bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbial Ecology of an Extreme Acidic Environment, the Tinto River
Authors: González-Toril E. , Llobet-Brossa E. , Casamayor E. O. , Amann R. , Amils R. 
Published: August 2003

ABSTRACTThe Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related toLeptospirillumspp.,Acidithiobacillus ferrooxidans,Acidiphiliumspp., “Ferrimicrobium acidiphilum,”Ferroplasma acidiphilum, andThermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domainBacteria, with only a minor fraction corresponding toArchaea. Members ofLeptospirillum ferrooxidans,Acidithiobacillus ferrooxidans, andAcidiphiliumspp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.
Directed Evolution of
Thermus
Maltogenic Amylase toward Enhanced Thermal Resistance
Authors: Kim Young-Wan , Choi Ji-Hye , Kim Jung-Wan , Park Cheonseok , Kim Jung-Woo , Cha Hyunju , Lee Soo-Bok , Oh Byoung-Ha , Moon Tae-Wha , Park Kwan-Hwa 
Published: August 2003

ABSTRACT

The thermostability of maltogenic amylase from
Thermus
sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.
High-Level Production of Porphyrins in Metabolically Engineered
Escherichia coli
: Systematic Extension of a Pathway Assembled from Overexpressed Genes Involved in Heme Biosynthesis
Authors: Kwon Seok Joon , de Boer Arjo L. , Petri Ralf , Schmidt-Dannert Claudia 
Published: August 2003

ABSTRACT

Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with
Escherichia coli
as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 μmol/liter, which is close to industrial vitamin B
12
production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed
Bacillus subtilis
ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of
E. coli
or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.
Influence of Seasonal Environmental Variables on the Distribution of Presumptive Fecal Coliforms around an Antarctic Research Station
Authors: Hughes Kevin A. 
Published: August 2003

ABSTRACT
Factors affecting fecal microorganism survival and distribution in the Antarctic marine environment include solar radiation, water salinity, temperature, sea ice conditions, and fecal input by humans and local wildlife populations. This study assessed the influence of these factors on the distribution of presumptive fecal coliforms around Rothera Point, Adelaide Island, Antarctic Peninsula during the austral summer and winter of February 1999 to September 1999. Each factor had a different degree of influence depending on the time of year. In summer (February), although the station population was high, presumptive fecal coliform concentrations were low, probably due to the biologically damaging effects of solar radiation. However, summer algal blooms reduced penetration of solar radiation into the water column. By early winter (April), fecal coliform concentrations were high, due to increased fecal input by migrant wildlife, while solar radiation doses were low. By late winter (September), fecal coliform concentrations were high near the station sewage outfall, as sea ice formation limited solar radiation penetration into the sea and prevented wind-driven water circulation near the outfall. During this study, environmental factors masked the effect of station population numbers on sewage plume size. If sewage production increases throughout the Antarctic, environmental factors may become less significant and effective sewage waste management will become increasingly important. These findings highlight the need for year-round monitoring of fecal coliform distribution in Antarctic waters near research stations to produce realistic evaluations of sewage pollution persistence and dispersal.
Single-Leaf Resolution of the Temporal Population Dynamics of
Aureobasidium pullulans
on Apple Leaves
Authors: Woody Scott T. , Spear Russell N. , Nordheim Erik V. , Ives Anthony R. , Andrews John H. 
Published: August 2003

ABSTRACT

The abundance of phylloplane microorganisms typically varies over several orders of magnitude among leaves sampled concurrently. Because the methods traditionally used to sample leaves are destructive, it has remained unclear whether this high variability is due to fixed differences in habitat quality among leaves or to asynchronous temporal variation in the microbial population density on individual leaves. We developed a novel semidestructive assay to repeatedly sample the same apple leaves from orchard trees over time by removing progressively more proximal ∼1-cm-wide transverse segments.
Aureobasidium pullulans
densities were determined by standard leaf homogenization and plating procedures and were expressed as CFU per square centimeter of segment. The
A. pullulans
population densities among leaves were lognormally distributed. The variability in
A. pullulans
population densities among subsections of a given leaf was one-third to one-ninth the variability among whole leaves harvested concurrently. Sequential harvesting of leaf segments did not result in detectable changes in
A. pullulans
density on residual leaf surfaces. These findings implied that we could infer whole-leaf
A. pullulans
densities over time by using partial leaves. When this successive sampling regimen was applied over the course of multiple 7- to 8-day experiments, the among-leaf effects were virtually always the predominant source of variance in
A. pullulans
density estimates. Changes in
A. pullulans
density tended to be synchronous among leaves, such that the rank order of leaves arrayed with respect to
A. pullulans
density was largely maintained through time. Occasional periods of asynchrony were observed, but idiosyncratic changes in
A. pullulans
density did not contribute appreciably to variation in the distribution of populations among leaves. This suggests that persistent differences in habitat (leaf) quality are primarily responsible for the variation in
A. pullulans
density among leaves in nature.
Remarkable Diversity of Phototrophic Purple Bacteria in a Permanently Frozen Antarctic Lake
Authors: Karr Elizabeth A. , Sattley W. Matthew , Jung Deborah O. , Madigan Michael T. , Achenbach Laurie A. 
Published: August 2003

ABSTRACT

Although anoxygenic photosynthesis is thought to play an important role in the primary productivity of permanently frozen lakes in the Antarctic dry valleys, the bacterial communities responsible for this metabolism remain uncharacterized. Here we report the composition and activity of phototrophic purple bacteria in Lake Fryxell, Antarctica, as determined by analysis of a photosynthesis-specific gene,
pufM
. The results revealed an extensive diversity and highly stratified distribution of purple nonsulfur bacteria in Lake Fryxell and showed which phylotypes produced
pufM
transcripts in situ. Enrichment cultures for purple bacteria yielded two morphotypes, each with a
pufM
signature identical to signatures detected by environmental screening. The isolates also contained gas vesicles, buoyancy structures previously unknown in purple nonsulfur bacteria, that may be necessary for these organisms to position themselves at specific depths within the nearly freezing water column.
Colonization of
Arabidopsis thaliana
with
Salmonella enterica
and Enterohemorrhagic
Escherichia coli
O157:H7 and Competition by
Enterobacter asburiae
Authors: Cooley Michael B. , Miller William G. , Mandrell Robert E. 
Published: August 2003

ABSTRACT

Enteric pathogens, such as
Salmonella enterica
and
Escherichia coli
O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed
Arabidopsis thaliana
(thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity,
S. enterica
serovar Newport and
E. coli
O157:H7 grew to 10
9
CFU g
−1
on
A. thaliana
roots and to 2 × 10
7
CFU g
−1
on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled
S. enterica
and
E. coli
O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of
S. enterica
were used. Survival of
S. enterica
serovar Newport and
E. coli
O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil.
Enterobacter asburiae
was isolated from soil-grown
A. thaliana
and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of
S. enterica
and
E. coli
O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor,
Enterobacter asburiae.
Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (
P
= 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.
Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library
Authors: Sebat Jonathan L. , Colwell Frederick S. , Crawford Ronald L. 
Published: August 2003

ABSTRACT
Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ∼1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.
Is the In Situ Accessibility of the 16S rRNA of
Escherichia coli
for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?
Authors: Behrens Sebastian , Fuchs Bernhard M. , Mueller Florian , Amann Rudolf 
Published: August 2003

ABSTRACT

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our
Escherichia coli
16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.
Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of
Escherichia coli
Isolates To Identify Nonpoint Fecal Sources
Authors: Seurinck Sylvie , Verstraete Willy , Siciliano Steven D. 
Published: August 2003

ABSTRACT

Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific
Escherichia coli
fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of
E. coli
isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267
E. coli
isolates from different fecal sources were typed with both techniques.
E. coli
was found to be highly diverse. Only two candidate source-specific
E. coli
fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific
E. coli
fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the
E. coli
population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of
E. coli
isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific
E. coli
fingerprints that were stable under the simulated environmental conditions.
Overexpression of
groESL
in
Clostridium acetobutylicum
Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program
Authors: Tomas Christopher A. , Welker Neil E. , Papoutsakis Eleftherios T. 
Published: August 2003

ABSTRACT

DNA array and Western analyses were used to examine the effects of
groESL
overexpression and host-plasmid interactions on solvent production in
Clostridium acetobutylicum
ATCC 824. Strain 824(pGROE1) was created to overexpress the
groESL
operon genes from a clostridial thiolase promoter. The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del). Overexpression of
groESL
resulted in increased final solvent titers 40% and 33% higher than those of the wild type and plasmid control strains, respectively. Active metabolism lasted two and one half times longer in 824(pGROE1) than in the wild type. Transcriptional analysis of 824(pGROE1) revealed increased expression of motility and chemotaxis genes and a decrease in the expression of the other major stress response genes. Decreased expression of the
dnaKJ
operon upon overexpression of
groESL
suggests that
groESL
functions as a modulator of the CIRCE regulon, which is shown here to include the
hsp90
gene. Analysis of the plasmid control strain 824(pSOS95del) revealed complex host-plasmid interactions relative to the wild-type strain, resulting in prolonged biphasic growth and metabolism. Decreased expression of four DNA gyrases resulted in differential expression of many key primary metabolism genes. The
ftsA
and
ftsZ
genes were expressed at higher levels in 824(pSOS95del), revealing an altered cell division and sporulation pattern. Both transcriptional and Western analyses revealed elevated stress protein expression in the plasmid-carrying strain.
Infectious
Cryptosporidium parvum
Oocysts in Final Reclaimed Effluent
Authors: Gennaccaro Angela L. , McLaughlin Molly R. , Quintero-Betancourt Walter , Huffman Debra E. , Rose Joan B. 
Published: August 2003

ABSTRACT

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious
Cryptosporidium parvum
by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious
C. parvum
oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious
C. parvum
.
Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for
Clostridium acetobutylicum
Authors: Girbal Laurence , Mortier-Barrière Isabelle , Raynaud Frédéric , Rouanet Céline , Croux Christian , Soucaille Philippe 
Published: August 2003

ABSTRACT

A sensitive gene expression reporter system was developed for
Clostridium acetobutylicum
ATCC 824 by using a customized
gusA
expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in
C. acetobutylicum
. Furthermore, a new inducible gene expression system was developed in
C. acetobutylicum
, based on the
Staphylococcus xylosus
xylose operon promoter-repressor regulatory system.
The Ti Plasmid of
Agrobacterium tumefaciens
Harbors an
attM
-Paralogous Gene,
aiiB
, Also Encoding
N
-Acyl Homoserine Lactonase Activity
Authors: Carlier A. , Uroz S. , Smadja B. , Fray R. , Latour X. , Dessaux Y. , Faure D. 
Published: August 2003

ABSTRACT

The
Agrobacterium tumefaciens
C58 genome contains three putative
N
-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of
Bacillus
. When expressed in
Escherichia coli
, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In
Erwinia
strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.