of 1,633 specimens) with pooled specimens (pools of 4 specimens each; reduced cutoff). Of the 1,633 specimens examined, 655 (40.1%) were found to contain one or more endocervical cells. The rate of positivity for
C. trachomatis
was 10.8% (71 of 655 specimens) among specimens containing endocervical cells, whereas it was 3.6% (35 of 978 specimens) among specimens lacking endocervical cells (
P
< 0.0001). There was no linear trend between the rate of positivity for
C. trachomatis
and the number of endocervical cells (
P
= 0.24). The rate of positivity for
C. trachomatis
was 5.4% (8 of 147 specimens) among specimens containing large numbers of erythrocytes (≥100 per high-power field), whereas it was 6.6% (98 of 1,486 specimens) among specimens containing less than 100 erythrocytes per high-power field (
P
= 0.59). These results show that the sensitivity of the Abbott
C. trachomatis
LCR test is affected by the presence of endocervical cells. Additionally, they indicate that the presence of a single endocervical cell is as good an indicator of specimen adequacy as the presence of many endocervical cells. The presence of a large number of erythrocytes was not associated with an increased rate of sensitivity of the LCR.
Pneumocystis carinii
f. sp.
hominis
DNA in Immunocompetent Health Care Workers in Contact with Patients with
P. carinii
Pneumonia
Authors: Miller Robert F. , Ambrose Helen E. , Wakefield Ann E. 
Published: July 2002

ABSTRACT

The possible transmission of
Pneumocystis carinii
f. sp.
hominis
from patients with
P. carinii
pneumonia to asymptomatic health care workers (HCW), with or without occupational exposure to human immunodeficiency virus (HIV)-infected patients with
P. carinii
pneumonia, was examined. HCW in a specialist inpatient HIV-AIDS facility and a control group in the general medical-respiratory service in the same hospital provided induced sputum and/or nasal rinse samples, which were analyzed for the presence of
P. carinii
f. sp.
hominis
DNA by using DNA amplification (at the gene encoding the mitochondrial large subunit rRNA [mt LSU rRNA]).
P. carinii
f. sp.
hominis
DNA was detected in some HCW samples; those with the closest occupational contact were more likely to have detectable
P. carinii
DNA.
P. carinii
DNA was detected in one HCW who carried out bronchoscopy over a 2-year period.
P. carinii
-positive samples were genotyped by using DNA sequence variations at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, along with bronchoalveolar lavage samples from patients with
P. carinii
pneumonia hospitalized at the same time. Genotyping identified 31 different
P. carinii
f. sp.
hominis
ITS genotypes, 26 of which were found in the patient samples. Five of the eight ITS genotypes detected in HCW samples were not observed in the patient samples. The results suggested that HCW in close occupational contact with patients who had
P. carinii
pneumonia may have become colonized with
P. carinii
. Carriage was asymptomatic and did not result in the development of clinical disease.
Molecular Epidemiology of Rotaviruses in Nigeria: Detection of Unusual Strains with G2P[6] and G8P[1] Specificities
Authors: Adah Mohammed I. , Wade Abel , Taniguchi Koki 
Published: July 2002

ABSTRACT
During an epidemiological study on rotaviruses among diarrheic children in the northeastern and middle belt regions of Nigeria, the distribution of G and P types was investigated in 127 stool specimens. By PCR G typing, the G type of rotaviruses in 97 samples was identified. Interestingly, an unusual G8 type, as well as common G1, G2, and G3 types, was detected more frequently (31 of 112; 27.7%). Eleven samples contained multiple G types, and a G9 strain (Bulumkutu) was identified for one of the probable mixed infections. In PCR P typing, P[6] was detected most frequently, P[8] being the second most common type, while the P type of 73 samples could not be identified. One rotavirus strain with a G8 type specificity could be cultivated in cell culture, and the P type of this strain was found to be P[1], which is usually carried by bovine strains. When the combinations of G and P types were examined, the unusual strains G2P[6] and G8P[1] were often identified. Sequence analysis was performed for the VP7 gene of the G9 strain Bulumkutu and the VP4 and VP7 genes of G8P[1] strain HMG035. The VP7 sequence of the Nigerian serotype G9 was more closely related to that of a Brazilian strain than to those of other African strains. The VP7 and VP4 genes of G8P[1] strain HMG035 were found to be very similar to that of a Thai bovine strain A5, suggesting that bovine strains may have been transmitted directly to humans. These results highlight an unexpected diversity among rotavirus strains in Nigeria and emphasize the need for further serological and genetic surveys on more rotavirus strains in African countries, including Nigeria.
Recombinant Flagellin A Proteins from
Borrelia burgdorferi
Sensu Stricto,
B
.
afzelii
, and
B. garinii
in Serodiagnosis of Lyme Borreliosis
Authors: Panelius Jaana , Lahdenne Pekka , Saxen Harri , Heikkilä Tero , Seppälä Ilkka 
Published: July 2002

ABSTRACT

Genes for flagellin A (FlaA) proteins from European borrelial strains of
Borrelia burgdorferi
sensu stricto,
B. afzelii
, and
B. garinii
were cloned and sequenced. An identity of 92 to 93% was observed in the
flaA
sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in
Escherichia coli
and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from
B. garinii
and
B. afzelii
than against rFlaA from
B. burgdorferi
sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from
Yersinia
enterocolitica
or beta-hemolytic
Streptococcus
infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.
Epstein-Barr Virus (EBV) DNA in Sera of Patients with Primary EBV Infection
Authors: Chan K. H. , Ng M. H. , Seto W. H. , Peiris J. S. M. 
Published: July 2002

ABSTRACT
Detection of Epstein-Barr Virus (EBV) DNA by PCR in serum had a sensitivity of 80%, a specificity of 94%, and positive and negative predictive values of 95 and 79%, respectively, for the diagnosis of primary EBV infection. We suggest that this is a useful addition to the panel of tests used for this purpose.
Comparison of Automated Ribotyping to Pulsed-Field Gel Electrophoresis for Genetic Fingerprinting of
Streptococcus pneumoniae
Authors: Quale John M. , Landman David , Flores Carlos , Ravishankar Jayashree 
Published: July 2002

ABSTRACT

Fifty-two isolates of
Streptococcus pneumoniae
were characterized by pulsed-field gel electrophoresis (PFGE) and automated ribotyping by using
Hin
dIII and
Pvu
II.
Hin
dIII ribotypes correlated well with PFGE.
Pvu
II produced fewer bands and was less discriminatory. Automated ribotyping with
Hin
dIII is an accurate method for genetic fingerprinting of
S. pneumoniae
and can complement PFGE.
Recovery of
Candida dubliniensis
and Other Yeasts from Human Immunodeficiency Virus-Associated Periodontal Lesions
Authors: Jabra-Rizk M. A. , Ferreira S. M. S. , Sabet M. , Falkler W. A. , Merz W. G. , Meiller T. F. 
Published: July 2002

ABSTRACT

Oral and subgingival samples from periodontal lesions were collected from 54 human immunodeficiency virus (HIV)-positive and 20 HIV-negative patients and cultured for yeast species. Of the 54 samples cultured from HIV-positive patients, 44 (82%) were positive for yeast species, of which 29 (66%) were subgingival. A total of 19 (48%) patients were positive for
Candida dubliniensis
, of which 15 (79%) were colonized in subgingival sites. Seven isolates of
Candida glabrata
, two isolates of
Candida parapsilosis
, and one isolate of
Saccharomyces cerevisiae
were recovered. This study reports for the first time the recovery of
C. dubliniensis
from subgingival intraoral sites and confirms the presence of
Candida
species in sites of periodontal disease associated with HIV.
Molecular Characterization of First Human
Bartonella
Strain Isolated in Italy
Authors: Ciervo Alessandra , Petrucca Andrea , Ciarrocchi Simonetta , Pinto Antonella , Bonazzi Lucio , Fabio Anna , Farnetti Enrico , Chomel Bruno B. , Ciceroni Lorenzo 
Published: July 2002

ABSTRACT

The aim of this study was to characterize a
Bartonella
strain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a
Bartonella quintana
strain, similar but not identical to
B. quintana
Oklahoma, which was used as a control strain.
Fatal Myocarditis Associated with Acute Parvovirus B19 and Human Herpesvirus 6 Coinfection
Authors: Rohayem Jacques , Dinger Jürgen , Fischer Rainer , Klingel Karin , Kandolf Reinhard , Rethwilm Axel 
Published: July 2002

ABSTRACT
We report on the case of a healthy young boy who developed a fulminant myocarditis due to acute coinfection with erythrovirus (parvovirus B19) and human herpesvirus 6 (HHV-6) in the absence of an antiviral immune response. We suggest that the HHV-6-induced immunosuppression enhanced dissemination of parvovirus B19, which led to fatal myocarditis.