bluuebunny/crossref_metadata_2025_split huggingface dataset:
Degradation of 4-Chloro-2-Methylphenoxyacetic Acid in Top- and Subsoil Is Quantitatively Linked to the Class III
tfdA
Gene
Authors: Bælum Jacob , Henriksen Trine , Hansen Hans Christian Bruun , Jacobsen Carsten Suhr 
Published: February 2006

ABSTRACT

The
tfdA
gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other
tfdA
-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of
tfdA
genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the
tfdA
-containing bacterial community, from 500 genes g
−1
soil to approximately 3 × 10
4
genes g
−1
soil and to 7 × 10
5
genes g
−1
soil for topsoil initially added to 2.3 mg MCPA kg
−1
(dry weight) soil and 20 mg MCPA kg
−1
(dry weight) soil, respectively. We analyzed the diversity of the
tfdA
gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant
tfdA
population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the
tfdA
genes responsible for the degradation of MCPA belonged to the class III
tfdA
genes, while the
tfdA
genes present in the soil before the occurrence of degradation belonged to the class I
tfdA
genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.
Pyrroloquinoline Quinone-Dependent Dehydrogenases fromKetogulonicigenium vulgareCatalyze the Direct Conversion ofl-Sorbosone tol-Ascorbic Acid
Authors: Miyazaki Taro , Sugisawa Teruhide , Hoshino Tatsuo 
Published: February 2006

ABSTRACTA novel enzyme,l-sorbosone dehydrogenase 1 (SNDH1), which directly convertsl-sorbosone tol-ascorbic acid (l-AA), was isolated fromKetogulonicigenium vulgareDSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and hemecas the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs producedl-AA, as well as 2-keto-l-gulonic acid (2KGA), froml-sorbosone, suggesting that tautomerization ofl-sorbosone causes the dual conversion by SNDHs. ThesndHgene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase fromAcinetobacter calcoaceticus.The C-terminal one-fifth of the sequence exhibited similarity to ac-type cytochrome with a heme-binding motif. A lysate ofEscherichia colicells expressingsndHexhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis ofK. vulgareindicated that all of the SNDH proteins are encoded by thesndHgene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.
In Vivo Functional Assay of a Recombinant Aquaporin inPichia pastoris
Authors: Daniels Mark J. , Wood Malcolm R. , Yeager Mark 
Published: February 2006

ABSTRACTThe water channel protein PvTIP3;1 (α-TIP) is a member of themajorintrinsicprotein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeastPichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-typeP. pastorisdisplayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts ofP. pastorisexpressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of theEscherichia coliglycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.
Colonization of Tomato Root by Pathogenic and Nonpathogenic
Fusarium oxysporum
Strains Inoculated Together and Separately into the Soil
Authors: Olivain Chantal , Humbert Claude , Nahalkova Jarmila , Fatehi Jamshid , L'Haridon Floriane , Alabouvette Claude 
Published: February 2006

ABSTRACT

In soil, fungal colonization of plant roots has been traditionally studied
by indirect methods such as microbial isolation that do not enable
direct observation of infection sites or of interactions between fungal
pathogens and their antagonists. Confocal laser scanning microscopy was
used to visualize the colonization of tomato roots in heat-treated soil
and to observe the interactions between a nonpathogenic strain, Fo47,
and a pathogenic strain, Fol8, inoculated onto tomato roots in soil.
When inoculated separately, both fungi colonized the entire root
surface, with the exception of the apical zone. When both strains were
introduced together, they both colonized the root surface and were
observed at the same locations. When Fo47 was introduced at a higher
concentration than Fol8, it colonized much of the root surface, but
hyphae of Fol8 could still be observed at the same location on the
root. There was no exclusion of the pathogenic strain by the presence
of the nonpathogenic strain. These results are not consistent with the
hypothesis that specific infection sites exist on the root for
Fusarium oxysporum
and instead support the hypothesis that
competition occurs for nutrients rather than for infection
sites.
Induction by
Klebsiella aerogenes
of a Melanin-Like Pigment in
Cryptococcus neoformans
Authors: Frases Susana , Chaskes Stuart , Dadachova Ekaterina , Casadevall Arturo 
Published: February 2006

ABSTRACT

While studying the interaction of
Cryptococcus neoformans
with
Dictyostelium discoideum
, we noticed that yeast colonies in agar with a feeder lawn of
Klebsiella aerogenes
were brown. This finding was intriguing because
C. neoformans
colonies are not pigmented unless they are provided with precursors for melanization. Strains of all
C. neoformans
serotypes produced brown pigment in response to
K. aerogenes
at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when
C. neoformans
was exposed to live
Escherichia coli
or heat-killed
K. aerogenes
. Analysis of
K. aerogenes
supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by
C. neoformans
. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for
C. neoformans
melanization in the environment.
Molecular Assays for DetectingAphanomyces invadansin Ulcerative Mycotic Fish Lesions
Authors: Vandersea Mark W. , Litaker R. Wayne , Yonnish Bryan , Sosa Emilio , Landsberg Jan H. , Pullinger Chris , Moon-Butzin Paula , Green Jason , Morris James A. , Kator Howard , Noga Edward J. , Tester Patricia A. 
Published: February 2006

ABSTRACTThe pathogenic oomyceteAphanomyces invadansis the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detectA. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n= 50) collected in September 2004 were positive forA. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence thatA. invadansis the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusivelyA. invadans.
Characterization of Metabolism in the Fe(III)-Reducing Organism
Geobacter sulfurreducens
by Constraint-Based Modeling
Authors: Mahadevan R. , Bond D. R. , Butler J. E. , Esteve-Nuñez A. , Coppi M. V. , Palsson B. O. , Schilling C. H. , Lovley D. R. 
Published: February 2006

ABSTRACT

Geobacter sulfurreducens
is a well-studied representative of the
Geobacteraceae
, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate
G. sulfurreducens
central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why
Geobacter
species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of
G. sulfurreducens
could synthesize amino acids more efficiently than that of
Escherichia coli
due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of
G. sulfurreducens
central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.
A Quorum-Quenching Approach To Investigate the Conservation of Quorum-Sensing-Regulated Functions within the
Burkholderia cepacia
Complex
Authors: Wopperer Julia , Cardona Silvia T. , Huber Birgit , Jacobi Christoph A. , Valvano Miguel A. , Eberl Leo 
Published: February 2006

ABSTRACT

Taxonomic studies of the past few years have shown that the
Burkholderia cepacia
complex, a heterogeneous group of
B. cepacia
-like organisms, consists of at least nine species.
B. cepacia
complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time,
B. cepacia
complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All
B. cepacia
complex species investigated thus far use quorum-sensing (QS) systems that rely on
N
-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the
Bacillus
sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described
B. cepacia
complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested
B. cepacia
complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and
Caenorhabditis elegans
killing efficiency was regulated by AHL in the large majority of strains investigated.
Functional and Structural Characterization of Thermostabled-Amino Acid Aminotransferases fromGeobacillusspp
Authors: Lee Seung-Goo , Hong Seung-Pyo , Song Jae Jun , Kim Su-Jin , Kwak Mi-Sun , Sung Moon-Hee 
Published: February 2006

ABSTRACTd-Amino acid aminotransferases (d-AATs) fromGeobacillus toebiiSK1 andGeobacillussp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly activeBacillienzymes, with respective turnover rates of 47 and 55 s−1at 50°C. TheGeobacillusenzymes were unique and shared limited sequence identities of below 45% withd-AATs from mesophilic and thermophilicBacillusspp., except for a hypothetical protein with a 72% identity from theG. kaustophilusgenome. Structural alignments showed that most key residues were conserved in theGeobacillusenzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence inBacillusd-AATs was 144-EYcY-147 in theGeobacillusd-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward α-ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from theGeobacillusd-AATs had a π/π interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.
Lack of Detrimental Effects of
Bacillus thuringiensis
Cry Toxins on the Insect Predator
Chrysoperla carnea
: a Toxicological, Histopathological, and Biochemical Analysis
Authors: Rodrigo-Simón Ana , de Maagd Ruud A. , Avilla Carlos , Bakker Petra L. , Molthoff Jos , González-Zamora Jose E. , Ferré Juan 
Published: February 2006

ABSTRACT

The effect of Cry proteins of
Bacillus thuringiensis
on the green lacewing (
Chrysoperla carnea
) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed
Helicoverpa armigera
larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated
H. armigera
larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from
Spodoptera exigua
but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.
Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by
Legionella pneumophila
Authors: Piao Zhenyu , Sze Chun Chau , Barysheva Oksana , Iida Ken-ichiro , Yoshida Shin-ichi 
Published: February 2006

ABSTRACT

Fifty strains representing 38 species of the genus
Legionella
were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of
Legionella pneumophila
, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of
L. pneumophila
biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a
recA
null mutant of
L. pneumophila
was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by
L. pneumophila
to compete for proliferation among the environmental microbiota is discussed.
Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester
Authors: Hori Tomoyuki , Haruta Shin , Ueno Yoshiyuki , Ishii Masaharu , Igarashi Yasuo 
Published: February 2006

ABSTRACT

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a
Methanoculleus
sp. and a
Methanothermobacter
sp.) and one species of aceticlastic methanogen (i.e., a
Methanosarcina
sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the
Methanoculleus
sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the
Methanothermobacter
sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the
Methanosarcina
sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.
Two Subpopulations of
Listeria monocytogenes
Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin
Authors: Hornbæk Tina , Brockhoff Per B. , Siegumfeldt Henrik , Budde Birgitte Bjørn 
Published: February 2006

ABSTRACT

In situ analyses of single
Listeria monocytogenes
cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (ΔpH), and the other consisted of cells that maintained ΔpH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate ΔpH for 90% of the cell population (ED
90
) was predicted. ED
90
increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED
90
decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED
90
. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.
Txp40, a Ubiquitous Insecticidal Toxin Protein from
Xenorhabdus
and
Photorhabdus
Bacteria
Authors: Brown S. E. , Cao A. T. , Dobson P. , Hines E. R. , Akhurst R. J. , East P. D. 
Published: February 2006

ABSTRACT

Xenorhabdus
and
Photorhabdus
are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from
Xenorhabdus nematophila
that was lethal to the larvae of insects such as
Galleria mellonella
and
Helicoverpa armigera
when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene
txp40
was identified in another 59 strains of
Xenorhabdus
and
Photorhabdus
, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species
G. mellonella
,
H. armigera
, and
Plodia interpunctella
and the dipteran species
Lucilia cuprina
. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from
H. armigera
larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.