bluuebunny/crossref_metadata_2025 huggingface dataset:
CynD, the Cyanide Dihydratase from
Bacillus pumilus
: Gene Cloning and Structural Studies
Authors: Jandhyala Dakshina , Berman Mark , Meyers Paul R. , Sewell B. Trevor , Willson Richard C. , Benedik Michael J.
Published: August 2003

The cyanide dihydratase in
Bacillus pumilus
was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in
Escherichia coli
. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has

K
m

and
V
max
values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative
Pseudomonas stutzeri
strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of
B
.
pumilus
(strain 8A3) was cloned and expressed. This suggests that
cynD
, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of
B
.
pumilus
and is not a reflection of its origin at a mining waste site.
Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate
Cyclospora
Species from
Eimeria
Species by Multiplex PCR
Authors: Orlandi Palmer A. , Carter Laurenda , Brinker Anna Marie , da Silva Alexandre J. , Chu Dan-My , Lampel Keith A. , Monday Steven R.
Published: August 2003

Cyclospora cayetanensis
is a coccidian parasite that causes protracted diarrheal illness in humans.
C. cayetanensis
is the only species of this genus thus far associated with human illness, although
Cyclospora
species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from
C. cayetanensis
and
Eimeria
species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between
C. cayetanensis
and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between
C. cayetanensis
, nonhuman primate species of
Cyclospora
, and
Eimeria
species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.
N
-Acetyl-
l
-Cysteine Affects Growth, Extracellular Polysaccharide Production, and Bacterial Biofilm Formation on Solid Surfaces
Authors: Olofsson Ann-Cathrin , Hermansson Malte , Elwing Hans
Published: August 2003

N
-Acetyl-
l
-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces.
Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism
Authors: Engebretson Jeff J. , Moyer Craig L.
Published: August 2003

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (
n
= 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined,
Bst
UI,
Dde
I,
Sau
96I, and
Msp
I had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).
Alteration of Chain Length Substrate Specificity of
Aeromonas caviae R
-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis
Authors: Tsuge Takeharu , Hisano Tamao , Taguchi Seiichi , Doi Yoshiharu
Published: August 2003

Aeromonas caviae R
-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ
Ac
) is capable of providing (
R
)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ
Ac
by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ
Ac
revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using
trans
-2-octenoyl-CoA (C
8
) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C
12
) was examined by using the mutated PhaJ
Ac
as a monomer supplier in recombinant
Escherichia coli
LS5218 harboring a PHA synthase gene from
Pseudomonas
sp. strain 61-3 (
phaC1
Ps
). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C
8
) and 3-hydroxydecanoate (C
10
) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.
The Biphenyl- and 4-Chlorobiphenyl-Catabolic Transposon Tn4371, a Member of a New Family of Genomic Islands Related to IncP and Ti Plasmids
Authors: Toussaint Ariane , Merlin Christophe , Monchy Sébastien , Benotmane M. Abderrafi , Leplae Raphaël , Mergeay Max , Springael Dirk
Published: August 2003

The nucleotide sequence of the biphenyl catabolic transposon Tn4371has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those ofAchromobacter georgiopolitanumKKS102, Tn4371carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome ofRalstonia solanacearumand on the yet-to-be-determined genomic sequences ofErwinia chrysanthemiandAzotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bphgenes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome ofRalstonia metalliduransCH34. Tn4371thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.
Dual Labeling of
Pseudomonas putida
with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid
Authors: Nancharaiah Y. Venkata , Wattiau Pierre , Wuertz Stefan , Bathe Stephan , Mohan S. Venkata , Wilderer Peter A. , Hausner Martina
Published: August 2003

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to
Pseudomonas putida
KT2442, which was chromosomally labeled with
dsRed
by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor
P. putida
cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.
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

The 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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Internal and External Mycoflora of the American Dog Tick,
Dermacentor variabilis
(Acari: Ixodidae), and Its Ecological Implications
Authors: Yoder Jay A. , Hanson Peter E. , Zettler Lawrence W. , Benoit Joshua B. , Ghisays Fiorella , Piskin Kurt A.
Published: August 2003

Scopulariopsis brevicaulis
, the anamorph of
Microascus brevicaulis
(Microascaceae, Ascomycota), has been identified in the body contents of the tick
Dermacentor variabilis
. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of
D. variabilis
with a novel technique used to recover potential biological control agents.
Microbial Isotopic Fractionation of Perchlorate Chlorine
Authors: Coleman Max L. , Ader Magali , Chaudhuri Swades , Coates John D.
Published: August 2003

Perchlorate contamination can be microbially respired to innocuous chloride and thus can be treated effectively. However, monitoring a bioremediative strategy is often difficult due to the complexities of environmental samples. Here we demonstrate that microbial respiration of perchlorate results in a significant fractionation (∼−15‰) of the chlorine stable isotope composition of perchlorate. This can be used to quantify the extent of biotic degradation and to separate biotic from abiotic attenuation of this contaminant.
Characterization ofAnaplasma marginaleIsolated from North American Bison
Authors: de la Fuente José , Golsteyn Thomas Elizabeth J. , Van Den Bussche Ronald A. , Hamilton Robert G. , Tanaka Elaine E. , Druhan Susan E. , Kocan Katherine M.
Published: August 2003

Anaplasma marginale(Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison,Bison bison, as being infected withA. marginale, the present study was undertaken to confirmA. marginaleinfection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of thisA. marginalebison isolate for cattle. The results of this study showed that theseA. marginaleisolates obtained from bison were similar to ones from naturally infected cattle.
Boundaries for Biofilm Formation: Humidity and Temperature
Authors: Else Terry Ann , Pantle Curtis R. , Amy Penny S.
Published: August 2003

Environmental conditions which define boundaries for biofilm production could provide useful ecological information for biofilm models. A practical use of defined conditions could be applied to the high-level nuclear waste repository at Yucca Mountain. Data for temperature and humidity conditions indicate that decreases in relative humidity or increased temperature severely affect biofilm formation on three candidate canister metals.
Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism
Authors: McIntosh F. M. , Williams P. , Losa R. , Wallace R. J. , Beever D. A. , Newbold C. J.
Published: August 2003

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (
P
< 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.
Chlorination and Cleavage of Lignin Structures by Fungal Chloroperoxidases
Authors: Ortiz-Bermúdez Patricia , Srebotnik Ewald , Hammel Kenneth E.
Published: August 2003

Two fungal chloroperoxidases (CPOs), the heme enzyme from
Caldariomyces fumago
and the vanadium enzyme from
Curvularia inaequalis
, chlorinated 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane, a dimeric model compound that represents the major nonphenolic structure in lignin. Both enzymes also cleaved this dimer to give 1-chloro-4-ethoxy-3-methoxybenzene and 1,2-dichloro-4-ethoxy-5-methoxybenzene, and they depolymerized a synthetic guaiacyl lignin. Since fungal CPOs occur in soils and the fungi that produce them are common inhabitants of plant debris, CPOs may have roles in the natural production of high-molecular-weight chloroaromatics and in lignin breakdown.
Structures of the Mating-Type Loci of
Cordyceps takaomontana
Authors: Yokoyama Eiji , Yamagishi Kenzo , Hara Akira
Published: August 2003

Nucleotide sequences of the mating-type loci
MAT1-1
and
MAT1-2
of
Cordyceps takaomontana
were determined, which is the first such report for the clavicipitaceous fungi.
MAT1-1
contains two mating-type genes,
MAT1-1-1
and
MAT1-1-2
, but
MAT1-1-3
could not be found. On the other hand,
MAT1-2
has
MAT1-2-1.
A pseudogene of
MAT1-1-1
is located next to
MAT1-2
.
Detection of Bacteria Carrying the
stx
2
Gene by In Situ Loop-Mediated Isothermal Amplification
Authors: Maruyama Fumito , Kenzaka Takehiko , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: August 2003

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect
stxA
2
in
Escherichia coli
O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of
E. coli
O157:H7 cells with an
stxA
2
gene. Higher-contrast images were obtained with this method than with in situ PCR.
Reduction of ExperimentalSalmonellaandCampylobacterContamination of Chicken Skin by Application of Lytic Bacteriophages
Authors: Goode D. , Allen V. M. , Barrow P. A.
Published: August 2003

Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated withSalmonella entericaserovar Enteritidis orCampylobacter jejuniat a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10units over 48 h. When the level ofSalmonellacontamination was low (< log102 per unit area of skin) and the MOI was 105, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 107), it was also possible to eliminate otherSalmonellastrains that showed high levels of resistance because of restriction but to which the phages were able to attach.
Fuzzy Connectives as a Combination Tool in a Hybrid Multi-Neural System
Authors: Canuto Anne , Fairhurst Michael , Howells Gareth
Published: August 2003

The set of fuzzy connectives can be seen as an important combination tool, such as in combining the antecedent sets of the rules, in multi-criteria decision making and in combining the outputs of neural classifiers in a multi-neural system. This papers investigates the performance of some fuzzy combination schemes applied to a multi hybrid neural system which is composed of neural and fuzzy neural networks. An empirical evaluation in a handwritten numeral recognition task is used to investigate the performance of the presented fuzzy methods with some existing combination methods.
Wavelet-Like Receptive Fields Emerges by Non-Linear Minimization of Neuron Error
Authors: Barros Allan Kardec , Cichocki Andrzej , Ohnishi Noboru
Published: August 2003

Redundancy reduction as a form of neural coding has been since the early sixties a topic of large research interest. A number of strategies has been proposed, but the one which is attracting most attention recently assumes that this coding is carried out so that the output signals are mutually independent. In this work we go one step further and suggest an strategy to deal also with non-orthogonal signals (i.e., ''dependent'' signals). Moreover, instead of working with the usual squared error, we design a neuron where the non-linearity is operating on the error. It is computationally more economic and, importantly, the permutation/scaling problem10 is avoided. The framework is given with a biological background, as we avocate throughout the manuscript that the algorithm fits well the single neuron and redundancy reduction doctrine.5 Moreover, we show that wavelet-like receptive fields emerges from natural images processed by this algorithm.
NSL: A Neuro–Symbolic Language for a Neuro–Symbolic Processor (NSP)
Authors: Burattini Ernesto , de Francesco Antonio , de Gregorio Massimo
Published: August 2003

A Neuro–Symbolic Language for monotonic and non–monotonic parallel logical inference by means of artificial neural networks (ANNs) is presented. Both the language and its compiler have been designed and implemented in order to translate the neural representation of a given problem into a VHDL software, which in turn can set devices such as FPGA. The result of this operation leads to an electronic circuit that we call NSP (Neuro–Symbolic Processor).
Artificial Neural Network Learning of Nonstationary Behavior in Time Series
Authors: Széliga María I. , Verdes Pablo F. , Granitto Pablo M. , Ceccatto H. Alejandro
Published: August 2003

We refine and complement a previously-proposed artificial neural network method for learning hidden signals forcing nonstationary behavior in time series. The method adds an extra input unit to the network and feeds it with the proposed profile for the unknown perturbing signal. The correct time evolution of this new input parameter is learned simultaneously with the intrinsic stationary dynamics underlying the series, which is accomplished by minimizing a suitably-defined error function for the training process. We incorporate here the use of validation data, held out from the training set, to accurately determine the optimal value of a hyperparameter required by the method. Furthermore, we evaluate this algorithm in a controlled situation and show that it outperforms other existing methods in the literature. Finally, we discuss a preliminary application to the real-world sunspot time series and link the obtained hidden perturbing signal to the secular evolution of the solar magnetic field.
Modeling a Particular Decision Process by Using a Modulatory Activation Function
Authors: Filho Jairo Diniz , Ludermir Teresa B.
Published: August 2003

Neuronal groups projecting widely in the brain are being experimentally associated to attention and mood changes. Those groups are known to exert a modulatory effect over other larger groups. On the other hand, some people think of the brain functions as being performed by specialized modular systems. In this work, we propose an architecture of modular nature to explore a particular decision process. We show the importance of the modulatory effect of a special evaluation segment in that process.