bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering
Escherichia coli
for Increased Productivity of Serine-Rich Proteins Based on Proteome Profiling
Authors: Han Mee-Jung , Jeong Ki Jun , Yoo Jong-Shin , Lee Sang Yup 
Published: October 2003

ABSTRACT

Variations in proteome profiles of
Escherichia coli
in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis. The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction. Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased. Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the
cysK
gene, encoding cysteine synthase A. By coexpression of the
cysK
gene, we were able to increase the cell growth rate by approximately twofold. Also, the specific leptin productivity could be increased by fourfold. In addition, we found that
cysK
coexpression can improve the production of another serine-rich protein, interleukin-12 β chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well. The approach taken in this study should be useful in designing a strategy for improving recombinant protein production.
Detection, Quantitation, and Phylogenetic Analysis of Noroviruses in Japanese Oysters
Authors: Nishida Tomoko , Kimura Hirokazu , Saitoh Mika , Shinohara Michiyo , Kato Masahiko , Fukuda Shinji , Munemura Tetsuya , Mikami Toshiyuki , Kawamoto Ayumi , Akiyama Miho , Kato Yumiko , Nishi Kanako , Kozawa Kunihisa , Nishio Osamu 
Published: October 2003

ABSTRACT

Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 10
2
copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.
Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate
Lactobacillus rhamnosus
BGT10
Authors: Pastar Irena , Tonic Ivana , Golic Natasa , Kojic Milan , van Kranenburg Richard , Kleerebezem Michiel , Topisirovic Ljubisa , Jovanovic Goran 
Published: October 2003

ABSTRACT

A novel proteinase, PrtR, produced by the human vaginal isolate
Lactobacillus rhamnosus
strain BGT10 was identified and genetically characterized. The
prtR
gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its α
S1
- and β-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all
L. rhamnosus
strains tested. Proteinase extracts of the BGT10 strain obtained with Ca
2+
-free buffer at pH 6.5 were proteolytically active. The
prtR
promoter-like sequence was determined, and the minimal promoter region was defined by use of
prtR-gusA
operon fusions. The
prtR
expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.
Establishment of an Arbitrary PCR for Rapid Identification of Tn
917
Insertion Sites in
Staphylococcus epidermidis
: Characterization of Biofilm-Negative and Nonmucoid Mutants
Authors: Knobloch Johannes K.-M. , Nedelmann Max , Kiel Kathrin , Bartscht Katrin , Horstkotte Matthias A. , Dobinsky Sabine , Rohde Holger , Mack Dietrich 
Published: October 2003

ABSTRACT

Transposon mutagenesis with the
Enterococcus faecalis
transposon Tn
917
is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn
917
insertion sites in
Staphylococcus epidermidis
with six independent, well-characterized biofilm-negative Tn
917
transposon mutants, which were clustered in the
icaADBC
gene locus or harbor Tn
917
in the regulatory gene
rsbU
. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn
917
insertion sites in the published
S. epidermidis
genomes. By using this technique, the Tn
917
insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn
917
is inserted into a gene homologous to the regulatory gene
purR
of
Bacillus subtilis
and
Staphylococcus aureus
. The Tn
917
insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of
B. subtilis
,
S. aureus
, and
Staphylococcus carnosus
, indicating an influence of the PTS on the mucoid phenotype in
S. epidermidis
.
Development of a PCR-Enzyme Immunoassay Oligoprobe Detection Method for
Toxoplasma gondii
Oocysts, Incorporating PCR Controls
Authors: Schwab Kellogg J. , McDevitt James J. 
Published: October 2003

ABSTRACT

Infections caused by
Toxoplasma gondii
are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of
T. gondii
infection.
T. gondii
has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to
T. gondii
oocysts. Because of the lack of information regarding the prevalence of
T. gondii
in surface waters, there is a clear need for a rapid, sensitive method to detect
T. gondii
from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of
T. gondii
nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for
T. gondii
oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-
N
-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a
T. gondii
oocyst PCR detection method will provide a useful technique to estimate the levels of
T. gondii
oocysts present in surface waters.
Membrane Fluidity Adjustments in Ethanol-Stressed
Oenococcus oeni
Cells
Authors: Da Silveira M. Graça , Golovina Elena A. , Hoekstra Folkert A. , Rombouts Frank M. , Abee Tjakko 
Published: October 2003

ABSTRACT

The effect of ethanol on the cytoplasmic membrane of
Oenococcus oeni
cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.
Substrate Preferences in Biodesulfurization of Diesel Range Fuels by
Rhodococcus
sp. Strain ECRD-1
Authors: Prince Roger C. , Grossman Matthew J. 
Published: October 2003

ABSTRACT

The range of sulfur compounds in fuel oil and the substrate range and preference of the biocatalytic system determine the maximum extent to which sulfur can be removed by biodesulfurization. We show that the biodesulfurization apparatus in
Rhodococcus
sp. strain ECRD-1 is able to attack all isomers of dibenzothiophene including those with at least four pendant carbons, with a slight preference for those substituted in the α-position. With somewhat less avidity, this apparatus is also able to attack substituted benzothiophenes with between two and seven pendant carbons. Some compounds containing sulfidic sulfur are also susceptible to desulfurization, although we have not yet been able to determine their molecular identities.
Anaerobic Degradation of Flavonoids by
Clostridium orbiscindens
Authors: Schoefer Lilian , Mohan Ruchika , Schwiertz Andreas , Braune Annett , Blaut Michael 
Published: October 2003

ABSTRACT

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as
Clostridium orbiscindens
by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated
C. orbiscindens
strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2′-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of
C. orbiscindens
in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of
C. orbiscindens
in 8 out of the 10 samples tested. The numbers ranged from 1.87 × 10
8
to 2.50 × 10
9
cells g of fecal dry mass
−1
, corresponding to a mean count of 4.40 × 10
8
cells g of dry feces
−1
.
Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from
Lactobacillus fermentum
BR11
Authors: Turner Mark S. , Hafner Louise M. , Walsh Terry , Giffard Philip M. 
Published: October 2003

ABSTRACT

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from
Lactobacillus fermentum
BR11 has been identified by using an antiserum raised against whole
L. fermentum
BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from
Streptococcus agalactiae
, while the other protein, Mlp, is similar to the mucus binding protein Mub from
Lactobacillus reuteri
. It was shown that multiple copies of
mlp
exist in the genome of
L. fermentum
BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in
L. fermentum
. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His
6
). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The
mlp
promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His
6
and CFTR peptides or His
6
peptide, respectively. Therefore, these newly described proteins from
L. fermentum
BR11 have potential as protein production and targeting vectors.
Identification of
Naegleria fowleri
in Domestic Water Sources by Nested PCR
Authors: Marciano-Cabral Francine , MacLean Rebecca , Mensah Alex , LaPat-Polasko Laurie 
Published: October 2003

ABSTRACT

The free-living amoeboflagellate
Naegleria fowleri
is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to
N. fowleri
and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for
N. fowleri
by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for
N. fowleri
by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for
N. fowleri
were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.
Mycotoxin Fumonisin B1Increases Intestinal Colonization by PathogenicEscherichia coliin Pigs
Authors: Oswald Isabelle P. , Desautels Clarisse , Laffitte Joëlle , Fournout Sylvie , Peres Sylvie Y. , Odin Marielle , Le Bars Pierrette , Le Bars Joseph , Fairbrother John M. 
Published: October 2003

ABSTRACTFumonisin B1(FB1) is a mycotoxin that commonly occurs in maize. FB1causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans. In the present study, we examined the effect of dietary exposure to FB1on intestinal colonization by pathogenicEscherichia coliassociated with extraintestinal infection. Three-week-old weaned pigs were given FB1by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days. On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenicE. colistrain. All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination. Ingestion of FB1had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile. However, colonization of the small and large intestines by an extraintestinal pathogenicE. colistrain was significantly increased. Our results show that FB1is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food.
Stimulating the In Situ Activity of
Geobacter
Species To Remove Uranium from the Groundwater of a Uranium-Contaminated Aquifer
Authors: Anderson Robert T. , Vrionis Helen A. , Ortiz-Bernad Irene , Resch Charles T. , Long Philip E. , Dayvault Richard , Karp Ken , Marutzky Sam , Metzler Donald R. , Peacock Aaron , White David C. , Lowe Mary , Lovley Derek R. 
Published: October 2003

ABSTRACT

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 μM in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of
Geobacter
species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and
Geobacter
species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of
Geobacter
species.
Effects of Iron Limitation on Adherence and Cell Surface Carbohydrates of
Corynebacterium diphtheriae
Strains
Authors: Moreira Lílian de Oliveira , Andrade Arnaldo Feitosa Braga , Vale Márcio Damasceno , Souza Sônia Maria Silva , Hirata Raphael , Asad Lídia Maria Oliveira Buarque , Asad Nasser Ribeiro , Monteiro-Leal Luiz Henrique , Previato José Osvaldo , Mattos-Guaraldi Ana Luiza 
Published: October 2003

ABSTRACT

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of
Corynebacterium diphtheriae
subsp.
mitis
and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl
3
. The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in
d
-mannose and
d
-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of
C. diphtheriae
surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for
d
-mannose,
d
-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For
C. diphtheriae
, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.
Role of Ectoine in
Vibrio cholerae
Osmoadaptation
Authors: Pflughoeft Kathryn J. , Kierek Katharine , Watnick Paula I. 
Published: October 2003

ABSTRACT

Vibrio cholerae
is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment,
V. cholerae
must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of
V. cholerae
to increases in environmental osmolarity. Optimal growth of
V. cholerae
in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted
V. cholerae
displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted
V. cholerae
displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for
V. cholerae
population dynamics when seawater and freshwater and their attendant microbes mix.