bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus
Trichoderma reesei
Requires a Carrier Polypeptide with an Intact Domain Structure
Authors: Paloheimo Marja , Mäntylä Arja , Kallio Jarno , Suominen Pirkko 
Published: December 2003

ABSTRACT

A
bacterial xylanase gene,
Nonomuraea flexuosa xyn11A
, was
expressed in the filamentous fungus
Trichoderma reesei
from
the strong cellobiohydrolase 1 promoter as fusions to a variety of
carrier polypeptides. By using single-copy isogenic transformants, it
was shown that production of this xylanase was clearly increased (up to
820 mg/liter) when it was produced as a fusion protein with a carrier
polypeptide having an intact domain structure compared to the
production (150 to 300 mg/liter) of fusions to the signal sequence
alone or to carriers having incomplete domain structures. The carriers
tested were the
T. reesei
mannanase I (Man5A, or
MANI) core-hinge and a fragment thereof and the cellulose
binding domain of
T. reesei
cellobiohydrolase II (Cel6A, or
CBHII) with and without the hinge region(s) and a fragment thereof. The
flexible hinge region was shown to have a positive effect on both the
production of Xyn11A and the efficiency of cleavage of the fusion
polypeptide. The recombinant Xyn11A produced had properties similar to
those of the native xylanase. It constituted 6 to 10% of the
total proteins secreted by the transformants. About three times more of
the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A
was observed. Even in the best Xyn11A producers, the levels of the
fusion mRNAs were only ∼10% of the level of
cel7A
(
cbh1
) mRNA in the untransformed host
strain.
Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus
Authors: González Tania , Terrón María C. , Zapico Ernesto J. , Téllez Alejandro , Yagüe Susana , Carbajo José M. , González Aldo E. 
Published: December 2003

ABSTRACT

Laccases
produced by white rot fungi are involved in the degradation of lignin
and a broad diversity of other natural and synthetic molecules, having
a great potential for biotechnological applications. They are
frequently encoded by gene families, as in the basidiomycete
Trametes
sp. strain I-62, from which the
lcc1
,
lcc2
, and
lcc3
laccase genes have been cloned and
sequenced. A multiplex reverse transcription-PCR method to
simultaneously study the expression of these genes was developed in
this study. The assay proved to be quick, simple, highly sensitive, and
reproducible and is particularly valuable when numerous samples are to
be analyzed and/or if the amount of initial mRNA is limited. It was
used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl
alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and
3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in
Trametes
sp. strain I-62. These aromatic compounds produced
different induction patterns despite their chemical similarity. We
found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase
activity while also producing the highest increase in gene expression;
3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript
amounts of each gene fluctuated dramatically in the presence of these
three inducers, while the total amounts of laccase mRNAs seemed to be
modulated by a coordinated regulation of the different
genes.
Nitrification and Nitrifying Bacteria in the LowerSeine River and Estuary(France)
Authors: Cébron Aurélie , Berthe Thierry , Garnier Josette 
Published: December 2003

ABSTRACT

The
Achères wastewater treatment plant, located just downstream of
Paris, discharges its effluents into the lower Seine River. The
effluents contain large numbers of heterotrophic bacteria, organic
matter, and ammonium and are a source of nitrifying bacteria. As a
result, degradation of organic matter by heterotrophic bacteria and
subsequent oxygen depletion occur immediately downstream of the
effluent outlet, whereas nitrifying bacteria apparently need to build
up a significant biomass before ammonium oxidation significantly
depletes the oxygen. We quantified the potential total nitrifying
activity and the potential activities of the ammonia- and
nitrite-oxidizing communities along the Seine River. In the summer, the
maximum nitrifying activity occurs in the upper freshwater estuary,∼
200 km downstream of Achères. The quantities of
nitrifying bacteria, based on
amoA
gene copy numbers, and of
Nitrobacter
organisms, based on 16S rRNA gene copy numbers,
were correlated with the potential nitrifying activities. The species
composition of ammonia-oxidizing bacteria was investigated at two
sites: the Triel station just downstream from Achères (km 84) and
the Seine freshwater estuary at the Duclair station (km 278). By means
of PCR primers targeting the
amoA
gene, a gene library was
created. Phylogenetic analysis revealed that the majority of the
analyzed clones at both sites were affiliated with the genus
Nitrosomonas
. The
Nitrosomonas oligotropha
- and
Nitrosomonas urea
-related clones represented nearly 81%
of the community of ammonia-oxidizing bacteria at Triel and 60%
at Duclair. Two other ammonia-oxidizing clusters of the β
subclass of the
Proteobacteria
, i.e.,
Nitrosomonas
europaea
- and
Nitrosospira
-like bacteria, were found in
smaller numbers. The major change in the ammonia-oxidizing community
between the two stations along the Seine River-upper estuary continuum
was the replacement of the
N. oligotropha
- and
N.
urea
-related bacteria by the
Nitrosospira
-affiliated
bacteria. Although the diversities of the ammonia oxidizers appear to
be similar for the two sites, only half of the restriction patterns are
common to both sites, which could be explained by the differences in
ammonium concentrations, which are much lower in the upper estuary than
in the river at the effluent outlet. These results imply a significant
immigration and/or selection of the ammonia-oxidizing bacterial
population along the continuum of the Seine River from Paris to the
estuary.
ControlledModulation of Folate Polyglutamyl Tail Length by Metabolic Engineeringof
Lactococcuslactis
Authors: Sybesma Wilbert , van den Born Erwin , Starrenburg Marjo , Mierau Igor , Kleerebezem Michiel , de Vos Willem M. , Hugenholtz Jeroen 
Published: December 2003

ABSTRACT

The
dairy starter bacterium
Lactococcus lactis
is able to
synthesize folate and accumulates >90% of the produced
folate intracellularly, predominantly in the polyglutamyl form.
Approximately 10% of the produced folate is released into the
environment. Overexpression of
folC
in
L. lactis
led
to an increase in the length of the polyglutamyl tail from the
predominant 4, 5, and 6 glutamate residues in wild-type cells to a
maximum of 12 glutamate residues in the folate synthetase overproducer
and resulted in a complete retention of folate in the cells.
Overexpression of
folKE
, encoding the bifunctional protein
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and
GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl
tail length, leading to enhanced excretion of folate. By simultaneous
overexpression of
folKE
and
folC
, encoding the enzyme
folate synthetase or polyglutamyl folate synthetase, the average
polyglutamyl tail length was increased, again resulting in normal
wild-type distribution of folate. The production of bioavailable
monoglutamyl folate and almost complete release of folate from the
bacterium was achieved by expressing the gene for γ-glutamyl
hydrolase from human or rat origin. These engineering studies clearly
establish the role of the polyglutamyl tail length in intracellular
retention of the folate produced. Also, the potential application of
engineered food microbes producing folates with different tail lengths
is
discussed.
Efficient Degradation of 2,4,6-TrichlorophenolRequires a Set of Catabolic Genes Related to
tcp
Genesfrom
Ralstonia eutropha
JMP134(pJP4)
Authors: Matus V. , Sánchez M. A. , Martínez M. , González B. 
Published: December 2003

ABSTRACT

2,4,6-Trichlorophenol
(2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are
known to degrade this compound. One of these,
Ralstonia
eutropha
JMP134(pJP4), a well-known, versatile chloroaromatic
compound degrader, is able to grow in 2,4,6-TCP by converting it to
2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate,
maleylacetate, and β-ketoadipate. Three enzyme activities
encoded by
tcp
genes, 2,4,6-TCP monooxygenase (
tcpA
),
6-chlorohydroxyquinol 1,2-dioxygenase (
tcpC
), and
maleylacetate reductase (
tcpD
), are involved in this catabolic
pathway. Here we provide evidence that all these
tcp
genes are
clustered in the
R. eutropha
JMP134(pJP4) chromosome, forming
the putative catabolic operon
tcpRXABCYD.
We studied the
presence of
tcp
-like gene sequences in several other
2,4,6-TCP-degrading bacterial strains and found two types of strains.
One type includes strains belonging to the
Ralstonia
genus and
possessing a set of
tcp
-like genes, which efficiently degrade
2,4,6-TCP and therefore grow in liquid cultures containing this
chlorophenol as a sole carbon source. The other type includes strains
belonging to the genera
Pseudomonas
,
Sphingomonas
, or
Sphingopixis
, which do not have
tcp
-like gene
sequences and degrade this pollutant less efficiently and which
therefore grow only as small colonies on plates with 2,4,6-TCP. Other
than strain JMP134, none of the bacterial strains whose genomes have
been sequenced possesses a full set of
tcp
-like gene
sequences.
Purification and Characterization ofα-l-Arabinopyranosidase andα-l-Arabinofuranosidase fromBifidobacteriumbreveK-110, a Human Intestinal AnaerobicBacterium Metabolizing Ginsenoside Rb2 andRc
Authors: Shin Ho-Young , Park Sun-Young , Sung Jong Hwan , Kim Dong-Hyun 
Published: December 2003

ABSTRACTTwo arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizingBifidobacterium breveK-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+andp-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted onp-nitrophenyl-β-galactopyranoside orp-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobicBifidobacteriumsp.
Inactivation ofGeobacillus stearothermophilusSpores by High-Pressure CarbonDioxideTreatment
Authors: Watanabe Taisuke , Furukawa Soichi , Hirata Junichi , Koyama Tetsuya , Ogihara Hirokazu , Yamasaki Makari 
Published: December 2003

ABSTRACTHigh-pressure CO2treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores ofBacillus coagulans,Bacillus subtilis,Bacillus cereus,Bacillus licheniformis, andGeobacillus stearothermophiluswere subjected to CO2treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except theG. stearothermophilusspores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores ofG. stearothermophiluswere not the most resistant to CO2treatment. We also investigated the influence of temperature on CO2inactivation ofG. stearothermophilus.Treatment with CO2and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO2treatment ofG. stearothermophilusspores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2treatment ofG. stearothermophilusspores, CO2treatment at 95°C was more effective than treatment at 95°C alone.
Molecular Surveillance of Enterovirus andNorwalk-Like Virus in Oysters Relocated to a Municipal-Sewage-ImpactedGulfEstuary
Authors: Shieh Y. Carol , Baric Ralph S. , Woods Jacquelina W. , Calci Kevin R. 
Published: December 2003

ABSTRACT

An
18-month survey was conducted to examine the prevalence of enteric
viruses and their relationship to indicators in environmentally
polluted shellfish. Groups of oysters, one group per 4 weeks, were
relocated to a coastal water area in the Gulf of Mexico that is
impacted by municipal sewage and were analyzed for enteroviruses,
Norwalk-like viruses (NLV), and indicator microorganisms (fecal
coliform,
Escherichia coli
, and male-specific coliphages). The
levels of indicator microorganisms were consistent with the expected
continuous pollution of the area. Fourteen of the 18 oyster samples
were found by reverse transcription (RT)-PCR to harbor NLV and/or
enterovirus sequences. Of the four virus-negative oysters, three had
exposure to water temperatures of >29°C. Concomitant
with these findings, two of these four oysters also accumulated the
lowest levels of coliphages. PCR primers targeting pan-enteroviruses
and the NLV 95/96-US common subset were utilized; NLV sequences were
detected more frequently than those of enteroviruses. Within the
12-month sampling period, NLV and enterovirus sequences were detected
in 58 and 42%, respectively, of the oysters (67% of the
oysters tested were positive for at least one virus) from a
prohibited shellfish-growing area approximately 30 m away
from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid
nucleotide sequences were heterogeneous among the clones derived from
naturally polluted oysters. Overall, enteric viral sequences were found
in the contaminated oysters throughout all seasons except hot summer,
with a higher prevalence of NLV than enterovirus. Although a high
percentage of the oysters harbored enteric viruses, the virus levels
were usually less than or equal to 2 logs of RT-PCR-detectable units
per gram of oyster
meat.
Real-TimePCR Analysis of
Vibrio vulnificus
fromOysters
Authors: Campbell Mark S. , Wright Anita C. 
Published: December 2003

ABSTRACT

Vibrio
vulnificus
is an opportunistic human pathogen commonly found in
estuarine environments. Infections are associated with raw oyster
consumption and can produce rapidly fatal septicemia in susceptible
individuals. Standard enumeration of this organism in shellfish or
seawater is laborious and inaccurate; therefore, more efficient assays
are needed. An oligonucleotide probe derived from the cytolysin gene,
vvhA
, was previously used for colony hybridizations to
enumerate
V. vulnificus
. However, this method requires
overnight growth, and vibrios may lack culturability under certain
conditions. In the present study, we targeted the same locus for
development of a TaqMan real-time PCR assay. Probe specificity was
confirmed by amplification of 28
V. vulnificus
templates and
by the lack of a PCR product with 22 non-
V. vulnificus
strains. Detection of
V. vulnificus
in pure cultures was
observed over a 6-log-unit linear range of concentration
(10
2
to 10
8
CFU ml
−1
), with a
lower limit of 72 fg of genomic DNA μl of PCR
mixture
−1
or the equivalent of six cells. Similar
sensitivity was observed in DNA extracted from mixtures of
V.
vulnificus
and
V. parahaemolyticus
cells. Real-time PCR
enumeration of artificially inoculated oyster homogenates correlated
well with colony hybridization counts (
r
2
=
0.97). Numbers of indigenous
V. vulnificus
cells in oysters by
real-time PCR showed no significant differences from numbers from plate
counts with probe (
t
test;
P
= 0.43). Viable
but nonculturable cells were also enumerated by real-time PCR and
confirmed by the
Bac
Light viability assay. These data indicate
that real-time PCR can provide sensitive species-specific detection and
enumeration of
V. vulnificus
in
seafood.
Identification and Characterization of BenzimidazoleResistance in
Monilinia fructicola
from Stone Fruit OrchardsinCalifornia
Authors: Ma Zhonghua , Yoshimura Michael A. , Michailides Themis J. 
Published: December 2003

ABSTRACT

Low
and high levels of resistance to the benzimidazole fungicides benomyl
and thiophanate-methyl were observed in field isolates of
Monilinia
fructicola
, which is the causative agent of brown rot of stone
fruit. Isolates that had low levels of resistance (hereafter referred
to as LR isolates) and high levels of resistance (hereafter referred to
as HR isolates) were also cold and heat sensitive, respectively.
Results from microsatellite DNA fingerprints showed that genetic
identities among the populations of sensitive (S), LR, and HR isolates
were very high (>0.96). Analysis of DNA sequences of theβ
-tubulin gene showed that the LR isolates had a point mutation
at codon 6, causing a replacement of the amino acid histidine by
tyrosine. Codon 198, which encodes a glutamic acid in S and LR
isolates, was converted to a codon for alanine in HR isolates. Based on
these point mutations in the β-tubulin gene,
allele-specific PCR assays were developed for rapid detection of
benzimidazole-resistant isolates of
M.
fructicola
from stone
fruit.
Prevalence and Antimicrobial Resistance of
Enterococcus
Species Isolated from RetailMeats
Authors: Hayes Joshua R. , English Linda L. , Carter Peggy J. , Proescholdt Terry , Lee Kyung Y. , Wagner David D. , White David G. 
Published: December 2003

ABSTRACT

From
March 2001 to June 2002, a total of 981 samples of retail raw meats
(chicken, turkey, pork, and beef) were randomly obtained from 263
grocery stores in Iowa and cultured for the presence of
Enterococcus
spp. A total of 1,357 enterococcal isolates were
recovered from the samples, with contamination rates ranging from
97% of pork samples to 100% of ground beef samples.
Enterococcus faecium
was the predominant species recovered
(61%), followed by
E. faecalis
(29%), and
E.
hirae
(5.7%).
E. faecium
was the predominant
species recovered from ground turkey (60%), ground beef
(65%), and chicken breast (79%), while
E.
faecalis
was the predominant species recovered from pork chops
(54%). The incidence of resistance to many production and
therapeutic antimicrobials differed among enterococci recovered from
retail meat samples. Resistance to quinupristin-dalfopristin, a human
analogue of the production drug virginiamycin, was observed in 54, 27,
9, and 18% of
E. faecium
isolates from turkey, chicken,
pork, and beef samples, respectively. No resistance to linezolid or
vancomycin was observed, but high-level gentamicin resistance was
observed in 4% of enterococci, the majority of which were
recovered from poultry retail meats. Results indicate that
Enterococcus
spp. commonly contaminate retail meats and that
dissimilarities in antimicrobial resistance patterns among enterococci
recovered from different meat types may reflect the use of approved
antimicrobial agents in each food animal production
class.
Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by
Pseudomonas fluorescens
Authors: de Souza Jorge T. , de Boer Marjan , de Waard Pieter , van Beek Teris A. , Raaijmakers Jos M. 
Published: December 2003

ABSTRACT

Zoospores
play an important role in the infection of plant and animal hosts by
oomycetes and other zoosporic fungi. In this study, six fluorescent
Pseudomonas
isolates with zoosporicidal activities were
obtained from the wheat rhizosphere. Zoospores of multiple oomycetes,
including
Pythium
species,
Albugo candida
, and
Phytophthora infestans
, were rendered immotile within
30 s of exposure to cell suspensions or cell culture
supernatants of the six isolates, and subsequent lysis occurred within
60 s. The representative strain SS101, identified as
Pseudomonas fluorescens
biovar II, reduced the surface tension
of water from 73 to 30 mN m
−1
. The application of
cell suspensions of strain SS101 to soil or hyacinth bulbs provided
significant protection against root rot caused by
Pythium
intermedium
. Five Tn
5
mutants of strain SS101lacked the abilities to reduce the surface tension of water and to
cause lysis of zoospores. Genetic characterization of two
surfactant-deficient mutants showed that the transposons had integrated
into condensation domains of peptide synthetases. A partially purified
extract from strain SS101 reduced the surface tension of water to 30 mN
m
−1
and reached the critical micelle concentration
at 25 μg ml
−1
. Reverse-phase
high-performance liquid chromatography yielded eight different
fractions, five of which had surface activity and caused lysis of
zoospores. Mass spectrometry and nuclear magnetic resonance analyses
allowed the identification of the main constituent as a cyclic
lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon
hydroxy fatty acid. The other four zoosporicidal fractions were closely
related to the main constituent, with molecular massesranging from 1,111 to 1,169
Da.