bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of an Intergenic Region in
Pseudomonas syringae
pv. Syringae B728a for Site-Directed Genomic Marking of Bacterial Strains for Field Experiments
Authors: Hirano Susan S. , Willis David K. , Clayton Murray K. , Upper Christen D. 
Published: July 2002

ABSTRACT

To construct differentially-marked derivatives of our model wild-type strain,
Pseudomonas syringae
pv. syringae B728a (a causal agent of bacterial brown spot disease in snap bean plants), for field experiments, we selected a site in the
gacS-cysM
intergenic region for site-directed insertion of antibiotic resistance marker cassettes. In each of three field experiments, population sizes of the site-directed chromosomally marked B728a derivatives in association with snap bean plants were not significantly different from that of the wild-type strain. Inserts of up to 7 kb of DNA in the intergenic region did not measurably affect fitness of B728a in the field. The site is useful for site-directed genomic insertions of single copies of genes of interest.
Growth Patterns of Two Marine Isolates: Adaptations to Substrate Patchiness?
Authors: Pernthaler Annelie , Pernthaler Jakob , Eilers Heike , Amann Rudolf 
Published: July 2002

ABSTRACT

During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus
Pseudoalteromonas
rapidly multiplied in substrate-amended North Sea water, whereas the densities of
Oceanospirillum
changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634–4640, 2000). We therefore studied the growth patterns of two isolates affiliating with
Pseudoalteromonas
and
Oceanospirillum
in batch culture. Upon substrate resupply,
Oceanospirillum
lagged threefold longer than
Pseudoalteromonas
but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved
Oceanospirillum
populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of
Oceanospirillum
were reduced three times as much as those
Pseudoalteromonas
, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the
Pseudoalteromonas
population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.
Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in
Lactococcus lactis
Authors: Le Loir Y. , Nouaille S. , Commissaire J. , Brétigny L. , Gruss A. , Langella P. 
Published: July 2002

ABSTRACT

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in
Lactococcus lactis
using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP
Nuc
) by that of
L. lactis
protein Usp45 (SP
Usp
) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP
Usp
than when directed by SP
Nuc
. This SP
Usp
effect on Nuc secretion is not due to a better antifolding activity, since SP
Usp
:Nuc precursor proteins display enzymatic activity in vitro, while SP
Nuc
:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in
L. lactis
(Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP
Usp
and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in
L. lactis
.
Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4
Listeria monocytogenes
and Identify Four Distinct Lineages in Serotype 4b
Authors: Tran Huyen L. , Kathariou Sophia 
Published: July 2002

ABSTRACT

Listeria monocytogenes
of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of
Listeria monocytogenes
had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of
Eco
RI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other
L. monocytogenes
strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of
L. monocytogenes
and may facilitate detection and population genetic analysis of specific lineages.
Continuous Steady-State Method Using Tenax for Delivering Tetrachloroethene to Chloro-Respiring Bacteria
Authors: Brennan Rachel A. , Sanford Robert A. 
Published: July 2002

ABSTRACT

Tenax-TA, a solid-phase sorbent, was used as an alternative to hexadecane for continuous delivery of tetrachloroethene (PCE) to
Desulfuromonas
strain BB1, a chloro-respiring microorganism. In both batch and bioreactor configurations, Tenax not only maintained low, steady-state concentrations of PCE in an active culture for several months but also adsorbed the product of dechlorination,
cis
-1,2-dichloroethene, before it approached toxic levels.
Uric Acid Is a Genuine Metabolite of
Penicillium cyclopium
and Stimulates the Expression of Alkaloid Biosynthesis in This Fungus
Authors: Helbig Florian , Steighardt Jörg , Roos Werner 
Published: July 2002

ABSTRACT

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in
Penicillium cyclopium
uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor
l
-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor
l
-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.
Cloning of
rel
from
Listeria monocytogenes
as an Osmotolerance Involvement Gene
Authors: Okada Yumiko , Makino Sou-ichi , Tobe Toru , Okada Nobuhiko , Yamazaki Shouji 
Published: July 2002

ABSTRACT

Transposon insertional mutants of
Listeria monocytogenes
were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named
rel
, was 2,214 bp in length and had very high homology to
relA
of
Bacillus subtilis
, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included
rel
of
L. monocytogenes
was introduced into the mutant. After exposure to methyl glucoside, the
rel
mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in
L. monocytogenes
and that its mechanism is different from that for the accumulation of compatible solutes.
The
AWA1
Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast
Authors: Shimoi Hitoshi , Sakamoto Kazutoshi , Okuda Masaki , Atthi Ratchanee , Iwashita Kazuhiro , Ito Kiyoshi 
Published: July 2002

ABSTRACT

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as
Saccharomyces cerevisiae
. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named
AWA1
and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an
awa1
disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.
Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute
Spiroplasma citri
Authors: Malembic Sylvie , Saillard Colette , Bové Joseph M. , Garnier Monique 
Published: July 2002

ABSTRACT

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of
Spiroplasma citri
in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on
S. citri
glucose metabolism but were as efficient as polyclonal antibodies in inhibiting
S. citri
growth in liquid medium. Inhibition of motility was also observed.
Oral Treatment with
Lactococcus lactis
Expressing
Staphylococcus hyicus
Lipase Enhances Lipid Digestion in Pigs with Induced Pancreatic Insufficiency
Authors: Drouault Sophie , Juste Catherine , Marteau Philippe , Renault Pierre , Corthier Gérard 
Published: July 2002

ABSTRACT

The
Staphylococcus hyicus lip
gene was cloned in
Lactococcus lactis
. Pancreatic insufficiency was induced by ligation of the pancreatic duct in pigs. In pigs who had undergone pancreatic ligation, the coefficient of fat absorption was higher after consumption of lipase-expressing
L. lactis
(91.9% ± 3.7%) than that after consumption of the inactive control strain (78.4% ± 2.4%).
Antibody Response to Lipopolysaccharide in Patients Colonized or Infected with an Endemic Strain of
Acinetobacter
Genomic Species 13 Sensu Tjernberg and Ursing
Authors: Pantophlet Ralph , Seifert Harald , Brade Lore , Brade Helmut 
Published: July 2002

ABSTRACT

The levels of antilipopolysaccharide (anti-LPS) antibodies in patients colonized with an endemic
Acinetobacter
strain were compared to those in patients with bloodstream infections. Seropositivity and seronegativity correlated with positive and negative blood cultures, respectively, indicating that determination of the level of anti-LPS antibodies is useful for diagnosing
Acinetobacter
infections.
Clinical Application of a Dot Blot Test for Diagnosis of Enteric Fever Due to
Salmonella enterica
Serovar Typhi in Patients with Typhoid Fever from Colombia and Peru
Authors: Cardona-Castro Nora , Gotuzzo Eduardo , Rodriguez Monica , Guerra Humberto 
Published: July 2002

ABSTRACT

Clinical application of a dot blot test to detect immunoglobulin G (IgG) (88% sensitivity and specificity) and IgM (12.1% sensitivity and 97% specificity) against flagellar antigen from
Salmonella enterica
serovar Typhi was performed in Peruvian and Colombian patients with typhoid fever. This test can be used as a good predictor of serovar Typhi infection in regions lacking laboratory facilities and in field studies.
Monoclonal Antibody Differentiation of
Mycoplasma mycoides
subsp.
mycoides
Small-Colony Strains Causing Contagious Bovine Pleuropneumonia from Less Important Large-Colony Strains
Authors: Rurangirwa Fred R. , Shompole Patrick S. , Wambugu Anderson N. , McGuire Travis C. 
Published: July 2002

ABSTRACT

Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine
Mycoplasma mycoides
subsp.
mycoides
small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for
M. mycoides
subsp.
mycoides
small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.
Cloning of the Bovine Immunodeficiency Virus
gag
Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay
Authors: Zheng Ling , Swanson Michelle , Liao Jinghua , Wood Charles , Kapil Sanjay , Snider Ron , Loughin Thomas A. , Minocha Harish C. 
Published: July 2002

ABSTRACT

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The
gag
coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from
Escherichia coli
. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.
Cloning and Expression of Immunoreactive Antigens from
Mycobacterium tuberculosis
Authors: Hong Lim Renee Lay , Kiang Tan Li , Fun Lau Wai , Ching Ming Maxey , Chung  , Dunn Roseanne , Phon Too Heng , Chan Lily 
Published: July 2002

ABSTRACT

Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with molecular weights ranging from 16,000 to 58,000, were observed from immunoblots of
Mycobacterium tuberculosis
total lysates screened with sera from individuals with active tuberculosis. These proteins were identified from microsequence analyses, and genes of proteins with the highest homology were PCR amplified and cloned into the pQE30 vector for expression studies. In addition, a 37.5-kDa protein, designated C17, was identified from a phage expression library of
M. tuberculosis
genomic DNA. Preliminary immunoblot assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and extrapulmonary tuberculosis. The overall ranges of sensitivities, specificities, positive predictive values, and negative predictive values for the recombinant antigens were 20 to 58, 88 to 100, 69 to 100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis.
Diagnosis of Human Immunodeficiency Virus Type 1 Infection with Different Subtypes Using Rapid Tests
Authors: Phillips Susan , Granade Timothy C. , Pau Chou-Pong , Candal Debra , Hu Dale J. , Parekh Bharat S. 
Published: July 2002

ABSTRACT

We evaluated six rapid tests for their sensitivity and specificity in diagnosing human immunodeficiency virus type 1 (HIV-1) infection using 241 specimens (172 HIV-1 positive, 69 HIV-1 negative) representing different HIV-1 subtypes (A [
n
= 40], B [
n
= 47], C [
n
= 28], E [
n
= 42], and F [
n
= 7]). HIVCHEK, Multispot, RTD and SeroStrip were 100% sensitive and specific. Capillus failed to identify two of eight subtype C specimens (overall sensitivity of 98.85%), while the SUDS test (the only test approved by the Food and Drug Administration) gave false-positive results for 5 of 69 seronegative specimens (specificity of 93.24%). Our results suggest that although rapid tests perform well in general, it may be prudent to evaluate a rapid test for sensitivity and specificity in a local population prior to its widespread use.
Diagnosis of Granulocytic Ehrlichiosis in Humans by Immunofluorescence Assay
Authors: Brouqui P. , Salvo E. , Dumler J. S. , Raoult D. 
Published: July 2002

ABSTRACT
Serodiagnostic tests are widely available for tick-borne diseases. We evaluated a cell-free antigen of the human granulocytic ehrlichiosis agent. Immunofluorescence assay (IFA) with this antigen is as efficient as with the MRL kit and allows a one-step IFA with other cell-free antigens that is useful when testing sera from patients bitten by ticks.
PCR-Based Detection, Restriction Endonuclease Analysis, and Transcription of
tonB
in
Haemophilus influenzae
and
Haemophilus parainfluenzae
Isolates Obtained from Children Undergoing Tonsillectomy and Adenoidectomy
Authors: Matar Ghassan M. , Chahwan Richard , Fuleihan Nabil , Uwaydah Marwan , Hadi Usamah 
Published: July 2002

ABSTRACT

We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the
tonB
gene of
Haemophilus influenzae
and
Haemophilus parainfluenzae
from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55
H. influenzae
isolates were recovered from different sites of 20 patients, and 32
H. parainfluenzae
isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the
tonB
gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using
Xho
I and
Bgl
II were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the
tonB
gene was detected in both species. It is known to encode a virulent protein, TonB, in
H. influenzae
; however, demonstration of its presence in
H. parainfluenzae
is novel. Nested-PCR and restriction endonuclease analysis have shown that the
tonB
gene is apparently structurally the same in both species, with possible differences that may exist in certain
H. parainfluenzae
isolates. RT-PCR done on selected numbers of
H. influenzae
and
H. parainfluenzae
have shown that the
tonB
gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in
H. parainfluenzae
since it is not needed for heme or heme complexes uptake as with
H. influenzae.
Are the Opsonophagocytic Activities of Antibodies in Infant Sera Measured by Different Pneumococcal Phagocytosis Assays Comparable?
Authors: Väkeväinen Merja , Jansen Wouter , Saeland Eirikur , Jonsdottir Ingileif , Snippe Harm , Verheul Andre , Käyhty Helena 
Published: July 2002

ABSTRACT

Host protection against
Streptococcus pneumoniae
is mainly mediated by opsonin-dependent phagocytosis. Several techniques for measuring opsonophagocytic activity (OPA) of antibodies to
S. pneumoniae
have been standardized and used. These include the viable cell-assay, flow-cytometric assays, and an assay utilizing radiolabeled bacteria. Using these different methods, we measured the OPA of antibodies to
S. pneumoniae
types 6B and 19F from the sera of infants immunized with a pneumococcal conjugate vaccine, PncCRM. Generally, the results obtained by the various techniques correlated well, although serotype-specific differences were found (6B,
r
= 0.78 to 0.95,
P
< 0.001; 19F,
r
= 0.50 to 0.84,
P
< 0.001). The same serotype-specific differences were observed for the relationship between the concentrations of specific immunoglobulin G antibodies measured by enzyme immunoassay and the OPA. Since the sensitivities of the OPA assays differed, the most prominent discrepancies between the techniques were found at low antibody concentrations.