bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean
Authors: Langlois Rebecca J. , LaRoche Julie , Raab Philipp A. 
Published: December 2005

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.