bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium
Azoarcus evansii
Authors: Schühle Karola , Jahn Martina , Ghisla Sandro , Fuchs Georg 
Published: July 2002

ABSTRACT

In the β-proteobacterium
Azoarcus evansii
, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a β-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by β-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.
O2R, a Novel Regulatory Element Mediating Rox1p-Independent O
2
and Unsaturated Fatty Acid Repression of
OLE1
in
Saccharomyces cerevisiae
Authors: Nakagawa Youji , Sugioka Shigemi , Kaneko Yoshinobu , Harashima Satoshi 
Published: July 2002

ABSTRACT

Fatty acid desaturation catalyzed by fatty acid desaturases requires molecular oxygen (O
2
).
Saccharomyces cerevisiae
cells derepress expression of
OLE1
encoding Δ9 fatty acid desaturase under hypoxic conditions to allow more-efficient use of limited O
2
. It has been proposed that aerobic conditions lead to repression of
OLE1
by well-established O
2
-responsive repressor Rox1p, since putative binding sequences for Rox1p are present in the promoter of
OLE1
. However, we revealed in this study that disruption of
ROX1
unexpectedly did not affect the O
2
repression of
OLE1
, indicating that a Rox1p-independent novel mechanism operates for this repression. We identified by promoter deletion analysis the 50-bp O
2
-regulated (O2R) element in the
OLE1
promoter approximately 360 bp upstream of the start codon. Site-directed mutagenesis of the O2R element showed that the putative binding motif (5′-GATAA-3′) for the GATA family of transcriptional factors is important for O
2
repression. Anaerobic derepression of
OLE1
transcription was repressed by unsaturated fatty acids (UFAs), and interestingly the O2R element was responsible for this UFA repression despite not being included within the fatty acid-regulated (FAR) element previously reported. The fact that such a short 50-bp O2R element responds to both O
2
and UFA signals implies that O
2
and UFA signals merge in the ultimate step of the pathways. We discuss the differential roles of FAR and O2R elements in the transcriptional regulation of
OLE1.
Vertical Transmission of Biosynthetic Plasmids in Aphid Endosymbionts (
Buchnera
)
Authors: Wernegreen Jennifer J. , Moran Nancy A. 
Published: July 2002

ABSTRACT

This study tested for horizontal transfer of plasmids among
Buchnera aphidicola
strains associated with ecologically and phylogenetically related aphid hosts (
Uroleucon
species). Phylogenetic congruence of
Buchnera
plasmid (
trpEG
and
leuABC
) and chromosomal (
dnaN
and
trpB
) genes supports strictly vertical long-term transmission of plasmids, which persist due to their contributions to host nutrition rather than capacity for infectious transfer. Synonymous divergences indicate elevated mutation on plasmids relative to chromosomal genes.
Functional Domains of Yeast Plasmid-Encoded Rep Proteins
Authors: Sengupta A. , Blomqvist K. , Pickett A. J. , Zhang Y. , Chew J. S. K. , Dobson M. J. 
Published: July 2002

ABSTRACTBoth of theSaccharomyces cerevisiae2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector withrep1deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.
Loss of Ribosomal Protein L11 Blocks Stress Activation of the
Bacillus subtilis
Transcription Factor ς
B
Authors: Zhang Shuyu , Scott Janelle M. , Haldenwang W. G. 
Published: July 2002

ABSTRACT

ς
B
, the general stress response sigma factor of
Bacillus subtilis
, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates ς
B
through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of ς
B
from an anti-ς
B
factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the ς
B
stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude
B. subtilis
extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of ς
B
.
B. subtilis
strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of ς
B
. Neither the energy-responsive activation of ς
B
nor stress-dependent chaperone gene induction (a ς
B
-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of ς
B
are shown to be active in the RplK
−
strain but fail to be triggered by stress. The data demonstrate that the
B. subtilis
ribosomes provide an essential input for the stress activation of ς
B
and suggest that the ribosomes may themselves be the sensors for stress in this system.
Low-Molecular-Weight Plasmid of
Salmonella enterica
Serovar Enteritidis Codes for Retron Reverse Transcriptase and Influences Phage Resistance
Authors: Rychlik I. , Sebkova A. , Gregorova D. , Karpiskova R. 
Published: July 2002

ABSTRACT

Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that
Salmonella enterica
serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly,
Salmonella
serovar Enteritidis-produced low-molecular-weight DNA was shown to be a double-stranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19- and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis.
Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in
Ralstonia eutropha
JMP134
Authors: Louie Tai Man , Webster Christopher M. , Xun Luying 
Published: July 2002

ABSTRACT

Ralstonia eutropha
JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH
2
)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (
tcpABC
) from JMP134 by using primers designed from conserved regions of FADH
2
-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that
tcpA
,
tcpB
, and
tcpC
encoded an FADH
2
-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The
tcpA
mutant failed to degrade 2,4,6-TCP, while both
tcpB
and
tcpC
mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of
tcpB
may have led to a polar effect on downstream
tcpC
, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH
2
was supplied by an
Escherichia coli
flavin reductase. TcpC produced in
E. coli
oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that
tcpB
may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.
Functional Analysis of HrpF, a Putative Type III Translocon Protein from
Xanthomonas campestris
pv. vesicatoria
Authors: Büttner Daniela , Nennstiel Dirk , Klüsener Birgit , Bonas Ulla 
Published: July 2002

ABSTRACT

Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen
Xanthomonas campestris
pv. vesicatoria, encoded by the
hrp
(hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an
hrpF
null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.
Development of Monoclonal Antibodies Which Specifically RecognizeEntamoeba histolyticain Preserved Stool Samples
Authors: Yau Yvonne C. W. , Crandall Ian , Kain Kevin C. 
Published: July 2002

ABSTRACTWe report the generation of monoclonal antibodies against a recombinant 170-kDa subunit of the Gal or GalNAc lectin ofEntamoeba histolyticathat specifically recognizeE. histolyticabut notEntamoeba disparin preserved stool samples. These antibodies do not cross-react with other bowel protozoa, includingEntamoeba coli,Giardia lamblia, andDientamoeba fragilis.
Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqMan Real-Time PCR Assay
Authors: Désiré Nathalie , Dehée Axelle , Schneider Véronique , Jacomet Christine , Goujon Christophe , Girard Pierre-Marie , Rozenbaum Willy , Nicolas Jean-Claude 
Published: July 2002

ABSTRACT

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log
10
. Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10
6
PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10
6
PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (
P
= 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5′ end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4
+
cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.
Combinatorial Use of Antibodies to Secreted Mycobacterial Proteins in a Host Immune System-Independent Test for Tuberculosis
Authors: Landowski Christopher P. , Godfrey Henry P. , Bentley-Hibbert Stuart I. , Liu Xinyan , Huang Zhishan , Sepulveda Ricardo , Huygen Kris , Gennaro Maria L. , Moy Fred H. , Lesley Scott A. , Haak-Frendscho Mary 
Published: July 2002

ABSTRACT

Laboratory diagnosis of tuberculosis is often difficult. Immunodetection of circulating
Mycobacterium tuberculosis
proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays. We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S. I. Bentley-Hibbert, X. Quan, T. Newman, K. Huygen, and H. P. Godfrey, Infect. Immun. 67:581–588, 1999). To extend these observations to a
Mycobacterium bovis
BCG-vaccinated population and to another secreted mycobacterial protein, Ag85 and PstS-1 (protein antigen B, p38 antigen) were quantified in sera from 97 Chilean tuberculosis patients and healthy controls (many of whom had received BCG as children) using dot immunobinding, mouse monoclonal anti-BCG Ag85 complex antibody, and chicken antipeptide antibodies reactive with
M. tuberculosis
Ag85B and PstS-1. The latter antibodies had been raised to peptide-derived immunogens expressed on a novel proprietary protein carrier in
Escherichia coli
. Median serum Ag85 levels measured by using either anti-Ag85 antibody were significantly higher in patients with active tuberculosis than in healthy controls (
P
, <0.001 to 0.01); the median serum PstS-1 levels were similar in patients and controls. The sensitivity of significantly elevated circulating Ag85 levels in patients with pulmonary tuberculosis measured by anti-Ag85 complex or anti-Ag85B antibodies was 60 and 55%, respectively, but increased to 77% when results obtained with both anti-Ag85 antibodies were considered jointly (
P
< 0.02). The corresponding specificities for individual and joint consideration were 95, 85, and 80%, respectively. These results indicate that elevated Ag85 levels can be detected in patients with active tuberculosis even after BCG vaccination and suggest that combinatorial use of antibodies directed at different epitopes of this protein could provide a viable strategy for developing new host immune response-independent diagnostic tests for tuberculosis.
Serological Differentiation of Experimentally Induced
Candida dubliniensis
and
Candida albicans
Infections
Authors: Moragues Marı́a D. , Omaetxebarria Miren J. , Elguezabal Natalia , Bikandi Joseba , Quindós Guillermo , Coleman David C. , Pontón José 
Published: July 2002

ABSTRACT

Using a rabbit model of systemic infection, we show that it is possible to differentiate infections caused by
Candida dubliniensis
and other
Candida
species by detecting the antibody response mounted by the infected animals. These results confirm our previous observation in a patient with
C. dubliniensis
candidemia and suggest that detection of
C. dubliniensis
-specific antibodies is useful in the diagnosis of invasive candidiasis caused by this yeast.
Detection of B Virus Antibody in Monkey Sera Using Glycoprotein D Expressed in Mammalian Cells
Authors: Tanabayashi Kiyoshi , Mukai Ryozaburo , Yamada Akio 
Published: July 2002

ABSTRACT

The gene encoding glycoprotein D (gD) of the monkey B virus (
Cercopithecine herpesvirus
1
) was cloned into a mammalian expression vector, pcDNA3.1(−), and the recombinant plasmid DNA was transfected into COS7 cells. The expression of gD in transfected COS7 cells was detected by indirect immunofluorescence assay or radioimmunoprecipitation analysis (RIPA). Although the expressed gD protein was revealed to react well with sera from monkeys naturally infected with B virus by RIPA, some sera showed reduced reactivity when analyzed by the Western blotting (WB) method. Some sera also showed relatively high background when the WB was performed using gD expressed from recombinant plasmid. The mutant gD protein lacking the transmembrane domain (TM) and cytoplasmic tail (CT) was next expressed in COS7 cells. The mutant protein was secreted into culture medium without apparent loss of the antigenicity. Using the secretory form of the gD protein as antigen in dot blot analysis, sera from B virus-infected monkeys were shown to react with the mutant protein without nonspecific reaction. Since the recombinant gD or its derivative lacking TM and CT could be expressed in mammalian cells with proper antigenicity, these antigens appeared to be useful for serological detection of B virus infection in monkeys.
Identification and Molecular Characterization of a Bovine G3 Rotavirus Which Causes Age-Independent Diarrhea in Cattle
Authors: El-Attar L. , Dhaliwal W. , Iturriza-Gómara M. , Bridger J. C. 
Published: July 2002

ABSTRACT
G3 rotaviruses have been reported rarely in cattle, and none have been characterized. We report the first genomic characterization of a bovine G3 rotavirus, CP-1, which had been biologically characterized in vivo and shown to cause age-independent diarrhea. CP-1 was a G3 rotavirus as its VP7 had 92 to 96% deduced amino acid identity to those of G3 rotaviruses. However, initially, CP-1 was identified as a G10 rotavirus by RT-PCR even though the CP-1 VP7 had only 81 to 85% deduced amino acid identity to those of G10 rotaviruses. Rotavirus CP-1 was of P[5] specificity, a type common in cattle, and had a bovine NSP1 and NSP4. These results added another animal species to those in which G3 rotaviruses have been found, characterized a bovine rotavirus which caused age-independent diarrhea in calves, and raised the possibility that bovine G3 rotaviruses may be misdiagnosed as G10 rotaviruses.
Detection of
Mycoplasma pneumoniae
in Spiked Clinical Samples by Nucleic Acid Sequence-Based Amplification
Authors: Loens K. , Ursi D. , Ieven M. , van Aarle P. , Sillekens P. , Oudshoorn P. , Goossens H. 
Published: July 2002

ABSTRACT

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of
Mycoplasma pneumoniae. M. pneumoniae
RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type
M. pneumoniae
RNA generated in vitro and 5 color-changing units (CCU) of
M. pneumoniae
. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 × 10
4
molecules of in vitro-generated RNA or 5 CCU of
M. pneumoniae
. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for
M. pneumoniae
by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of
M. pneumoniae
in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.