bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stable Transformation of the
Xylella fastidiosa
Citrus Variegated Chlorosis Strain with
oriC
Plasmids
Authors: Monteiro Patrı́cia B. , Teixeira Diva C. , Palma Renê R. , Garnier Monique , Bové Joseph-Marie , Renaudin Joël 
Published: July 2002

ABSTRACT

Xylella fastidiosa
is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of
X. fastidiosa
is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of
X. fastidiosa
were constructed by combining the chromosomal replication origin (
oriC
) of
X. fastidiosa
with a gene which confers resistance to kanamycin (Kan
r
). In plasmid p16KdAori, the
oriC
fragment comprised the
dnaA
gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the
oriC
fragment was restricted to the
dnaA
-
dnaN
intergenic region, which contains
dnaA
-box like sequences and AT-rich clusters. In plasmid p16K, no
oriC
sequence was present. In the three constructs, the promoter region of one of the two
X. fastidiosa
rRNA operons was used to drive the transcription of the Kan
r
gene to optimize the expression of kanamycin resistance in
X. fastidiosa
. Five CVC
X. fastidiosa
strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in
X. fastidiosa
. Integration of
oriC
plasmids into the
X. fastidiosa
chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.
Development and Validation ofCorynebacteriumDNA Microarrays
Authors: Loos Andrea , Glanemann Christoph , Willis Laura B. , O'Brien Xian M. , Lessard Philip A. , Gerstmeir Robert , Guillouet Stéphane , Sinskey Anthony J. 
Published: July 2002

ABSTRACTWe have developed DNA microarray techniques for studyingCorynebacterium glutamicum. A set of 52C. glutamicumgenes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of aC. glutamicumfermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement ofC. glutamicumamino acid-producing strains.
Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen
Flavobacterium psychrophilum
Authors: Secades P. , Alvarez B. , Guijarro J. A. 
Published: July 2002

ABSTRACT

Flavobacterium psychrophilum
is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (

F

.

p

sychrophilum

p

rotease
1
), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by
F. psychrophilum
. Consequently, we suggest that environmental host factors govern Fpp1 production.
Characterization of a Granulovirus Isolated from
Epinotia aporema
Wals. (Lepidoptera: Tortricidae) Larvae
Authors: Sciocco-Cap Alicia , Parola Alejandro D. , Goldberg Alina V. , Ghiringhelli Pablo D. , Romanowski Vı́ctor 
Published: July 2002

ABSTRACT

A granulovirus (GV) isolated from
Epinotia aporema
(Lepidoptera: Tortricidae)—a major soybean pest—was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.
Identification and Characterization of Bacteria in a Selenium-Contaminated Hypersaline Evaporation Pond
Authors: de Souza M. P. , Amini A. , Dojka M. A. , Pickering I. J. , Dawson S. C. , Pace N. R. , Terry N. 
Published: July 2002

ABSTRACT

Solar evaporation ponds are commonly used to reduce the volume of seleniferous agricultural drainage water in the San Joaquin Valley, Calif. These hypersaline ponds pose an environmental health hazard because they are heavily contaminated with selenium (Se), mainly in the form of selenate. Se in the ponds may be removed by microbial Se volatilization, a bioremediation process whereby toxic, bioavailable selenate is converted to relatively nontoxic dimethylselenide gas. In order to identify microbes that may be used for Se bioremediation, a 16S ribosomal DNA phylogenetic analysis of an aerobic hypersaline pond in the San Joaquin Valley showed that a previously unaffiliated group of uncultured bacteria (belonging to the order
Cytophagales
) was dominant, followed by a group of cultured γ-
Proteobacteria
which was closely related to
Halomonas
species. Se K-edge X-ray absorption spectroscopy of selenate-treated bacterial isolates showed that they accumulated a mixture of predominantly selenate and a selenomethionine-like species, consistent with the idea that selenate was assimilated via the S assimilation pathway. One of these bacterial isolates (
Halomonas
-like strain MPD-51) was the best candidate for the bioremediation of hypersaline evaporation ponds contaminated with high Se concentrations because it tolerated 2 M selenate and 32.5% NaCl, grew rapidly in media containing selenate, and accumulated and volatilized Se at high rates (1.65 μg of Se g of protein
−1
h
−1
), compared to other cultured bacterial isolates.
Self-Protection against Cell Wall Hydrolysis in
Streptococcus milleri
NMSCC 061 and Analysis of the Millericin B Operon
Authors: Beukes Mervyn , Hastings John W. 
Published: July 2002

ABSTRACT

Streptococcus milleri
NMSCC 061 produces an endopeptidase, millericin B, which hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. The nucleotide sequence of a 4.9-kb chromosomal region showed three open reading frames (ORFs) and a putative tRNA
Leu
sequence. The three ORFs encode a millericin B preprotein (MilB), a putative immunity protein (MilF), and a putative transporter protein (MilT). The
milB
gene encodes a 277-amino-acid preprotein with an 18-amino-acid signal peptide with a consensus IIGG cleavage motif. The predicted protein encoded by
milT
is homologous to ABC (ATP-binding cassette) transporters of several bacteriocin systems and to proteins implicated in the signal-sequence-independent export of
Escherichia coli
hemolysin A. These similarities strongly suggest that the
milT
gene product is involved in the translocation of millericin B. The gene
milF
encodes a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of
Staphylococcus aureus
, which are involved in the addition of glycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell wall mucopeptide of
S. milleri
NMSCC 061(resistant to lysis by millericin B) and
S. milleri
NMSCC 051(sensitive) showed a single amino acid difference. Serial growth of
S. milleri
NMSCC 051 in a cell wall minimal medium containing an increased concentration of leucine resulted in the in vivo substitution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of
S. milleri
NMSCC 051 (sensitive) that contained an amino acid substitution (leucine for threonine) within its peptidoglycan cross bridge showed partial susceptibility to millericin B. The putative tRNA
Leu
sequence located upstream of
milB
may be a cell wall-specific tRNA and could together with the
milF
protein, play a potential role in the addition of leucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.
Ammonia- and Nitrite-Oxidizing Bacterial Communities in a Pilot-Scale Chloraminated Drinking Water Distribution System
Authors: Regan John M. , Harrington Gregory W. , Noguera Daniel R. 
Published: July 2002

ABSTRACT

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of
Nitrosomonas
in each of the distribution systems, with a considerably smaller peak attributable to
Nitrosospira
-like AOB. Sequences of AOB amplification products aligned within the
Nitrosomonas oligotropha
cluster and were closely related to
N. oligotropha
and
Nitrosomonas ureae
. The nitrite-oxidizing communities were comprised primarily of
Nitrospira
, although
Nitrobacter
was detected in some samples. These results suggest a possible selection of AOB related to
N. oligotropha
and
N. ureae
in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.
Differential Expression of Methanogenesis Genes of
Methanothermobacter thermoautotrophicus
(Formerly
Methanobacterium thermoautotrophicum
) in Pure Culture and in Cocultures with Fatty Acid-Oxidizing Syntrophs
Authors: Luo Hong-Wei , Zhang Hui , Suzuki Toshihiko , Hattori Satoshi , Kamagata Yoichi 
Published: July 2002

ABSTRACT

The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen,
Methanothermobacter thermoautotrophicus
strain TM, was investigated both in a pure culture sufficiently supplied with H
2
plus CO
2
and in a coculture with an acetate-oxidizing hydrogen-producing bacterium,
Thermacetogenium phaeum
strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the
mcr
gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas
mcr
and
mrt
, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (
mtd
and
mth
) for
N
5
,
N
10
-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (
frh
and
mvh
) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for
Methanothermobacter thermoautotrophicus
strain ΔH cocultured with a thermophilic butyrate-oxidizing syntroph,
Syntrophothermus lipocalidus
strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of
M. thermoautotrophicus
strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.
Microscale Biosensor for Measurement of Volatile Fatty Acids in Anoxic Environments
Authors: Meyer Rikke Louise , Larsen Lars Hauer , Revsbech Niels Peter 
Published: July 2002

ABSTRACT

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO
3
−
to N
2
O. A culture of denitrifying bacteria deficient in N
2
O reductase was immobilized in front of the tip of an electrochemical N
2
O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N
2
O sensor. The signal of the sensor, which corresponded to the rate of N
2
O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O
2
, N
2
O, NO
2
−
, H
2
S, and NO
3
−
. Interference from NO
3
−
was low and could be quantified and accounted for. The detection limit was equivalent to about 1 μM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32‰. The functioning of the sensor over a temperature span of 7 to 30°C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5°C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.
Identification of a Salt-Induced Primary Transporter for Glycine Betaine in the Methanogen
Methanosarcina mazei
Gö1
Authors: Roeßler M. , Pflüger K. , Flach H. , Lienard T. , Gottschalk G. , Müller V. 
Published: July 2002

ABSTRACT

The salt adaptation of the methanogenic archaeon
Methanosarcina mazei
Gö1 was studied at the physiological and molecular levels. The freshwater organism
M. mazei
Gö1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in
M. mazei
Gö1, and inhibitor studies argue for a primary transport device. Analysis of the genome of
M. mazei
Gö1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of
Bacillus subtilis
. Northern blot analysis of
otaC
revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in
Archaea
.
A Gene from
Aspergillus nidulans
with Similarity to
URE2
of
Saccharomyces cerevisiae
Encodes a Glutathione
S
-Transferase Which Contributes to Heavy Metal and Xenobiotic Resistance
Authors: Fraser James A. , Davis Meryl A. , Hynes Michael J. 
Published: July 2002

ABSTRACT

Aspergillus nidulans
is a saprophytic ascomycete that utilizes a wide variety of nitrogen sources. We identified a sequence from
A. nidulans
similar to the glutathione
S
-transferase-like nitrogen regulatory domain of
Saccharomyces cerevisiae
Ure2. Cloning and sequencing of the gene, designated
gstA
, revealed it to be more similar to
URE2
than the
S. cerevisiae
glutathione
S
-transferases. However, creation and analysis of a
gstA
deletion mutant revealed that the gene does not participate in nitrogen metabolite repression. Instead, it encodes a functional theta class glutathione
S
-transferase that is involved in resistance to a variety of xenobiotics and metals and confers susceptibility to the systemic fungicide carboxin. Northern analysis showed that
gstA
transcription is strongly activated upon exposure to 1-chloro-2,4-dinitrobenzene and weakly activated by oxidative stress or growth on galactose as a carbon source. These results suggest that nitrogen metabolite repression in
A. nidulans
does not involve a homolog of the
S. cerevisiae URE2
gene and that the global nitrogen regulatory system differs significantly in these two fungi.
Combined Ribotyping and Random Multiprimer DNA Analysis To Probe the Population Structure of
Listeria monocytogenes
Authors: Mereghetti L. , Lanotte P. , Savoye-Marczuk V. , Marquet-Van Der Mee N. , Audurier A. , Quentin R. 
Published: July 2002

ABSTRACT

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated
Listeria monocytogenes
strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that
L. monocytogenes
strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.
Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner
Authors: Kurita-Ochiai Tomoko , Ochiai Kuniyasu , Fukushima Kazuo 
Published: July 2002

ABSTRACTOur previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.