bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Carbon and Electron Flow in
Clostridium butyricum
VPI 3266 Grown on Glucose-Glycerol Mixtures
Authors: Saint-Amans Sylvie , Girbal Laurence , Andrade Jose , Ahrens Kerstin , Soucaille Philippe 
Published: July 2002

ABSTRACT

The metabolism of
Clostridium butyricum
was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for
Clostridium acetobutylicum
(L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of
C. butyricum
is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H
+
ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
Evolutionary Divergence of an Elongation Factor 3 from
Cryptococcus neoformans
Authors: Blakely Greg , Hekman James , Chakraburtty Kalpana , Williamson Peter R. 
Published: July 2002

ABSTRACT

Elongation factor 3 (EF3) is considered a promising drug target for the control of fungal diseases because of its requirement for protein synthesis and survival of fungi and a lack of EF3 in the mammalian host. However, EF3 has been characterized only in ascomycete yeast. In order to understand the role of EF3 in a basidiomycete yeast, we cloned the gene encoding EF3 from
Cryptococcus neoformans
(
CnEF3
), an important fungal pathogen in immunocompromised patients, including those infected with human immunodeficiency virus.
CnEF3
was found to encode a 1,055-amino-acid protein and has 44% identity with EF3 from
Saccharomyces cerevisiae
(
YEF3
). Expressed CnEF3 exhibited ATPase activity that was only modestly stimulated by ribosomes from
S. cerevisiae
. In contrast, CnEF3 showed tight binding to cryptococcal ribosomes, as shown by an inability to be removed under conditions which successfully remove
Saccharomyces
EF3 from ribosomes (0.5 M KCl or 2 M LiCl).
CnEF3
also poorly complemented a
YEF3
defect in a diploid null mutant and two temperature-sensitive mutants which have been shown previously to be complemented well by
EF3
from other ascomycetes, such as
Candida albicans
. These data clearly identify the presence of a functioning
EF3
in the basidiomycete yeast
C. neoformans
, which demonstrates an evolutionary divergence from
EF3
of ascomycete yeast.
Development of a New Integration Site within the
Bacillus subtilis
Chromosome and Construction of Compatible Expression Cassettes
Authors: Härtl Barbara , Wehrl Wolfgang , Wiegert Thomas , Homuth Georg , Schumann Wolfgang 
Published: July 2002

ABSTRACT

The
Bacillus subtilis lacA
gene, coding for β-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the
lacA
locus was able to control the expression of a transcriptional fusion at the
amyE
locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.
Mapping of the Rsd Contact Site on the Sigma 70 Subunit of
Escherichia coli
RNA Polymerase
Authors: Jishage Miki , Dasgupta Dipak , Ishihama Akira 
Published: July 2002

ABSTRACT

Rsd (regulator of sigma D) is an anti-sigma factor for the
Escherichia coli
RNA polymerase ς
70
subunit. The contact site of Rsd on ς
70
was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-
p
-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted ς
70
and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter −35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and ς
70
contact transcription factors.
Utilization of
l
-Ascorbate by
Escherichia coli
K-12: Assignments of Functions to Products of the
yjf-sga
and
yia-sgb
Operons
Authors: Yew Wen Shan , Gerlt John A. 
Published: July 2002

ABSTRACT

Escherichia coli
K-12 can ferment
l
-ascorbate. The operon encoding catabolic enzymes in the utilization of
l
-ascorbate (
ula
) has been identified; this operon of previously unknown function had been designated the
yif-sga
operon. Three enzymes in the pathway that produce
d
-xylulose 5-phosphate have been functionally characterized: 3-keto-
l
-gulonate 6-phosphate decarboxylase (UlaD),
l
-xylulose 5-phosphate 3-epimerase (UlaE), and
l
-ribulose 5-phosphate 4-epimerase (UlaF). Several products of the
yia-sgb
operon were also functionally characterized, although the substrate and physiological function of the operon remain unknown: 2,3-diketo-
l
-gulonate reductase (YiaK), 3-keto-
l
-gulonate kinase (LyxK), 3-keto-
l
-gulonate 6-phosphate decarboxylase (SgbH), and
l
-ribulose 5-phosphate 4-epimerase (SgbE).
Functional Interaction of Region 4 of the Extracytoplasmic Function Sigma Factor FecI with the Cytoplasmic Portion of the FecR Transmembrane Protein of the
Escherichia coli
Ferric Citrate Transport System
Authors: Mahren Susanne , Enz Sabine , Braun Volkmar 
Published: July 2002

ABSTRACT

Transcriptional regulation of the ferric citrate transport genes of
Escherichia coli
is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the
fecABCDE
genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and
fecB-lacZ
transcription. The cloned region 4 of FecI interfered with
fecB-lacZ
transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of
Pseudomonas aeruginosa
was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the
P. aeruginosa
proteins and between a
P. aeruginosa
FecI homolog and
E. coli
FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.
Diversity of Tn4001Transposition Products: the Flanking IS256Elements Can Form Tandem Dimers and IS Circles
Authors: Prudhomme M. , Turlan C. , Claverys J.-P. , Chandler M. 
Published: July 2002

ABSTRACTWe show that both flanking IS256elements carried by transposon Tn4001are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256belonging to one Tn4001copy attacks an IS256end carried by the second Tn4001copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis inEscherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such asStreptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.
Mutations in the GTPase Center of
Escherichia coli
23S rRNA Indicate Release Factor 2-Interactive Sites
Authors: Xu Wenbing , Pagel Frances T. , Murgola Emanuel J. 
Published: July 2002

ABSTRACT

Mutations in the GTPase center of
Escherichia coli
23S rRNA were characterized in vivo as UGA-specific nonsense suppressors. Some site-directed mutations did not exhibit suppressor activity and were interspersed among suppressor mutations. Our results demonstrate the involvement of the two adjacent loops of this conserved rRNA structure in UGA-dependent translation termination and, taken with previous in vitro analyses and with consideration of the crystal structure of the GTPase center RNA, indicate that nucleotides 1067, 1093, 1094, and 1095 are sites of interaction with release factor 2.
An Extracellular Matrix-Associated Zinc Metalloprotease Is Required for Dilauroyl Phosphatidylethanolamine Chemotactic Excitation in
Myxococcus xanthus
Authors: Kearns Daniel B. , Bonner Pamela J. , Smith Daniel R. , Shimkets Lawrence J. 
Published: July 2002

ABSTRACT

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium
Myxococcus xanthus
is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of
fibA
had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the
fibA
mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.
Force and Velocity of
Mycoplasma mobile
Gliding
Authors: Miyata Makoto , Ryu William S. , Berg Howard C. 
Published: July 2002

ABSTRACT

The effects of temperature and force on the gliding speed of
Mycoplasma mobile
were examined. Gliding speed increased linearly as a function of temperature from 0.46 μm/s at 11.5°C to 4.0 μm/s at 36.5°C. A polystyrene bead was attached to the tail of
M. mobile
using a polyclonal antibody raised against whole
M. mobile
cells. Cells attached to beads glided at the same speed as cells without beads. When liquid flow was applied in a flow chamber, cells reoriented and moved upstream with reduced speeds. Forces generated by cells at various gliding speeds were calculated by multiplying their estimated frictional drag coefficients with their velocities relative to the liquid. The gliding speed decreased linearly with force. At zero speed, the force measurements extrapolated to 26 pN at 22.5 and 27.5°C. At zero force, the speed extrapolated to 2.3 and 3.3 μm/s at 22.5 and 27.5°C, respectively—the same speeds as those observed for free gliding cells. Cells attached to beads were also trapped by an optical tweezer, and the stall force was measured to be 26 to 28 pN (17.5 to 27.5°C). The gliding speed depended on temperature, but the maximum force did not, suggesting that the mechanism is composed of at least two steps, one that generates force and another that allows displacement. Other implications of these results are discussed.
Single-Tube Single-Enzyme Reverse Transcriptase PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pooled Bovine Serum
Authors: Weinstock Daniel , Bhudevi Bodreddigari , Castro Anthony E. 
Published: July 2002

ABSTRACTA reverse transcriptase PCR (RT-PCR) was developed for use as a diagnostic screening test for the detection of bovine viral diarrhea virus (BVDV) in pooled bovine serum samples. Individual serum samples from 60 dairy cattle herds located in Pennsylvania were evaluated by the microplate virus isolation method, and pooled sera were analyzed by RT-PCR. RT-PCR was sensitive and specific and detected a single viremic serum sample in up to 100 pooled serum samples. RT-PCR analysis of pooled sera provides a rapid and cost-effective method for the screening of cattle herds for the presence of animals persistently infected with BVDV.
Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes
Authors: Lindsley Mark D. , Hurst Steven F. , Iqbal Naureen J. , Morrison Christine J. 
Published: July 2002

ABSTRACTSpecific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA fromHistoplasma capsulatum,Blastomyces dermatitidis,Coccidioides immitis,Paracoccidioides brasiliensis,Penicillium marneffei,Sporothrix schenckii,Cryptococcus neoformans, fiveCandidaspecies, andPneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA fromH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis, andP.marneffeibut not with DNA from nondimorphic fungi. Specific probes forH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis,P.marneffei,S.schenckii,C.neoformans, andP.cariniihybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for theB.dermititidisprobe used againstC.immitisDNA and for theH.capsulatumprobe used againstCandida albicansDNA. However, theC.immitisprobe did not cross-react withB.dermititidisDNA, nor did the dimorphic probe hybridize withC.albicansDNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.
Epidemiology and Susceptibility of 3,051Staphylococcus aureusIsolates from 25 University Hospitals Participating in the European SENTRY Study
Authors: Fluit A. C. , Wielders C. L. C. , Verhoef J. , Schmitz F.-J. 
Published: July 2002

ABSTRACTA total of 3,051 methicillin-susceptibleStaphylococcus aureus(MSSA) isolates and methicillin-resistantS. aureus(MRSA) isolates in Europe were compared. MRSA isolates constituted 25% of all isolates and were more prevalent in southern Europe. MRSA isolates appeared to be more prevalent in intensive care units than in outpatient departments. Only a small minority of MSSA isolates were multidrug resistant, whereas the majority of MRSA isolates were multidrug resistant.
Characterization ofNeospora caninumSurface Protein NcSRS2 Based on Baculovirus Expression System and Its Application for Serodiagnosis ofNeosporaInfection
Authors: Nishikawa Yoshifumi , Kousaka Yuko , Tragoolpua Khajornsak , Xuan Xuenan , Makala Levi , Fujisaki Kozo , Mikami Takeshi , Nagasawa Hideyuki 
Published: July 2002

ABSTRACTThe baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have characterized theNeospora caninumsurface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis ofNeosporainfection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera againstToxoplasma gondii,Hammondia heydorni, orN. caninum. Anti-N. caninummouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera againstN. caninumfrom IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis ofN. caninuminfection.
First European Pediatric Case of Human Granulocytic Ehrlichiosis
Authors: Arnež Maja , Petrovec Miroslav , Lotrič-Furlan Stanka , Zupanc Tatjana Avsic , Strle Franc 
Published: July 2002

ABSTRACT

Herein we report on the first confirmed pediatric case of acute human granulocytic ehrlichiosis in Europe. Presentation in this 11-year-old girl was comparable to clinical findings seen in adult European patients with human granulocytic ehrlichiosis; i.e., she had self-limited febrile illness with leukopenia, thrombocytopenia, and elevated serum C-reactive protein concentration. It is of interest that the patient not only had a fourfold change in antibody titer to
Ehrlichia phagocytophila
but also developed antibodies to
Ehrlichia chaffeensis
and that her PCR test result was positive on the third as well as on the 22nd day after the onset of illness, that is, 16 days after spontaneous defervescence.
Dogs as Sentinels for Human Lyme Borreliosis in The Netherlands
Authors: Goossens H. A. T. , van den Bogaard A. E. , Nohlmans M. K. E. 
Published: July 2002

ABSTRACTSerum samples from hunters (n= 440), their hunting dogs (n= 448), and hunters without dog ownership (n= 53) were collected in The Netherlands at hunting dog trials and were tested for antibodies againstBorrelia burgdorferiby a whole-cell enzyme-linked immunosorbent assay. Additionally, 75 healthy pet dogs were tested. The results of this study indicate that the seroprevalence among hunting dogs (18%) was of the same order as the seroprevalence among pet dogs (17%) and hunters (15%). The seropositivity of a hunting dog was not a significant indicator of increased risk of Lyme borreliosis for its owner. No significant rise in seroprevalence was found in dogs older than 24 months. This indicated that seropositivity after an infection withB. burgdorferiin dogs is rather short, approximately 1 year. In humans this is considerably longer but is also not lifelong. Therefore, the incidence ofB. burgdorferiinfections among dogs was greater than that among hunters, despite a similar prevalence of seropositivity among hunters and their hunting dogs. Because no positive correlation was observed between the seropositivity of a hunter and the seropositivity of the hunter's dog, direct transfer of ticks between dog and hunter does not seem important and owning a dog should not be considered a risk factor for Lyme borreliosis.
Comparison of Cultivation and PCR-Hybridization for Detection ofSalmonellain Porcine Fecal and Water Samples
Authors: Feder Ingrid , Nietfeld Jerome C. , Galland John , Yeary Teresa , Sargeant Jan M. , Oberst Richard , Tamplin Mark L. , Luchansky John B. 
Published: July 2002

ABSTRACTA total of 150 fecal and water samples from four swine farms were tested for the presence ofSalmonella entericausing different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37°C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viableSalmonellabacteria. PresumptiveSalmonellaisolates were biochemically and serologically confirmed. For the PCR detection ofSalmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to theinvAandinvEgenes were confirmed asSalmonellaspecific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for theSalmonella invAgene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone.Salmonellabacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement betweenSalmonelladetection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement betweenSalmonelladetection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, whileSalmonellabacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detectingSalmonellain swine feces or water samples from swine farms when using the medium combinations evaluated in this study.