bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractal characterization of brain lesions in CT images
Authors: Jauhari Rajnish K. , Trivedi Rashmi , Munshi Prabhat , Sahni Kamal
Published: December 2005

Fractal Dimension (FD) is a parameter used widely for classification, analysis, and pattern recognition of images. In this work we explore the quantification of CT (computed tomography) lesions of the brain by using fractal theory. Five brain lesions, which are portions of CT images of diseased brains, are used for the study. These lesions exhibit self‐similarity over a chosen range of scales, and are broadly characterized by their fractal dimensions.
A novel method for automatic detection of patient out‐of‐plane rotation by comparing a single portal image to a reference image
Authors: Jabbari Keyvan , Pistorius Stephen
Published: December 2005

A novel method for detecting out‐of‐plane patient rotation by comparing a single portal image to its reference image is presented. Out‐of‐plane rotation results in an apparent distortion of the anatomy in a portal image. This distortion can be mathematically predicted with the magnification varying at each point in the image. While scaling of points at equal depth is invariant under in‐plane rotation or translation, and changes equally in both dimensions for an axial shift of the patient, a change of scaling in only one dimension can be ascribed to an out‐of‐plane rotation. For the two conditions that are used in this study, it is shown that out‐of‐plane rotation yields a different scaling of the image in two perpendicular directions and therefore it is feasible to calculate the scale factors as a function of out‐of‐plane rotation. Conversely the recovery of scale factors in two different directions at the same time would enable the magnitude of the out‐of‐plane rotation to be recovered. The properties of the Fourier transform of the image are used to align the portal image with the reference image (a simulator image or first approved portal image) prior to the recovery of the scale factors. Correlating the Fourier transform of the portal image on a log‐scale with that of the reference image enables the scale factors to be automatically extracted from a single portal image. In the two approaches investigated, out‐of‐plane rotations of up to and (respectively) have been recovered with a maximum error of . This technique could be used to automatically detect patient roll or tilt prior to or during a treatment session.
M: A sub‐minute Monte Carlo dose calculation engine for prostate implants
Authors: Chibani Omar , Williamson Jeffrey F.
Published: December 2005

An accelerated Monte Carlo code [Monte Carlo dose calculation for prostate implant (MCPI)] is developed for dose calculation in prostate brachytherapy. MCPI physically simulates a set of radioactive seeds with arbitrary positions and orientations, merged in a three‐dimensional (3D) heterogeneous phantom representing the prostate and surrounding tissue. MCPI uses a phase space data source‐model to account for seed self‐absorption and seed anisotropy. A “hybrid geometry” model (full 3D seed geometry merged in 3D mesh of voxels) is used for rigorous treatment of the interseed attenuation and tissue heterogeneity effects. MCPI is benchmarked against the MCNP5 code for idealized and real implants, for and seeds. MCPI calculates the dose distribution (2‐mm voxel mesh) of a implant (83 seeds) with 2% average statistical uncertainty in 59 s using a single Pentium 4 PC (2.4 GHz). MCPI is more than and times faster than MCNP5 for prostate dose calculations using 2‐ and 1‐mm voxels, respectively. To illustrate its usefulness, MCPI is used to quantify the dosimetric effects of interseed attenuation, tissue composition, and tissue calcifications. Ignoring the interseed attenuation effect or slightly varying the prostate tissue composition may lead to 6% decreases of , the dose delivered to 100% of the prostate. The presence of calcifications, covering 1%–5% of the prostate volume, decreases , , and by up to 32%, 37%, and 58%, respectively. In conclusion, sub‐minute dose calculations, taking into account all dosimetric effects, are now possible for more accurate dose planning and dose assessment in prostate brachytherapy.
Gated CT imaging using a free‐breathing respiration signal from flow‐volume spirometry
Authors: D'souza Warren D. , Kwok Young , Deyoung Chad , Zacharapoulos Nicholas , Pepelea Mark , Klahr Paul , Yu Cedric X.
Published: December 2005

Respiration‐induced tumor motion is known to cause artifacts on free‐breathing spiral CT images used in treatment planning. This leads to inaccurate delineation of target volumes on planning CT images. Flow‐volume spirometry has been used previously for breath‐holds during CT scans and radiation treatments using the active breathing control (ABC) system. We have developed a prototype by extending the flow‐volume spirometer device to obtain gated CT scans using a PQ 5000 single‐slice CT scanner. To test our prototype, we designed motion phantoms to compare image quality obtained with and without gated CT scan acquisition. Spiral and axial (nongated and gated) CT scans were obtained of phantoms with motion periods of 3–5 s and amplitudes of 0.5–2 cm. Errors observed in the volume estimate of these structures were as much as 30% with moving phantoms during CT simulation. Application of motion‐gated CT with active breathing control reduced these errors to within 5%. Motion‐gated CT was then implemented in patients and the results are presented for two clinical cases: lung and abdomen. In each case, gated scans were acquired at end‐inhalation, end‐exhalation in addition to a conventional free‐breathing (nongated) scan. The gated CT scans revealed reduced artifacts compared with the conventional free‐breathing scan. Differences of up to 20% in the volume of the structures were observed between gated and free‐breathing scans. A comparison of the overlap of structures between the gated and free‐breathing scans revealed misalignment of the structures. These results demonstrate the ability of flow‐volume spirometry to reduce errors in target volumes via gating during CT imaging.
Cloning and Characterization of Two Xyloglucanases from
Paenibacillus
sp. Strain KM21
Authors: Yaoi Katsuro , Nakai Tomonori , Kameda Yoshiro , Hiyoshi Ayako , Mitsuishi Yasushi
Published: December 2005

Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium
Paenibacillus
sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in
Escherichia coli
, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.
Protamylasse, a Residual Compound of Industrial Starch Production, Provides a Suitable Medium for Large-Scale Cyanophycin Production
Authors: Elbahloul Yasser , Frey Kay , Sanders Johan , Steinbüchel Alexander
Published: December 2005

Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain ofEscherichia coliDH1 harboring plasmid pMa/c5-914::cphA6803, which carries the cyanophycin synthetase structural gene (cphA) fromSynechocystissp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.
Characterization of a Forest Soil Metagenome Clone That Confers Indirubin and Indigo Production on
Escherichia coli
Authors: Lim He Kyoung , Chung Eu Jin , Kim Jin-Cheol , Choi Gyung Ja , Jang Kyoung Soo , Chung Young Ryun , Cho Kwang Yun , Lee Seon-Woo
Published: December 2005

A microbial community analysis of forest soil from Jindong Valley, Korea, revealed that the most abundant rRNA genes were related to
Acidobacteria
, a major taxon with few cultured representatives. To access the microbial genetic resources of this forest soil, metagenomic libraries were constructed in fosmids, with an average DNA insert size of more than 35 kb. We constructed 80,500 clones from Yuseong and 33,200 clones from Jindong Valley forest soils. The double-agar-layer method allowed us to select two antibacterial clones by screening the constructed libraries using
Bacillus subtilis
as a target organism. Several clones produced purple or brown colonies. One of the selected antibacterial clones, pJEC5, produced purple colonies. Structural analysis of the purified pigments demonstrated that the metagenomic clone produced both the pigment indirubin and its isomer, indigo blue, resulting in purple colonies. In vitro mutational and subclonal analyses revealed that two open reading frames (ORFs) are responsible for the pigment production and antibacterial activity. The ORFs encode an oxygenase-like protein and a putative transcriptional regulator. Mutations of the gene encoding the oxygenase canceled both pigment production and antibacterial activity, whereas a subclone carrying the two ORFs retained pigment production and antibacterial activity. This finding suggests that these forest soil microbial genes are responsible for producing the pigment with antibacterial activity.
Effects of Long-Term Storage on Plasmid Stability in
Bacillus anthracis
Authors: Marston Chung K. , Hoffmaster Alex R. , Wilson Kathy E. , Bragg Sandra L. , Plikaytis Brian , Brachman Philip , Johnson Scott , Kaufmann Arnold F. , Popovic Tanja
Published: December 2005

The plasmid profiles of 619 cultures of
Bacillus anthracis
which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (
P
< 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (
P
= 0.25), but there was a statistical interdependence between the two plasmids (
P
= 0.004). Loss of viability of
B. anthracis
cultures stored on agar slants is also discussed.
Effect of Immersion Solutions Containing Enterocin AS-48 on
Listeria monocytogenes
in Vegetable Foods
Authors: Cobo Molinos Antonio , Abriouel Hikmate , Ben Omar Nabil , Valdivia Eva , Lucas López Rosario , Maqueda Mercedes , Cañamero Magdalena Martínez , Gálvez Antonio
Published: December 2005

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of
Listeria monocytogenes
CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 μg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 μg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15°C, as well as green asparagus at 15°C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22°C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate,
n
-propyl
p-
hydroxybenzoate,
p
-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.
Metabolic Engineering of
Escherichia coli
for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation
Authors: Lee Sang Jun , Lee Dong-Yup , Kim Tae Yong , Kim Byung Hun , Lee Jinwon , Lee Sang Yup
Published: December 2005

Comparative analysis of the genomes of mixed-acid-fermenting
Escherichia coli
and succinic acid-overproducing
Mannheimia succiniciproducens
was carried out to identify candidate genes to be manipulated for overproducing succinic acid in
E. coli
. This resulted in the identification of five genes or operons, including
ptsG
,
pykF
,
sdhA
,
mqo
, and
aceBA
, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of
E. coli
. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in
E. coli
. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes,
ptsG
,
pykF
, and
pykA
, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in
E. coli.
These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.
Sensitive Detection of Botulinum Neurotoxin Types C and D with an Immunoaffinity Chromatographic Column Test
Authors: Gessler Frank , Hampe Katrin , Böhnel Helge
Published: December 2005

A sensitive and specific immunoassay for the simultaneous detection ofClostridium botulinumtype C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml−1was achieved in culture supernatants.
Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean
Authors: Langlois Rebecca J. , LaRoche Julie , Raab Philipp A.
Published: December 2005

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the
nifH
gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of
nifH
genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial
nifH
phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial
nifH
sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous
nifH
sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.
Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil
Authors: Rønhede Stig , Jensen Bo , Rosendahl Søren , Kragelund Birthe B. , Juhler René K. , Aamand Jens
Published: December 2005

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [
N
-(4-isopropylphenyl)-
N
′,
N
′-dimethylurea] to
N
-(4-(2-hydroxy-1-methylethyl)phenyl)-
N
′,
N
′
-
dimethylurea and
N
-(4-(1-hydroxy-1-methylethyl)phenyl)-
N
′,
N
′-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to
N
-(4-isopropylphenyl)-
N
′-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of
Mortierella
sp. strain Gr4,
Phoma
cf.
eupyrena
Gr61, and
Alternaria
sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding
N
-(4-(2-hydroxy-1-methylethyl)phenyl)-
N
′,
N
′
-
dimethylurea, while
Mucor
sp. strain Gr22 hydroxylated the molecule at the second position, yielding
N
-(4-(1-hydroxy-1-methylethyl)phenyl)-
N
′,
N
′-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite
N
-(4-isopropylphenyl)-
N
′-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations.
Clonostachys
sp. strain Gr141 and putative
Tetracladium
sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent.
Mortierella
sp. strain Gr4 also produced
N
-(4-(2-hydroxy-1-methylethyl)phenyl)-
N
′-methylurea, which is the product resulting from combined N demethylation and hydroxylation.
Metabolic and Transcriptomic Adaptation of
Lactococcus lactis
subsp.
lactis
Biovar diacetylactis in Response to Autoacidification and Temperature Downshift in Skim Milk
Authors: Raynaud Sandy , Perrin Rémi , Cocaign-Bousquet Muriel , Loubiere Pascal
Published: December 2005

For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy
Lactococcus lactis
subsp.
lactis
biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.
Role of Spontaneous Current Oscillations during High-Efficiency Electrotransformation of Thermophilic Anaerobes
Authors: Tyurin Michael V. , Sullivan Charles R. , Lynd Lee R.
Published: December 2005

Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes
Clostridium thermocellum
ATCC 27405,
C. thermocellum
DSM 1313, and
Thermoanaerobacterium saccharolyticum
YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both
C. thermocellum
strains only at field strengths of ≥12 kV/cm, and ET was only observed above the same threshold. Similarly, for
T. saccharolyticum
, oscillations were only observed at field strengths of ≥10 kV/cm, and ET was only observed above the same threshold. When a passive electrical filter consisting of an inductor and resistor in parallel was added to the system to prevent the development of oscillations, ET efficiencies were reduced dramatically for all three strains at all field strengths tested. The maximum tested field strength, 25 kV/cm, resulted in the maximum measured transformation efficiency for all three strains. At this field strength, the efficiency of ET in the absence of oscillations was decreased compared to that observed in the presence of oscillations by 500-fold for
C. thermocellum
ATCC 27405, 2,500-fold for
C. thermocellum
DSM 1313, and 280-fold for
T. saccharolyticum
. Controls using the same apparatus with
Escherichia coli
cells or a resistor with a value representative of the direct current resistance of typical cell samples did not develop oscillations, and ET efficiencies obtained with
E. coli
were the same with or without the electrical filter included in the pulse generator circuit. The results are interpreted to indicate that spontaneously arising oscillations have a large beneficial effect on transformation efficiency in the system employed here and that the development of oscillations in this system is affected by the cell species present.
Evidence of Intense Archaeal and Bacterial Methanotrophic Activity in the Black Sea Water Column
Authors: Durisch-Kaiser Edith , Klauser Lucia , Wehrli Bernhard , Schubert Carsten
Published: December 2005

In the northwestern Black Sea, methane oxidation rates reveal that above shallow and deep gas seeps methane is removed from the water column as efficiently as it is at sites located off seeps. Hence, seeps should not have a significant impact on the estimated annual flux of ∼4.1 × 10
9
mol methane to the atmosphere [W. S. Reeburgh, B. B. Ward, S. C. Wahlen, K. A. Sandbeck, K. A. Kilatrick, and L. J. Kerkhof, Deep-Sea Res.
38
(Suppl. 2)
:
S1189-S1210, 1991]. Both the stable carbon isotopic composition of dissolved methane and the microbial community structure analyzed by fluorescent in situ hybridization provide strong evidence that microbially mediated methane oxidation occurs. At the shelf, strong isotope fractionation was observed above high-intensity seeps. This effect was attributed to bacterial type I and II methanotrophs, which on average accounted for 2.5% of the DAPI (4′,6′-diamidino-2-phenylindole)-stained cells in the whole oxic water column. At deep sites, in the oxic-anoxic transition zone, strong isotopic fractionation of methane overlapped with an increased abundance of
Archaea
and
Bacteria
, indicating that these organisms are involved in the oxidation of methane. In underlying anoxic water, we successfully identified the archaeal methanotrophs ANME-1 and ANME-2, eachof which accounted for 3 to 4% of the total cell counts. ANME-1 and ANME-2 appear as single cells in anoxicwater, compared to the sediment, where they may form cell aggregates with sulfate-reducing bacteria (A. Boetius, K. Ravenschlag, C. J. Schubert, D. Rickert, F. Widdel, A. Giesecke, R. Amann, B. B. Jørgensen, U. Witte, and O. Pfannkuche, Nature 407:623-626, 2000; V. J. Orphan, C. H. House, K.-U. Hinrichs, K. D. McKeegan, and E. F. DeLong, Proc. Natl. Acad. Sci. USA 99:7663-7668, 2002).
Construction of Engineered Bifunctional Enzymes and Their Overproduction in
Aspergillus niger
for Improved Enzymatic Tools To Degrade Agricultural By-Products
Authors: Levasseur Anthony , Navarro David , Punt Peter J. , Belaïch Jean-Pierre , Asther Marcel , Record Eric
Published: December 2005

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in
Aspergillus niger
. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of
A. niger
. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a

K
d

of 9.9 × 10
−8
M for the dissociation constant and 0.98 μmol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an
A. niger
-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.
High-Temperature Fluorescent In Situ Hybridization for Detecting
Escherichia coli
in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry
Authors: Tang Ying Zhong , Gin Karina Yew Hoong , Lim Tok Hoon
Published: December 2005

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of
Escherichia coli
in seawater spiked with
E.coli
culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to
E. coli
-spiked seawater samples, i.e., obtained flow cytometric signatures where the
E. coli
population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked
E. coli
cells (90%).
Influence of Fermentation Medium Composition on Physicochemical Surface Properties of
Lactobacillus acidophilus
Authors: Schär-Zammaretti Prisca , Dillmann Marie-Lise , D'Amico Nicola , Affolter Michael , Ubbink Job
Published: December 2005

The effect of the simple and complex basic components of a fermentation medium on the surface properties of
Lactobacillus acidophilus
NCC2628 is studied by physicochemical methods, such as electrophoresis, interfacial adhesion, and X-ray photonelectron spectroscopy, and by transmission electron microscopy. Starting from an optimized complete medium, the effect of carbohydrates, peptones, and yeast extracts on the physicochemical properties of the cell wall is systematically investigated by consecutively omitting one of the principal components from the fermentation medium at the time. The physicochemical properties and structure of the bacterial cell wall remain largely unchanged if the carbohydrate content of the fermentation medium is strongly reduced, although the concentration of surface proteins increases slightly. Both peptone and yeast extract have a considerable influence on the bacterial cell wall, as witnessed by changes in surface charge, hydrophobicity, and the nitrogen-to-carbon ratio. Both zeta potential and the cell wall hydrophobicity show a positive correlation with the nitrogen-to-carbon ratio of the bacterial surfaces, indicative of the important role of surface proteins in the overall surface physical chemistry. The hydrophobicity of the cell wall, which is low for the cultures grown in the complete medium and in the absence of carbohydrates, becomes fairly high for the cultures grown in the medium without peptones and the medium without yeast extract. UV spectrophotometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry are used to analyze the effect of medium composition on LiCl-extractable cell wall proteins, confirming the major change in protein composition of the cell wall for the culture fermented in the medium without peptones. In particular, it is found that expression of the S-layer protein is dependent on the protein source of the fermentation medium.
Expression of the
nifH
Gene of a
Herbaspirillum
Endophyte in Wild Rice Species: Daily Rhythm during the Light-Dark Cycle
Authors: You Mu , Nishiguchi Tomohiro , Saito Asami , Isawa Tsuyoshi , Mitsui Hisayuki , Minamisawa Kiwamu
Published: December 2005

The expression of nitrogenase genes of
Herbaspirillum
sp. B501 associated in shoot (leaf and stem) of wild rice,
Oryza officinalis
, was studied by means of reverse transcription-PCR (RT-PCR) targeted at the
nifH
gene. RT-PCR analyses indicate that
nifH
transcript was detected exclusively from nitrogen-fixing cells of
gfp
-tagged strain B501
gfp
1 in both free-living and endophytic states by using a constitutive
gfp
gene transcript as a positive control. Transcription of
nifH
and nitrogen fixation in free-living cells were induced maximally at a 2% O
2
concentration and repressed in free air (21% O
2
).
nifH
transcription was monitored in the endophytic cells by using total RNA extracted from B501
gfp
1-inoculated wild rice plants during daily light-dark cycles. The level of
nifH
transcription in planta varied dramatically, with a maximum during the light period. Moreover, the light radiation enhanced
nifH
expression even in free-living cells grown in culture. These results suggest that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment.
Improvement of Xylose Uptake and Ethanol Production in Recombinant
Saccharomyces cerevisiae
through an Inverse Metabolic Engineering Approach
Authors: Jin Yong-Su , Alper Hal , Yang Yea-Tyng , Stephanopoulos Gregory
Published: December 2005

We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant
Saccharomyces cerevisiae
. Specifically, we created a genomic fragment library from
Pichia stipitis
and introduced it into recombinant
S. cerevisiae
expressing
XYL1
and
XYL2
. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the
XYL3
gene, thus confirming the previous finding that
XYL3
is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a
XYL1 XYL2 XYL3
background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated
PsTAL1
based on a high level of homology with
S. cerevisiae TAL1
. To further investigate whether the newly identified
PsTAL1
ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the
PsTAL1
ORF under the control of a constitutive promoter and transformed it into an
S. cerevisiae
recombinant expressing
XYL1
,
XYL2
, and
XYL3
. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of
PsTAL1
did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the
ScTAL1
gene. These results suggest that
PsTAL1
is a better gene target for engineering of the pentose phosphate pathway in recombinant
S. cerevisiae
.
Timing of Induction of Osmotically Controlled Genes in
Salmonella enterica
Serovar Typhimurium, Determined with Quantitative Real-Time Reverse Transcription-PCR
Authors: Balaji Boovaraghan , O'Connor Kathleen , Lucas Jeffrey R. , Anderson Joseph M. , Csonka Laszlo N.
Published: December 2005

The signals that control the transcription of osmoregulated genes are not understood satisfactorily. The “turgor control model” suggested that the primary osmoregulatory signal in
Enterobacteriaceae
is turgor loss, which induces the
kdp
K
+
transport operon and activates the Trk K
+
permease. The ensuing increase in cytoplasmic K
+
concentration was proposed to be the signal that turns on all secondary responses, including the induction of the
proU
(proline-glycine betaine transport) operon. The “ionic strength model” proposed that the regulatory signal for all osmotically controlled responses is the increase in the cytoplasmic ionic strength or macromolecular crowding after an osmotic upshift. The assumption in the turgor control model that the induction of
kdp
is a primary response to osmotic shock predicts that this response should precede all secondary responses. Both models predict that the induction of all osmotically activated responses should be independent of the chemical nature of the solute used to impose osmotic stress. We tested these predictions by quantitative real-time reverse transcription-PCR analysis of the expression of six osmotically regulated genes in
Salmonella enterica
serovar Typhimurium. After shock with 0.3 M NaCl,
proU
was induced at 4 min,
proP
and
rpoS
were induced at 4 to 6 min,
kdp
was induced at 8 to 9 min, and
otsB
and
ompC
were induced at 10 to 12 min. After an equivalent osmotic shock with 0.6 M sucrose,
proU
was induced with kinetics similar to those seen with NaCl, but induction of
kdp
was reduced 150-fold in comparison to induction by NaCl. Our results are inconsistent with both the turgor control and the ionic strength control models.
Activity and Composition of the Denitrifying Bacterial Community Respond Differently to Long-Term Fertilization
Authors: Enwall Karin , Philippot Laurent , Hallin Sara
Published: December 2005

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO
3
)
2
, (NH
4
)
2
SO
4
, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The
nosZ
and
narG
genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of
nosZ
and restriction fragment length polymorphism of
narG
, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the
nosZ
and
narG
genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH
4
)
2
SO
4
and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO
3
)
2
or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.
Sequence and Expression Analyses of
Cytophaga
-Like Hydrolases in a Western Arctic Metagenomic Library and the Sargasso Sea
Authors: Cottrell Matthew T. , Yu Liying , Kirchman David L.
Published: December 2005

Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by
Cytophaga
-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of
Cytophaga
-like bacteria and hydrolase genes revealed a cellulase gene (
celM
) most similar to the gene found in
Cytophaga hutchinsonii
. A BLAST search of the entire Sargasso Sea WGS data set indicated that
celM
was the most abundant cellulase-like gene in the Sargasso Sea. However, the similarity between CelM-like cellulases and peptidases belonging to metalloprotease family M42 led us to question whether CelM is involved in the degradation of polysaccharides or proteins. PCR primers were designed for the
celM
genes in the Sargasso Sea WGS data set and used to identify
celM
in a fosmid library constructed with prokaryotic DNA from the western Arctic Ocean. Expression analysis of the
Cytophaga
-like Arctic CelM, which is 63% identical and 77% similar to CelM in
C. hutchinsonii
, indicated that there was peptidase activity, whereas cellulase activity was not detected. Our analysis suggests that the
celM
gene plays a role in the degradation of protein by
Cytophaga
-like bacteria. The abundance of peptidase genes in the
Cytophaga
-like fosmid clone provides further evidence for the importance of
Cytophaga
-like bacteria in the degradation of protein in high-molecular-weight dissolved organic matter.
Enantioselective Transformation of α-Hexachlorocyclohexane by the Dehydrochlorinases LinA1 and LinA2 from the Soil Bacterium
Sphingomonas paucimobilis
B90A
Authors: Suar Mrutyunjay , Hauser Andrea , Poiger Thomas , Buser Hans-Rudolf , Müller Markus D. , Dogra Charu , Raina Vishakha , Holliger Christof , van der Meer Jan Roelof , Lal Rup , Kohler Hans-Peter E.
Published: December 2005

Sphingomonas paucimobilis
B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol.
68:
6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of
S. paucimobilis
UT26. Incubation of chiral α-hexachlorocyclohexane (α-HCH) with
Escherichia coli
BL21 expressing functional LinA1 and LinA2
S
-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (−) enantiomer. Concurrent formation and subsequent dissipation of β-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3
S
,4
S
,5
R
,6
R
)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3
R
,4
R
,5
S
,6
S
)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of
S. paucimobilis
B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral α-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of α-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the
linA
genes, such as
S. paucimobilis
UT26, prevail.
Waterborne Pathogen Detection by Use of Oligonucleotide-Based Microarrays
Authors: Maynard Christine , Berthiaume Frédéric , Lemarchand Karine , Harel Josée , Payment Pierre , Bayardelle Paul , Masson Luke , Brousseau Roland
Published: December 2005

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and
cpn60
genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets,
wecE
-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the
Enterobacteriaceae
family. Universal PCR primers were used to amplify variable regions of 16S rRNA,
cpn60
, and
wecE
genes directly in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium genomic DNA mixtures (binary);
E. coli
,
S. enterica
serovar Typhimurium, and
Yersinia enterocolitica
genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for
S. enterica
serovar Typhimurium was estimated to be on the order of 0.1% (10
4
S. enterica
genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as
wecE
genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.