bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of the Incidental Production of a Melanin-Like Pigment during 6-Demethylchlortetracycline Production in
Streptomyces aureofaciens
Authors: Nakano Tetsuo , Miyake Koichiro , Ikeda Masato , Mizukami Toru , Katsumata Ryoichi 
Published: July 2002

ABSTRACT

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by
Streptomyces aureofaciens
, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using
S. aureofaciens
NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.
Expression of the Antifeeding Gene
anfA1
in
Serratia entomophila
Requires RpoS
Authors: Giddens Stephen R. , Tormo Antonio , Mahanty H. Khris 
Published: July 2002

ABSTRACT

The
rpoS
gene of
Serratia entomophila
BC4B was cloned and used to create
rpoS
-mutant strain BC4BRS. Larvae of the New Zealand grass grub
Costelytra zealandica
infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene
anfA1
in
trans
was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional
rpoS
gene is vital for full expression of
anfA1
and for development of the antifeeding component of amber disease.
Cloning and Sequencing of the Gene Encoding an Aldehyde Dehydrogenase That Is Induced by Growing
Alteromonas
sp. Strain KE10 in a Low Concentration of Organic Nutrients
Authors: Maeda Toshimichi , Yoshinaga Ikuo , Shiba Tsuneo , Murakami Masatada , Wada Akira , Ishida Yuzaburou 
Published: July 2002

ABSTRACT

The protein composition of
Alteromonas
sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter
−1
) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD
+
-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in
Escherichia coli
, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.
Simultaneous Immunofluorescent Detection of Coentrapped Cells in Gel Beads
Authors: Prioult Guenolee , Lacroix Christophe , Turcotte Carl , Fliss Ismaïl 
Published: July 2002

ABSTRACT
An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.
New Insights into Methyl Bromide Cooxidation by
Nitrosomonas europaea
Obtained by Experimenting with Moderately Low Density Cell Suspensions
Authors: Duddleston Khrystyne N. , Bottomley Peter J. , Porter Angela J. , Arp Daniel J. 
Published: July 2002

ABSTRACT

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (∼6 × 10
7
cells ml
−1
) of the NH
3
-oxidizing bacterium
Nitrosomonas europaea
. In the presence of 10 mM NH
4
+
and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH
4
+
, 18% of the NH
4
+
, and 35% of the NH
4
+
, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH
4
+
-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells
−1
). Approximately 90% of the NH
3
-dependent O
2
uptake activity and the NO
2
−
-producing activity were lost after
N. europaea
was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO
2
−
production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO
2
−
accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO
2
−
-producing and MeBr-oxidizing activities could not be attributed directly to NH
4
+
or NH
3
limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO
2
−
and formaldehyde) in the medium. Additional cooxidation-related studies of
N. europaea
are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH
3
-oxidizing activity.
Rhizosphere Soil Aggregation and Plant Growth Promotion of Sunflowers by an Exopolysaccharide-Producing
Rhizobium
sp. Strain Isolated from Sunflower Roots
Authors: Alami Younes , Achouak Wafa , Marol Christine , Heulin Thierry 
Published: July 2002

ABSTRACT

Root-adhering soil (RAS) forms the immediate environment where plants take up water and nutrients for their growth. We report the effect of an exopolysaccharide (EPS)-producing rhizobacterium (strain YAS34) on the physical properties of sunflower (
Helianthus annuus
L.) RAS, associated with plant growth promotion, under both water stress and normal water supply conditions. Strain YAS34 was isolated as a major EPS-producing bacterium from the rhizoplane of sunflowers grown in a French dystric cambisol. Strain YAS34 was assigned to the
Rhizobium
genus by 16S ribosomal DNA gene sequencing. Inoculation of sunflower seeds and soil with strain YAS34 caused a significant increase in RAS per root dry mass (dm) (up to 100%) and a significant increase in soil macropore volume (12 to 60 μm in diameter). The effect of inoculation on sunflower shoot dm (up to +50%) and root dm (up to +70%) was significant under both normal and water stress conditions. Inoculation with strain YAS34 modified soil structure around the root system, counteracting the negative effect of water deficit on growth. Using [
15
N]nitrate, we showed that inoculation made the use of fertilizer more effective by increasing nitrogen uptake by sunflower plantlets.
Relationship of Exo-β-
d
-Galactofuranosidase Kinetic Parameters to the Number of Phosphodiesters in
Penicillium fellutanum
Peptidophosphogalactomannan: Enzyme Purification and Kinetics of Glycopeptide and Galactofuran Chain Hydrolysis
Authors: Tuekam Brigitte A. , Park Yong-Il , Unkefer Clifford J. , Gander John E. 
Published: July 2002

ABSTRACT

Extracellular
Penicillium fellutanum
exo-β-
d
-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP
2
GM
ii
and pP
25
GM
ii
(containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-
O
-methyl-β-
d
-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP
2
GM
ii
or pP
25
GM
ii
. The
k
cat
/

K
m

value for pP
25
GM
ii
is 1.7 × 10
3
M
−1
s
−1
, that for 1-
O
-methyl-β-
d
-galactofuranoside is 1.1 × 10
4
M
−1
s
−1
, that for pP
2
GM
ii
is 1.7 × 10
4
M
−1
s
−1
, and those for 5-
O
-β-
d
-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 10
5
and 4.1 × 10
5
M
−1
s
−1
, respectively. Variability in the
k
cat
/

K
m

values is due primarily to differences in

K
m

values; the
k
−1
/
k
1
ratio likely provides the most influence on

K
m
. k

cat
increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP
x
GM
ii
added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml
−1
h
−1
. No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP
x
GM
ii
and of related polymers is proportional to the
k
cat
/

K
m

value of each polymer. The kinetic data show that the
k
cat
/

K
m

value increases as the number of phosphodiesters of pP
x
GM
ii
decreases, also resulting in an increase in the activity of exo-β-
d
-galactofuranosidase.
Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1
Authors: Østergaard Solvej , Brøndsted Lone , Vogensen Finn K. 
Published: July 2002

ABSTRACT

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by
orf13
and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within
orf13
and was shown to confer a TP901-1 resistance phenotype when present in
trans
. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by
orf13
and the repeats within
orf13
were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that
orf13
encodes a replication protein and that the repeats within the gene are the origin of replication.
Cloning of Genomic DNA of
Lactococcus lactis
That Restores Phage Sensitivity to an Unusual Bacteriophage sk1-Resistant Mutant
Authors: Kraus Jennifer , Geller Bruce L. 
Published: July 2002

ABSTRACT

An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of
Lactococcus lactis
C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (β-1,4-
N
-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of
Lactobacillus
and
Streptococcus
and also revealed DNA homologous to noncoding sequences of temperate phage of
L. lactis
, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to
sun
and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.
Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in
Escherichia coli
Authors: Manosroi Jiradej , Tayapiwatana Chatchai , Götz Friedrich , Werner Rolf G. , Manosroi Aranya 
Published: July 2002

ABSTRACT

The DNA fragment coding for kringle 2 plus serine protease domains (
K2S
) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-
K2S
, the
K2S
gene was fused to
gpIII
of ΦM13 and linked to the OmpA signal sequence. The resulting gene,
rK2S-gpIII,
was inducibly expressed in
Escherichia coli
XL-1 Blue. The protein was presented on the phage particle. To stop the expression of
gpIII,
a stop codon between
K2S
and the
gpIII
gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-
K2S
, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-
d
-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.
Increased Enterocyte Production in Gnotobiotic Rats Mono-Associated with
Lactobacillus rhamnosus
GG
Authors: Banasaz M. , Norin E. , Holma R. , Midtvedt T. 
Published: July 2002

ABSTRACT

There is increasing scientific and commercial interest in using beneficial microorganisms (i.e., probiotics) to enhance intestinal health. Of the numerous microbial strains examined,
Lactobacillus rhamnosus
GG has been most extensively studied. Daily intake of
L. rhamnosus
GG shortens the course of rotavirus infection by mechanisms that have not been fully elucidated. Comparative studies with germfree and conventional rats have shown that the microbial status of an animal influences the intestinal cell kinetics and morphology. The present study was undertaken to study whether establishment of
L. rhamnosus
GG as a mono-associate in germfree rats influences intestinal cell kinetics and morphology.
L. rhamnosus
GG was easily established in germfree rats. After 3 days of mono-association, the rate of mitoses in the upper part of the small intestine (jejunum 1) increased as much as 14 and 22% compared to the rates in germfree and conventional counterparts, respectively. The most striking alteration in morphology was an increase in the number of cells in the villi. We hypothesis that the compartmentalized effects of
L. rhamnosus
GG may represent a reparative event for the mucosa.
Variability and Immunogenicity of Human Immunodeficiency Virus Type 1 p24 Gene Quasispecies
Authors: Iroegbu Johanna , Birk Markus , Lazdina Una , Sönnerborg Anders , Sällberg Matti 
Published: July 2002

ABSTRACTDespite the conserved nature of the human immunodeficiency virus type 1 (HIV-1)gaggene, multiple quasispecies of the p24 gene coexist in HIV-1-infected patients. We cloned and sequenced 31 p24 genes from four HIV-1-infected patients. The intrapatient homology between the p24 genes ranged from 97.1 to 99.1%, whereas the interpatient homology ranged from 91.5 to 93.8%, suggesting a host-specific evolution. Synonymous and nonsynonymous nucleotide changes were evenly distributed in the p24 gene, with 27 and 28%, respectively, located within host human leukocyte antigen class I recognition sites. This would suggest only a minor influence from the host cytotoxic T-cell response on the evolution of the p24 gene. The importance of minor variations within p24 was analyzed by designing DNA-based immunogens from two distinct p24 quasispecies genes simultaneously derived from one patient. In plasmid-immunizedH-2b,H-2d, andH-2khaplotype mice, a clear influence from the host major histocompatibility complex was noted on the immune responses, fully consistent with those noted when a recombinant p24 protein is used as the immunogen. The two p24 DNA immunogens did not differ in their immunogenicity, indicating that the limited genetic variability (<1%) had little influence on the immune responses.
Neutralization Profiles of Sera from Human Immunodeficiency Virus (HIV)-Infected Individuals: Relationship to HIV Viral Load and CD4 Cell Count
Authors: Nokta Mostafa , Turk Patricia , Loesch Kimberly , Pollard Richard B. 
Published: July 2002

ABSTRACT

The relationship of the neutralizing activity (NA) profile of sera from human immunodeficiency virus (HIV)-infected individuals to the HIV viral load and the absolute CD4 count was examined. The NA of 24 serum samples against autologous isolates (AI) and HIV type 1 strain MN was examined. Three NA patterns were recognized. Nine sera neutralized both AI and MN (+/+), six sera neutralized MN but not AI (−/+), and nine sera failed to neutralize both AI and MN (−/−). The identification of the three neutralization patterns (+/+, −/+, and −/−) indicated that resistance to neutralization was progressive. A reciprocal relationship between the viral burden of the patients and the NA profiles was observed. The nine subjects with a −/− NA profile had a plasma viral load of ≥5 × 10
4
copies/ml and a cellular viral burden of ≥1,122 infectious units per million viable cells, which were significantly different from those of the other groups (
P
< 0.02). These patterns were independent of the phenotypic characteristics of the virus. Longitudinally, subjects with a −/− profile at baseline gained their HIV-specific NA by 24 weeks of antiretroviral therapy when this was associated with a ≥1-log
10
decline in the plasma HIV viral load. The sera from week 24 from some patients were able to neutralize both the 24-week and the baseline dominant virus isolates. A change in CD4 cell count of 50 or more in either direction predicted a −/− or +/+ profile. The verification of the autologous NA profile might be important in selecting patients who may benefit from immune-based therapies involving neutralizing monoclonal antibodies.
Identification and Strain Differentiation of
Vibrio cholerae
by Using Polyclonal Antibodies against Outer Membrane Proteins
Authors: Martı́nez-Govea A. , Ambrosio J. , Gutiérrez-Cogco L. , Flisser A. 
Published: July 2002

ABSTRACT

Cholera is caused only by O1 and O139
Vibrio cholerae
strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from
V. cholerae
O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal
Vibrio
antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for
V. cholerae
detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.
Serum Antibody Response to Polysaccharides in Children with Recurrent Respiratory Tract Infections
Authors: López-Yap Alberto , Abdelnour Arturo , Lomonte Bruno , Porras Oscar 
Published: July 2002

ABSTRACT

We evaluated children (15-months old and older) with recurrent upper respiratory tract infections and normal levels of immunoglobulins in serum for specific polysaccharide immunodeficiency using an enzyme-linked immunosorbent assay method. Results showed that of 12 patients vaccinated with Act-HIB vaccine, one did not develop specific antibodies to
Haemophilus influenzae
type b, demonstrating that such immunodeficiency is present in Costa Rican children.