bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of
Lactococcus lactis
Authors: Palmfeldt Johan , Paese Marco , Hahn-Hägerdal Bärbel , van Niel Ed W. J. 
Published: September 2004

ABSTRACT

Lactococcus lactis
grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose,
L. lactis
65.1 and MG1363, showed more pronounced mixed acid product formation during growth than
L. lactis
ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P
i
was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of
L. lactis
65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in
L. lactis
is discussed.
Regulation of the
N
-Acyl Homoserine Lactone-Dependent Quorum-Sensing System in Rhizosphere
Pseudomonas putida
WCS358 and Cross-Talk with the Stationary-Phase RpoS Sigma Factor and the Global Regulator GacA
Authors: Bertani Iris , Venturi Vittorio 
Published: September 2004

ABSTRACT

Quorum sensing is a cell population-density dependent regulatory system which in gram-negative bacteria often involves the production and detection of
N
-acyl homoserine lactones (AHLs). Some
Pseudomonas putida
strains have been reported to produce AHLs, and one quorum-sensing locus has been identified. However, it appears that the majority of strains do not produce AHLs. In this study we report the identification and regulation of the AHL-dependent system of rhizosphere
P. putida
WCS358. This system is identical to the recently identified system of
P. putida
strain IsoF and very similar to the
las
system of
Pseudomonas aeruginosa
. It is composed of three genes, the
luxI
family member
ppuI
, the putative repressor
rsaL
, and the
luxR
family member
ppuR
. A genomic
ppuR
::Tn
5
mutant of strain WCS358 was identified by its inability to produce AHLs when it was cross-streaked in close proximity to an AHL biosensor, whereas an
rsaL
::Tn
5
genomic mutant was identified by its ability to overproduce AHL molecules. Using transcriptional promoter fusions, we studied expression profiles of the
rsaL
,
ppuI
, and
ppuR
promoters in various genetic backgrounds. At the onset of the stationary phase, the autoinducer synthase
ppuI
gene expression is under positive regulation by PpuR-AHL and under negative regulation by RsaL, indicating that the molecules could be in competition for binding at the
ppuI
promoter. In genomic
rsaL
::Tn
5
mutants
ppuI
expression and production of AHL levels increased dramatically; however, both processes were still under growth phase regulation, indicating that RsaL is not involved in repressing AHL production at low cell densities. The roles of the global response regulator GacA and the stationary-phase sigma factor RpoS in the regulation of the AHL system at the onset of the stationary phase were also investigated. The
P. putida
WCS358
gacA
gene was cloned and inactivated in the genome. It was determined that the three global regulatory systems are closely linked, with quorum sensing and RpoS regulating each other and GacA positively regulating
ppuI
expression. Studies of the regulation of AHL quorum-sensing systems have lagged behind other studies and are important for understanding how these systems are integrated into the overall growth phase and metabolic status of the cells.
Phylogenetic Analysis of Polyketide Synthase I Domains from Soil Metagenomic Libraries Allows Selection of Promising Clones
Authors: Ginolhac Aurélien , Jarrin Cyrille , Gillet Benjamin , Robe Patrick , Pujic Petar , Tuphile Karine , Bertrand Hélène , Vogel Timothy M. , Perrière Guy , Simonet Pascal , Nalin Renaud 
Published: September 2004

ABSTRACT
The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.
Genetic Structure of a
Spodoptera frugiperda
Nucleopolyhedrovirus Population: High Prevalence of Deletion Genotypes
Authors: Simón Oihane , Williams Trevor , López-Ferber Miguel , Caballero Primitivo 
Published: September 2004

ABSTRACT

A Nicaraguan field isolate (SfNIC) of
Spodoptera frugiperda
nucleopolyhedrovirus was purified by plaque assay on Sf9 cells. Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles. Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate. Physical maps were generated for each of the variants. The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5. This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments. Eight genotypes presented a deletion in their genome compared with SfNIC-B. Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones. These variants were not infectious per os but retained infectivity by injection into
S. frugiperda
larvae. Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type. The variants also differed in their speed of kill. Noninfectious variants SfNIC-C and -D lacked the
pif
and
pif-2
genes. Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both
pif
and
pif-2
. Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed. Transcription was not detected in SfNIC-C or -D-inoculated larvae. We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing
pif
and
pif-2
, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests.
Alkaline Anaerobic Respiration: Isolation and Characterization of a Novel Alkaliphilic and Metal-Reducing Bacterium
Authors: Ye Qi , Roh Yul , Carroll Susan L. , Blair Benjamin , Zhou Jizhong , Zhang Chuanlun L. , Fields Matthew W. 
Published: September 2004

ABSTRACT

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with
Alkaliphilus transvaalensis
and
Alkaliphilus crotonatoxidans
, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name
Alkaliphilus metalliredigens
sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.
Raw Cow Milk Bacterial Population Shifts Attributable to Refrigeration
Authors: Lafarge Véronique , Ogier Jean-Claude , Girard Victoria , Maladen Véronique , Leveau Jean-Yves , Gruss Alexandra , Delacroix-Buchet Agnès 
Published: September 2004

ABSTRACT

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (∼150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as
Listeria
spp. or
Aeromonas hydrophila
is demonstrated.
Metabolic Primers for Detection of (Per)chlorate-Reducing Bacteria in the Environment and Phylogenetic Analysis of
cld
Gene Sequences
Authors: Bender Kelly S. , Rice Melissa R. , Fugate William H. , Coates John D. , Achenbach Laurie A. 
Published: September 2004

ABSTRACT

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (
cld
) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were
cld
gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one
cld
phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of
cld
genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the
cld
and 16S ribosomal DNA (rDNA) gene trees indicated that the
cld
gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.
Microbial Species Involved in Production of 1,2-
sn
-Diacylglycerol and Effects of Phosphatidylcholine on Human Fecal Microbiota
Authors: Vulevic Jelena , McCartney Anne L. , Gee Jennifer M. , Johnson Ian T. , Gibson Glenn R. 
Published: September 2004

ABSTRACT

1,2-
sn
-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and
Escherichia coli
multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of
Bifidobacterium
spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly
Clostridium bifermentans
(8 of 12), followed by
Escherichia coli
(2 of 12). Interestingly, one DAG-producing strain was
Bifidobacterium infantis
, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.
Increasing the Carbon Flux toward Synthesis of Short-Chain-Length—Medium-Chain-Length Polyhydroxyalkanoate in the Peroxisome of
Saccharomyces cerevisiae
through Modification of the β-Oxidation Cycle
Authors: de Oliveira Valeria Cora , Maeda Isamu , Delessert Syndie , Poirier Yves 
Published: September 2004

ABSTRACT

Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in
Saccharomyces cerevisiae
from intermediates of the β-oxidation cycle by expressing the polyhydroxyalkanoate synthases from
Aeromonas caviae
and
Ralstonia eutropha
in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the β-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward
R
-3-hydroxybutyryl coenzyme A.
Fluorogenic Selective and Differential Medium for Isolation of
Enterobacter sakazakii
Authors: Oh Se-Wook , Kang Dong-Hyun 
Published: September 2004

ABSTRACT

4-Methylumbelliferyl-α-
d
-glucoside, the fluorogenic substrate of α-glucosidase, was used as a selective marker to develop a differential medium for
Enterobacter sakazakii
. This bacterium showed strong fluorogenic characteristics clearly distinguishable from other microorganisms. On the basis of reducing background noise, an optimum basal medium and nitrogen source were selected. Incubation conditions were optimized.
Production of Minicellulosomes from
Clostridium cellulovorans
in
Bacillus subtilis
WB800
Authors: Cho Hee-Yeon , Yukawa Hideaki , Inui Masayuki , Doi Roy H. , Wong Sui-Lam 
Published: September 2004

ABSTRACT

Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of
Clostridium cellulovorans
were constructed and coexpressed in
Bacillus subtilis
WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into
B. subtilis
cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.
Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412
Authors: Stone Richard M. , DeAngelo Daniel J. , Klimek Virginia , Galinsky Ilene , Estey Eli , Nimer Stephen D. , Grandin Wilson , Lebwohl David , Wang Yanfeng , Cohen Pamela , Fox Edward A. , Neuberg Donna , Clark Jennifer , Gilliland D. Gary , Griffin James D. 
Published: September 2004

Abstract
Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to &lt; 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.
Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophilacells
Authors: Reavy Brian , Dawson Sheila , Canto Tomas , MacFarlane Stuart A 
Published: September 2004

Abstract
Background
RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system.

Results
Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein.

Conclusion
These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.
Structure, isolation, composition and reconstitution of the neuronal fusion pore
Authors: Cho Won Jin , Jeremic Aleksandar , Rognlien Kathy T. , Zhvania Mzia G. , Lazrishvili Ilia , Tamar Bikashvili , Jena Bhanu P. 
Published: August 2004

AbstractNeuronal communication is dependent on the fusion of 40–50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5–8 Å resolution, the presence of 8–10 nm in diameter cup‐shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.
Vesicle swelling regulates content expulsion during secretion
Authors: Kelly Marie L. , Cho Won Jin , Jeremic Aleksandar , Abu‐Hamdah Rania , Jena Bhanu P. 
Published: August 2004

AbstractThe involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.