bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Markers Unique to
Listeria monocytogenes
Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages
Authors: Evans Matthew R. , Swaminathan Bala , Graves Lewis M. , Altermann Eric , Klaenhammer Todd R. , Fink Ryan C. , Kernodle Sheri , Kathariou Sophia 
Published: April 2004

ABSTRACT

A small number of closely related strains of
Listeria monocytogenes
serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to
inlA
, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.
Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by
Sphingobium chlorophenolicum
ATCC 39723
Authors: Dai MingHua , Copley Shelley D. 
Published: April 2004

ABSTRACT

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by
Sphingobium chlorophenolicum
, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and
S. chlorophenolicum
cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by
S. chlorophenolicum
. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.
Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria
Authors: Rahman Mokhlasur , Kühn Inger , Rahman Motiur , Olsson-Liljequist Barbro , Möllby Roland 
Published: April 2004

ABSTRACT

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log
2
dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.
Spatial Distribution of Marine Crenarchaeota Group I in the Vicinity of Deep-Sea Hydrothermal Systems
Authors: Takai Ken , Oida Hanako , Suzuki Yohey , Hirayama Hisako , Nakagawa Satoshi , Nunoura Takuro , Inagaki Fumio , Nealson Kenneth H. , Horikoshi Koki 
Published: April 2004

ABSTRACTDistribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.
Identification, Detection, and Spatial Resolution of
Clostridium
Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor
Authors: Burrell P. C. , O'Sullivan C. , Song H. , Clarke W. P. , Blackall L. L. 
Published: April 2004

ABSTRACT

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by
Firmicutes
phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to
Clostridium stercorarium
), clone group 2 (most closely related to
Clostridium thermocellum
), and clone group 5 (most closely related to
Bacteroides cellulosolvens
) comprised sequences in
Clostridium
group III. Clone group 3 sequences were in
Clostridium
group XIVa (most closely related to
Clostridium
sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with
Clostridium
group VI. This monophyletic group comprises a new
Clostridium
cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.
Relationship of Species-Specific Filament Levels to Filamentous Bulking in Activated Sludge
Authors: Liao Jiangying , Lou Inchio , de los Reyes Francis L. 
Published: April 2004

ABSTRACT

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament
Sphaerotilus natans
were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with
S. natans
cells to stimulate bulking. Instead of
S. natans
, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 × 10
8
μm ml
−1
resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the “substrate diffusion limitation” hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.
Exploring Nitrilase Sequence Space for Enantioselective Catalysis
Authors: Robertson Dan E. , Chaplin Jennifer A. , DeSantis Grace , Podar Mircea , Madden Mark , Chi Ellen , Richardson Toby , Milan Aileen , Miller Mark , Weiner David P. , Wong Kelvin , McQuaid Jeff , Farwell Bob , Preston Lori A. , Tan Xuqiu , Snead Marjory A. , Keller Martin , Mathur Eric , Kretz Patricia L. , Burk Mark J. , Short Jay M. 
Published: April 2004

ABSTRACT

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10
6
to 10
10
members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.
Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts
Authors: Martinez Asuncion , Kolvek Steven J. , Yip Choi Lai Tiong , Hopke Joern , Brown Kara A. , MacNeil Ian A. , Osburne Marcia S. 
Published: April 2004

ABSTRACT

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an
Escherichia coli
library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in
E. coli
. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include
Streptomyces lividans
and
Pseudomonas putida
as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of
S. lividans
that contain complete and unmarked deletions of the
act
and
red
endogenous pigment gene clusters, a derivative of
P. putida
that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from
E. coli
to both
S. lividans
and
P. putida
by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.
Phylogeny and Strain Typing ofEscherichiacoli, Inferred from Variation at Mononucleotide RepeatLoci
Authors: Diamant Eran , Palti Yniv , Gur-Arie Riva , Cohen Helit , Hallerman Eric M. , Kashi Yechezkel 
Published: April 2004

ABSTRACTMultilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains ofEscherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensalE. colistrains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of theE. colireference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.
Method for Collecting Air-Water Interface Microbes Suitable for Subsequent Microscopy and Molecular Analysis in both Research and Teaching Laboratories
Authors: Henk Margaret C. 
Published: April 2004

ABSTRACT
A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.
Chlorophenol Production by Anaerobic Microorganisms: Transformation of a Biogenic Chlorinated Hydroquinone Metabolite
Authors: Milliken C. E. , Meier G. P. , Sowers K. R. , May H. D. 
Published: April 2004

ABSTRACT

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as
Desulfitobacterium
spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol.
70:
385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of
Desulfitobacterium
sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions.
Desulfitobacterium
sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.
Fate of
Salmonella enterica
Serovar Typhimurium on Carrots and Radishes Grown in Fields Treated with Contaminated Manure Composts or Irrigation Water
Authors: Islam Mahbub , Morgan Jennie , Doyle Michael P. , Phatak Sharad C. , Millner Patricia , Jiang Xiuping 
Published: April 2004

ABSTRACT

Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of
Salmonella enterica
serovar Typhimurium at 10
7
CFU g
−1
and 10
5
CFU ml
−1
, respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 × 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively.
Salmonella
survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of
Salmonella
on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.
Frequency and Distribution of Tetracycline Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical
Escherichia coli
Strains Isolated from Diverse Human and Animal Sources
Authors: Bryan Andrew , Shapir Nir , Sadowsky Michael J. 
Published: April 2004

ABSTRACT

Nonselected and natural populations of
Escherichia coli
from 12 animal sources and humans were examined for the presence and types of 14 tetracycline resistance determinants. Of 1,263 unique
E. coli
isolates from humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, 31% were highly resistant to tetracycline. More than 78, 47, and 41% of the
E. coli
isolates from pigs, chickens, and turkeys were resistant or highly resistant to tetracycline, respectively. Tetracycline MICs for 61, 29, and 29% of
E. coli
isolates from pig, chickens, and turkeys, respectively, were ≥233 μg/ml. Muliplex PCR analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes [
tetA
,
tetB
,
tetC
,
tetD
,
tetE
,
tetG
,
tetK
,
tetL
,
tetM
,
tetO
,
tetS
,
tetA(P)
,
tetQ
, and
tetX
] examined. While the most common genes found in these isolates were
tetB
(63%) and
tetA
(35%),
tetC
,
tetD
, and
tetM
were also found.
E. coli
isolates from pigs and chickens were the only strains to have
tetM
. To our knowledge, this represents the first report of
tetM
in
E. coli
.
Growth and Sporulation of
Bacillus cereus
ATCC 14579 under Defined Conditions: Temporal Expression of Genes for Key Sigma Factors
Authors: de Vries Ynte P. , Hornstra Luc M. , de Vos Willem M. , Abee Tjakko 
Published: April 2004

ABSTRACT

An airlift fermentor system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of
Bacillus cereus
ATCC 14579. Sporulation was complete and synchronous. Expression of
sigA
,
sigB
,
sigF
, and
sigG
was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of
Bacillus subtilis
. This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further gene expression studies of cellular differentiation of
B. cereus
.
Geobacter sulfurreducens
Can Grow with Oxygen as a Terminal Electron Acceptor
Authors: Lin W. C. , Coppi M. V. , Lovley D. R. 
Published: April 2004

ABSTRACT

Geobacter sulfurreducens
, previously classified as a strict anaerobe, tolerated exposure to atmospheric oxygen for at least 24 h and grew with oxygen as the sole electron acceptor at concentrations of 10% or less in the headspace. These results help explain how
Geobacter
species may survive in oxic subsurface environments, being poised to rapidly take advantage of the development of anoxic conditions.
ω-Amino Acid:Pyruvate Transaminase from
Alcaligenes denitrificans
Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines
Authors: Yun Hyungdon , Lim Seongyop , Cho Byung-Kwan , Kim Byung-Gee 
Published: April 2004

ABSTRACT

Alcaligenes denitrificans
Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (
aptA
) was cloned. The gene was functionally expressed in
Escherichia coli
BL21 by using an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for
l
-β-amino-
n
-butyric acid (
l
-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent

K
m

and
V
max
for
l
-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM
l
-β-ABA, the apparent

K
m

and
V
max
for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM
d,l
-β-ABA, producing optically pure
d
-β-ABA (99% enantiomeric excess) with 53% conversion.