bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by
Archaeoglobus fulgidus
Strain Z
Authors: Habicht Kirsten S. , Salling Lilian , Thamdrup Bo , Canfield Donald E. 
Published: July 2005

ABSTRACT

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for
Archaeoglobus fulgidus
strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO
2
. This is the first time that the production of formate has been reported for
A. fulgidus
. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO
2
produced at high concentrations. We suggest different pathways for lactate oxidation by
A. fulgidus
at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.
Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study
Authors: Hendrickx Barbara , Dejonghe Winnie , Boënne Wesley , Brennerova Maria , Cernik Miroslav , Lederer Tomas , Bucheli-Witschel Margarete , Bastiaens Leen , Verstraete Willy , Top Eva M. , Diels Ludo , Springael Dirk 
Published: July 2005

ABSTRACT

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with
tmoA
- and
xylM/xylE1
-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only
tmoA
-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of
Proteobacteria
. At the uncontaminated location,
Actinobacteria
and
Proteobacteria
were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.
Diversity of Methanotrophic Bacteria in Tropical Upland Soils under Different Land Uses
Authors: Knief Claudia , Vanitchung Supika , Harvey Narumon W. , Conrad Ralf , Dunfield Peter F. , Chidthaisong Amnat 
Published: July 2005

ABSTRACT

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of
pmoA
gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that
Methylobacter
spp. and
Methylocystis
spp. were present. Sequences of the “forest soil cluster” or “upland soil cluster α,” which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the
Gammaproteobacteria
were also detected.
Definition of Genetically Distinct Attenuation Mechanisms in Naturally Virulence-Attenuated
Listeria monocytogenes
by Comparative Cell Culture and Molecular Characterization
Authors: Roberts Angela , Chan Yvonne , Wiedmann Martin 
Published: July 2005

ABSTRACT

Listeria monocytogenes
is a foodborne pathogen able to cause serious disease in humans and animals. Not all isolates are equally pathogenic, however, and several isolates have been characterized as naturally virulence attenuated. We sought to identify the genetic basis of natural virulence attenuation using cell culture assays and molecular techniques. By comparing the phenotypes of naturally virulence-attenuated isolates to those of defined virulence gene mutants in plaque, cytotoxicity, and hemolysis assays and by characterizing selected virulence genes and their expression using DNA sequencing and TaqMan reverse transcriptase PCR, we classified virulence-attenuated isolates into four categories. Both group A and group B isolates were noncytotoxic and nonhemolytic; however, group A isolates underexpressed listeriolysin O (LLO, encoded by
hlyA
), while group B isolates produced LLO proteins that were inactive. The single isolate in group C was fully cytotoxic, had higher than wild-type hemolytic activity, and was, therefore, likely virulence attenuated due to overexpression of LLO. Group D isolates were characterized by normal cytotoxicity, hemolytic activity, and
hlyA
expression but had reduced intracellular growth. The genetic mechanisms causing virulence-attenuated phenotypes among the group D isolates could not be determined definitively but may involve defects in the expression of
actA
or the function of the ActA protein. Our results show (i) that the combination of cell culture assays and molecular techniques used in this study allows for identification and characterization of naturally virulence-attenuated isolates and (ii) that multiple distinct genetic mechanisms are responsible for natural virulence attenuation in
L. monocytogenes
.
Reduction of Perchlorate and Nitrate by Microbial Communities in Vadose Soil
Authors: Nozawa-Inoue Mamie , Scow Kate M. , Rolston Dennis E. 
Published: July 2005

ABSTRACT
Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.
Evaluation of Lateral-Flow
Clostridium botulinum
Neurotoxin Detection Kits for Food Analysis
Authors: Sharma Shashi K. , Eblen Brian S. , Bull Robert L. , Burr Donald H. , Whiting Richard C. 
Published: July 2005

ABSTRACT

The suitability and sensitivity of two in vitro lateral-flow assays for detecting
Clostridium botulinum
neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a
C. botulinum
contamination event.
Cloning and Expression of Islandisin, a New Thermostable Subtilisin from
Fervidobacterium islandicum
, in
Escherichia coli
Authors: Gödde Carolin , Sahm Kerstin , Brouns Stan J. J. , Kluskens Leon D. , van der Oost John , de Vos Willem M. , Antranikian Garabed 
Published: July 2005

ABSTRACT

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium
Fervidobacterium islandicum
(DSMZ 5733) was cloned and actively expressed in
Escherichia coli
. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in
E. coli
BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°C.
Improvement of the Fungal Biocontrol Agent
Trichoderma atroviride
To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance
Authors: Brunner Kurt , Zeilinger Susanne , Ciliento Rosalia , Woo Sheridian L. , Lorito Matteo , Kubicek Christian P. , Mach Robert L. 
Published: July 2005

ABSTRACT

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of
Trichoderma atroviride
, which expresses the
Aspergillus niger
glucose oxidase-encoding gene,
goxA
, under a homologous chitinase (
nag1
) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate.
goxA
expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less
N
-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of
Botrytis cinerea
. The transgenic strain also more quickly overgrew and lysed the plant pathogens
Rhizoctonia solani
and
Pythium ultimum
. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic
Trichoderma
strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen
B. cinerea
. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as
Trichoderma
spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.
Fate of
14
C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms
Authors: Okabe Satoshi , Kindaichi Tomonori , Ito Tsukasa 
Published: July 2005

ABSTRACT

The cross-feeding of microbial products derived from
14
C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [
14
C]bicarbonate, biofilm samples were incubated with and without NH
4
+
as a sole energy source for 10 days. The transfer of
14
C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-
Proteobacteria
showed significant uptake of
14
C-labeled microbial products. In particular, the members of the
Chloroflexi
were strongly MAR positive in the culture without NH
4
+
addition, in which nitrifying bacteria tended to decay. This indicated that the members of the
Chloroflexi
preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the
Cytophaga
-
Flavobacterium
cluster gradually utilized
14
C-labeled products in the culture with NH
4
+
addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of
14
C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.
Protein Engineering of Epoxide Hydrolase from
Agrobacterium radiobacter
AD1 for Enhanced Activity and Enantioselective Production of (
R
)-1-Phenylethane-1,2-Diol
Authors: Rui Lingyun , Cao Li , Chen Wilfred , Reardon Kenneth F. , Wood Thomas K. 
Published: July 2005

ABSTRACT

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of
Agrobacterium radiobacter
AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (
R
)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (
R
)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (
R
)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (
R
) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.
Development and Application of an Insertional System for Gene Delivery and Expression in
Campylobacter jejuni
Authors: Karlyshev A. V. , Wren B. W. 
Published: July 2005

ABSTRACT

The genetic investigation of
Campylobacter jejuni
, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for
Escherichia coli
and other bacteria cannot be maintained in
Campylobacter
cells. Additionally, gene expression in
Campylobacter
requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other
Campylobacter
strains. The presence of a
Campylobacter
-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of
Campylobacter
. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing
Campylobacter
allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.
Susceptibility of Biofilms to
Bdellovibrio bacteriovorus
Attack
Authors: Kadouri Daniel , O'Toole George A. 
Published: July 2005

ABSTRACT

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium
Bdellovibrio bacteriovorus
to control and reduce an existing
Escherichia coli
biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of
Bdellovibrio
as low as 10
2
PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of
B. bacteriovorus
to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh
Bdellovibrio
; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by
Bdellovibrio
but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments
B. bacteriovorus
was able to decrease the biomass of both
E. coli
and
Pseudomonas fluorescens
biofilms as determined by phase-contrast and epifluorescence microscopy.
Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens
Authors: Sela Shlomo , Nestel David , Pinto Riky , Nemny-Lavy Esther , Bar-Joseph Moshe 
Published: July 2005

ABSTRACT

The Mediterranean fruit fly (
Ceratitis capitata
) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged
Escherichia coli
to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged
E. coli
were similarly contaminated and were capable of transmitting
E. coli
to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the
E. coli
. Flies inoculated with
E. coli
harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of
E. coli
was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.
Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR Assays
Authors: Fierer Noah , Jackson Jason A. , Vilgalys Rytas , Jackson Robert B. 
Published: July 2005

ABSTRACT
Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure.