bluuebunny/crossref_metadata_2025 huggingface dataset:
Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by
Mycobacterium
sp. Strain RJGII-135
Authors: Miyata Naoyuki , Iwahori Keisuke , Foght Julia M. , Gray Murray R.
Published: January 2004

The mechanism of uptake of phenanthrene by
Mycobacterium
sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-
14
C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state
14
C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide
m
-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (

K
t

) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated
14
C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (

K
d

) of 41 ± 21 nM (mean ± standard deviation). Given the low values of

K
t

and

K
d

,
Mycobacterium
sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.
Overproduction of Trehalose: Heterologous Expression of
Escherichia coli
Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in
Corynebacterium glutamicum
Authors: Padilla Leandro , Krämer Reinhard , Stephanopoulos Gregory , Agosin Eduardo
Published: January 2004

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of
Corynebacterium glutamicum
. This paper describes the heterologous expression of
Escherichia coli
trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in
C. glutamicum
, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in
C. glutamicum
to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in
C. glutamicum
.
Longitudinal Study of Fecal Shedding of
Escherichia coli
O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover
Authors: LeJeune J. T. , Besser T. E. , Rice D. H. , Berg J. L. , Stilborn R. P. , Hancock D. D.
Published: January 2004

Identification of the sources and methods of transmission of
Escherichia coli
O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of
E. coli
O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation,
E. coli
O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of
E. coli
O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of
Xba
I-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of
E. coli
O157:H7 on farms.
Microbial Anaerobic Demethylation and Dechlorination of Chlorinated Hydroquinone Metabolites Synthesized by Basidiomycete Fungi
Authors: Milliken C. E. , Meier G. P. , Watts J. E. M. , Sowers K. R. , May H. D.
Published: January 2004

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed thatDesulfitobacterium hafniensestrains DCB2 and PCP1,Desulfitobacterium chlororespiransstrain Co23, andDesulfitobacterium dehalogenansJW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.
Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific
Authors: Nakagawa Tatsunori , Ishibashi Jun-Ichiro , Maruyama Akihiko , Yamanaka Toshiro , Morimoto Yusuke , Kimura Hiroyuki , Urabe Tetsuro , Fukui Manabu
Published: January 2004

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with theAquificaeand γ- and ε-Proteobacteriaas well as the occurrence of archaeal phylotypes affiliated with theThermococcalesand of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genusThiomicrospira, andPelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.
Growth of
Escherichia coli
Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source
Authors: McLoughlin Sean Yu , Jackson Colin , Liu Jian-Wei , Ollis David L.
Published: January 2004

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The
Escherichia coli
strain DH10B expressing the phosphotriesterase from
Agrobacterium radiobacter
P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source.
Enterobacter aerogenes
is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled
E. coli
to grow when dimethyl phosphate is the sole phosphorus source.
E. coli
coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled
E. coli
to use phosphotriesters as a sole source of phosphorus.
Periplasmic Cytochrome
c
3
of
Desulfovibrio vulgaris
Is Directly Involved in H
2
-Mediated Metal but Not Sulfate Reduction
Authors: Elias Dwayne A. , Suflita Joseph M. , McInerney Michael J. , Krumholz Lee R.
Published: January 2004

Kinetic parameters and the role of cytochrome
c
3
in sulfate, Fe(III), and U(VI) reduction were investigated in
Desulfovibrio vulgaris
Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (

K
m

= 220 μM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H
2
and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H
2
and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine
c
-type cytochrome involvement in electron acceptor reduction.
c
-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H
2
, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the
c
-type cytochrome in lactate- or pyruvate-reduced, but not H
2
-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H
2
was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H
2
was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome
c
3
is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.
Supplementation of
Perkinsus marinus
Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity
Authors: Earnhart Christopher G. , Vogelbein Mary Ann , Brown Gwynne D. , Reece Kimberly S. , Kaattari Stephen L.
Published: January 2004

The protozoan oyster parasite
Perkinsus marinus
can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of
P. marinus
-susceptible (
Crassostrea virginica
) and -tolerant (
Crassostrea gigas
,
Crassostrea ariakensis
) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.
Measurement of Biologically Available Naphthalene in Gas and Aqueous Phases by Use of a
Pseudomonas putida
Biosensor
Authors: Werlen Christoph , Jaspers Marco C. M. , van der Meer Jan Roelof
Published: January 2004

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent
Pseudomonas putida
naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the
sal
promoter and the
luxAB
genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 μM). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.
Cultivation and Growth Characteristics of a Diverse Group of Oligotrophic Marine
Gammaproteobacteria
Authors: Cho Jang-Cheon , Giovannoni Stephen J.
Published: January 2004

Forty-four novel strains of
Gammaproteobacteria
were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine
Gammaproteobacteria
(OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day
−1
), and none of them grew in high-nutrient media (>351 mg of C liter
−1
). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 μm
3
). This cultivation study revealed that sporadically detected
Gammaproteobacteria
gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.
Differential Expression of Pine and
Cronartium quercuum
f. sp.
fusiforme
Genes in Fusiform Rust Galls
Authors: Warren Jaimie M. , Covert Sarah F.
Published: January 2004

Cronartium quercuum
f. sp.
fusiforme
is the causative agent of fusiform rust disease of southern pines in the United States. This disease is characterized by the formation of woody branch and stem galls. Differential display was used to identify pine genes whose expression is altered by
C. quercuum
f. sp.
fusiforme
infection and to identify
C. quercuum
f. sp.
fusiforme
genes that are expressed in fusiform rust galls. Six pine cDNAs that appeared to be differentially expressed in galled and healthy stems and 13
C. quercuum
f. sp.
fusiforme
cDNAs expressed in galled tissues were identified. A probe that hybridizes specifically to
C. quercuum
f. sp.
fusiforme
18S rRNA was used to estimate that 14% of the total RNA in fusiform rust galls was from
C. quercuum
f. sp.
fusiforme
. This finding was used to calibrate gene expression levels in galls when comparing them to expression levels in uninfected pines or in isolated
C. quercuum
f. sp.
fusiforme
cultures. According to Northern analysis and reverse transcriptase PCR analysis, all six of the pine clones were expressed at lower levels in galls than in healthy tissues. Seven of the nine
C. quercuum
f. sp.
fusiforme
clones that were assayed were expressed at higher levels in galls than in axenic culture. A number of the cDNAs encode proteins that are similar to those that play roles in plant development, plant defense, or fungal stress responses.
Phylogenetic Analysis of the Hypervariable Region of the 18S rRNA Gene ofCryptosporidiumOocysts in Feces of Canada Geese (Branta canadensis): Evidence for Five Novel Genotypes
Authors: Jellison Kristen L. , Distel Daniel L. , Hemond Harold F. , Schauer David B.
Published: January 2004

To assess genetic diversity inCryptosporidiumoocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive forCryptosporidiumspp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reportedCryptosporidium18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that ofCryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that ofC. baileyi. Phylogenetic analysis provided evidence for new genotypes ofCryptosporidiumspecies in Canada geese. Results of this study suggest that the taxonomy ofCryptosporidiumspecies in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.
Characterization of Secretory Genes
ypt1
/
yptA
and
nsf1
/
nsfA
from Two Filamentous Fungi: Induction of Secretory Pathway Genes of
Trichoderma reesei
under Secretion Stress Conditions
Authors: Saloheimo Markku , Wang Huaming , Valkonen Mari , Vasara Tuija , Huuskonen Anne , Riikonen Marjukka , Pakula Tiina , Ward Michael , Penttilä Merja
Published: January 2004

Two genes involved in protein secretion, encoding the Rab protein YPT1/YPTA and the general fusion factor NSFI/NSFA, were characterized from two filamentous fungi,
Trichoderma reesei
and
Aspergillus niger
var.
awamori
. The isolated genes showed a high level of conservation with their
Saccharomyces cerevisiae
and mammalian counterparts, and
T. reesei ypt1
was shown to complement yeast Ypt1p depletion. The transcriptional regulation of the
T. reesei ypt1
,
nsf1
, and
sar1
genes, involved in protein trafficking, was studied with mycelia treated with the folding inhibitor dithiothreitol (DTT) and with brefeldin A, which inhibits membrane traffic between the endoplasmic reticulum and Golgi complex. The well-known inducer of the yeast and
T. reesei
unfolded protein response (UPR), DTT, induced the
nsf1
gene and the protein disulfide isomerase gene,
pdi1
, in both of the experiments, and
sar1
mRNA increased in only one experiment under strong UPR induction. The
ypt1
mRNA did not show a clear increase during DTT treatment. Brefeldin A strongly induced
pdi1
and all of the intracellular trafficking genes studied. These results suggest the possibility that the whole secretory pathway of
T. reesei
could be induced at the transcriptional level by stress responses caused by protein accumulation in the secretory pathway.
Bacterial Diversity in Agricultural Soils during Litter Decomposition
Authors: Dilly Oliver , Bloem Jaap , Vos An , Munch Jean Charles
Published: January 2004

Denaturing gradient gel electrophoresis (DGGE) of amplified fragments of genes coding for 16S rRNA was used to study the development of bacterial communities during decomposition of crop residues in agricultural soils. Ten strains were tested, and eight of these strains produced a single band. Furthermore, a mixture of strains yielded distinguishable bands. Thus, DGGE DNA band patterns were used to estimate bacterial diversity. A field experiment performed with litter in nylon bags was used to evaluate the bacterial diversity during the decomposition of readily degradable rye and more refractory wheat material in comparable luvisols and cambisols in northern, central, and southern Germany. The amount of bacterial DNA in the fresh litter was small. The DNA content increased rapidly after the litter was added to the soil, particularly in the rapidly decomposing rye material. Concurrently, diversity indices, such as the Shannon-Weaver index, evenness, and equitability, which were calculated from the number and relative abundance (intensity) of the bacterial DNA bands amplified from genes coding for 16S rRNA, increased during the course of decomposition. This general trend was not significant for evenness and equitability at any time. The indices were higher for the more degradation-resistant wheat straw than for the more easily decomposed rye grass. Thus, the DNA band patterns indicated that there was increasing bacterial diversity as decomposition proceeded and substrate quality decreased. The bacterial diversity differed for the sites in northern, central, and southern Germany, where the same litter material was buried in the soil. This shows that in addition to litter type climate, vegetation, and indigenous microbes in the surrounding soil affected the development of the bacterial communities in the litter.
Species Differentiation of a Diverse Suite of
Bacillus
Spores by Mass Spectrometry-Based Protein Profiling
Authors: Dickinson Danielle N. , La Duc Myron T. , Haskins William E. , Gornushkin Igor , Winefordner James D. , Powell David H. , Venkateswaran Kasthuri
Published: January 2004

In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of
Bacillus
spores. MALDI-TOFMS protein profiles of 11 different strains of
Bacillus
spores, encompassing nine different species, were evaluated.
Bacillus
species selected for MALDI-TOFMS analysis represented the spore-forming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine
Bacillus
species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of
Bacillus subtilis
168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the
Bacillus
genus.
Molecular and Metabolic Characterization of Cold-Tolerant Alpine Soil
Pseudomonas
Sensu Stricto
Authors: Meyer A. F. , Lipson D. A. , Martin A. P. , Schadt C. W. , Schmidt S. K.
Published: January 2004

Alpine soils undergo dramatic temporal changes in their microclimatic properties, suggesting that the bacteria there encounter uncommon shifting selection gradients. Pseudomonads constitute important members of the alpine soil community. In order to characterize the alpine
Pseudomonas
community and to assess the impact of shifting selection on this community, we examined the ability of cold-tolerant
Pseudomonas
isolates to grow on a variety of carbon sources, and we determined their phylogenetic relationships based on 16S ribosomal DNA sequencing. We found a high prevalence of
Pseudomonas
in our soil samples, and isolates from these soils exhibited extensive metabolic diversity. In addition, our data revealed that many of our isolates form a unique cold-adapted clade, representatives of which are also found in the Swedish tundra and Antarctica. Our data also show a lack of concordance between the metabolic properties and 16S phylogeny, indicating that the metabolic diversity of these organisms cannot be predicted by phylogeny.
Nutrient Shock and Incubation Atmosphere Influence Recovery of Culturable
Helicobacter pylori
from Water
Authors: Azevedo N. F. , Pacheco A. P. , Keevil C. W. , Vieira M. J.
Published: January 2004

Three different media—Columbia agar, Wilkins-Chalgren agar, and
Helicobacter pylori
special peptone agar—were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering
H. pylori
from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed
H. pylori
organisms recovered. An atmosphere of 5% CO
2
, 5% O
2
, and 3% H
2
is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of
H. pylori
, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water.
Improved Method for Polynucleotide Probe-Based Cell Sorting, Using DNA-Coated Microplates
Authors: Zwirglmaier K. , Ludwig W. , Schleifer K.-H.
Published: January 2004

We developed an improved method for cultivation-independent sorting of bacterial cells. The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes. Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells. Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence. The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits.
Rapid Detection ofVibrio vulnificusin Shellfish and Gulf of Mexico Water by Real-Time PCR
Authors: Panicker Gitika , Myers Michael L. , Bej Asim K.
Published: January 2004

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species withvvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection ofVibrio vulnificusin oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for thevvhgene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains ofV. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 102V. vulnificuscells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to oneV. vulnificuscell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell ofV. vulnificusin 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection ofV. vulnificuswould ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.
Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic
Methylobacterium
sp. Associated with Poplar Tissues (
Populus deltoides
×
nigra
DN34)
Authors: Van Aken Benoit , Yoon Jong Moon , Schnoor Jerald L.
Published: January 2004

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (
Populus deltoides
×
nigra
DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a
Methylobacterium
sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus
Methylobacterium
. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-
14
C]TNT (25 mg liter
−1
) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of
14
CO
2
was recorded from [
14
C]TNT. In addition, the isolated methylotroph was shown to transform [U-
14
C]RDX (20 mg liter
−1
) and [U-
14
C]HMX (2.5 mg liter
−1
) in less than 40 days. After 55 days of incubation, 58.0% of initial [
14
C]RDX and 61.4% of initial [
14
C]HMX were mineralized into
14
CO
2
. The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [
14
C]RDX and [
14
C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus
Methylobacterium
are distributed in a wide diversity of natural environments and are very often associated with plants,
Methylobacterium
sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.
In Vitro and In Vivo Inhibition of
Helicobacter pylori
by
Lactobacillus casei
Strain Shirota
Authors: Sgouras D. , Maragkoudakis P. , Petraki K. , Martinez-Gonzalez B. , Eriotou E. , Michopoulos S. , Kalantzopoulos G. , Tsakalidou E. , Mentis Α.
Published: January 2004

We studied the potential inhibitory effect of
Lactobacillus casei
strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on
Helicobacter pylori
by using (i) in vitro inhibition assays with
H. pylori
SS1 (Sydney strain 1) and nine
H. pylori
clinical isolates and (ii) the in vivo
H. pylori
SS1 mouse model of infection over a period of 9 months. In vitro activity against
H. pylori
SS1 and all of the clinical isolates was observed in the presence of viable
L. casei
strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of
L. casei
strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with
H. pylori
SS1 (study group;
n
= 25). Appropriate control groups of
H. pylori
-infected but untreated animals (
n
= 25) and uninfected animals given
L. casei
strain Shirota (
n
= 25) also were included in the study.
H. pylori
colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of
H. pylori
colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the
H. pylori
-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-
H. pylori
immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.
Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria
Authors: Oremland Ronald S. , Herbel Mitchell J. , Blum Jodi Switzer , Langley Sean , Beveridge Terry J. , Ajayan Pulickel M. , Sutto Thomas , Ellis Amanda V. , Curran Seamus
Published: January 2004

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria,Sulfurospirillum barnesii,Bacillus selenitireducens, andSelenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ∼300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H2Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.
Rapid Detection of
Escherichia coli
O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage
Authors: Oda Masahito , Morita Masatomo , Unno Hajime , Tanji Yasunori
Published: January 2004

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell,
Escherichia coli
O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around
soc
was amplified by PCR and sequenced. The gene alignment of
soc
and its upstream region was
g56-soc.2-soc.1-soc
, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the
E. coli
cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable
E. coli
cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive
E. coli
K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on
E. coli
O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C,
E. coli
O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for
E. coli
O157:H7 detection.
Bradyrhizobium elkanii rtxC
Gene Is Required for Expression of Symbiotic Phenotypes in the Final Step of Rhizobitoxine Biosynthesis
Authors: Okazaki Shin , Sugawara Masayuki , Minamisawa Kiwamu
Published: January 2004

We disrupted the
rtxC
gene on the chromosome of
Bradyrhizobium elkanii
USDA94 by insertion of a nonpolar
aph
cartridge. The
rtxC
mutant, designated Δ
rtxC
, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the
rtxC
gene into the Δ
rtxC
mutant complemented rhizobitoxine production, suggesting that
rtxC
is involved in the final step of rhizobitoxine biosynthesis in
B. elkanii
USDA94.
Glycine max
cv. Lee inoculated with Δ
rtxC
or with a null mutant, Δ
rtx
::Ω1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis. The two mutants showed significantly less nodulation competitiveness than the wild-type strain on
Macroptilium atropurpureum
. These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes. Thus, desaturation of dihydrorhizobitoxine by
rtxC
-encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta. In addition, complementation analysis of
rtxC
by cosmids differing in
rtxC
transcription levels suggested that rhizobitoxine production correlates with the amount of
rtxC
transcript.
Role of Two G-Protein Alpha Subunits, TgaA and TgaB, in the Antagonism of Plant Pathogens by
Trichoderma virens
Authors: Mukherjee Prasun K. , Latha Jagannathan , Hadar Ruthi , Horwitz Benjamin A.
Published: January 2004

G-protein α subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein α subunit genes,
tgaA
and
tgaB
, from the biocontrol fungus
Trichoderma virens. tgaA
belongs to the fungal Gαi class, while
tgaB
belongs to the class defined by
gna-2
of
Neurospora crassa
. We compared loss-of-function mutants of
tgaA
and
tgaB
with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of
Rhizoctonia solani
and
Sclerotium rolfsii
in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The
tgaA
mutants germinated by straight unbranched germ tubes, while
tgaB
mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both
tgaA
and
tgaB
mutants and the wild type overgrew, coiled, and lysed the mycelia of
R. solani
, but
tgaA
mutants had reduced ability to colonize
S. rolfsii
colonies. In the soil plate assay, both mutants parasitized the sclerotia of
R. solani
, but
tgaA
mutants were unable to parasitize the sclerotia of
S. rolfsii
. Thus,
tgaA
is involved in antagonism against
S. rolfsii
, but neither G protein subunit is involved in antagonism against
R. solani. T. virens
, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.
Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice
Authors: Junge Karen , Eicken Hajo , Deming Jody W.
Published: January 2004

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O
2
-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of
Bacteria
,
Cytophaga
-
Flavobacteria
-
Bacteroides
(CFB), and
Archaea
. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of
Bacteria
) and
Archaea
(0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.
Genotypic and Phenotypic Studies of Murine Intestinal Lactobacilli: Species Differences in Mice with and without Colitis
Authors: Peña J. A. , Li S. Y. , Wilson P. H. , Thibodeau S. A. , Szary A. J. , Versalovic J.
Published: January 2004

Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota and are useful as probiotics, functional foods, and dairy products. This study includes systematic polyphasic analyses of murine intestinal
Lactobacillus
isolates and correlation of taxonomic findings with data from cytokine production assays. Lactobacilli were recovered from mice with microbiota-dependent colitis (interleukin-10 [IL-10]-deficient C57BL/6 mice) and from mice without colitis (Swiss Webster and inducible nitric oxide synthetase-deficient C57BL/6 mice). Polyphasic analyses were performed to elucidate taxonomic relationships among 88 reference and murine gastrointestinal lactobacilli. Genotypic tests included single-locus analyses (16S ribosomal DNA sequencing and 16S-23S rRNA intergenic spacer region PCR) and genomic DNA profiling (repetitive DNA element-based PCR), and phenotypic analyses encompassed more than 50 tests for carbohydrate utilization, enzyme production, and antimicrobial resistance. From 20 mice without colitis, six
Lactobacillus
species were recovered; the majority of the mice were colonized with
L. reuteri
or
L. murinus
(72% of isolates). In contrast, only,
L. johnsonii
was isolated from 14 IL-10-deficient mice. Using an in vitro assay, we screened murine isolates for their ability to inhibit tumor necrosis factor alpha (TNF-α) secretion by lipopolysaccharide-activated macrophages. Interestingly, a subpopulation of lactobacilli recovered from mice without colitis displayed TNF-α inhibitory properties, whereas none of the
L. johnsonii
isolates from IL-10-deficient mice exhibited this effect. We propose that differences among intestinal
Lactobacillus
populations in mammals, combined with host genetic susceptibilities, may account partly for variations in host mucosal responses.