bluuebunny/crossref_metadata_2025_split huggingface dataset:
Species Abundance and Diversity of
Burkholderia cepacia
Complex in the Environment
Authors: Ramette Alban , LiPuma John J. , Tiedje James M. 
Published: March 2005

ABSTRACT

Despite considerable interest in studying
Burkholderia cepacia
complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of
B. cepacia
isolates. To better describe the ecology and diversity of
B. cepacia
complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a
B. cepacia
complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting
B. cepacia
complex-specific
recA
and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of
recA
for a total of 285 confirmed isolates of the
B. cepacia
complex, up to seven
B. cepacia
complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of
B. cepacia
complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.
Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil
Authors: Singleton David R. , Powell Sabrina N. , Sangaiah Ramiah , Gold Avram , Ball Louise M. , Aitken Michael D. 
Published: March 2005

ABSTRACT

[
13
C
6
]salicylate, [U-
13
C]naphthalene, and [U-
13
C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from
Escherichia coli
K-12 was added to each sample as an internal indicator of separation efficiency. While
E. coli
was not detected in most analyses of heavy DNA, a low number of
E. coli
sequences was recovered in the clone libraries associated with the heavy DNA fraction of [
13
C]phenanthrene incubations. The number of
E. coli
clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the
Pseudomonas
and
Ralstonia
genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the
Acidovorax
genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the
13
C during the incubation did not appear to be an issue in this experiment.
Functional Analysis of Three Plasmids from
Lactobacillus plantarum
Authors: van Kranenburg Richard , Golic Natasa , Bongers Roger , Leer Rob J. , de Vos Willem M. , Siezen Roland J. , Kleerebezem Michiel 
Published: March 2005

ABSTRACT

Lactobacillus plantarum
WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes
Lactobacillus
species and
Lactococcus lactis
, while that of the pWCFS102 replicon also includes
Carnobacterium maltaromaticum
and
Bacillus subtilis
. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to
L. plantarum
. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to
L. plantarum
NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.
Characterization of the
cro-ori
Region of the
Streptococcus thermophilus
Virulent Bacteriophage DT1
Authors: Lamothe Geneviève , Lévesque Céline , Bissonnette Frédéric , Cochu Armelle , Vadeboncoeur Christian , Frenette Michel , Duplessis Martin , Tremblay Denise , Moineau Sylvain 
Published: March 2005

ABSTRACT

The virulent
cos
-type
Streptococcus thermophilus
phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative
ori
of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between
orf36
and
orf37
. As the replication ability of the putative
ori
and flanking genes could not be established, its ability to confer phage resistance was tested. When
ori
is cloned on a high-copy-number plasmid, it provides protection to
S. thermophilus
strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the
cro
-like gene (
orf29
) and the
ori
. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the
cro-ori
region in the three
S. thermophilus cos
-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of
orf29
was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the
S. thermophilus
operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the
pts
promoter in
Lactococcus lactis
and in
Escherichia coli
.
Degradation of Wheat Straw by
Fibrobacter succinogenes
S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study
Authors: Matulova M. , Nouaille R. , Capek P. , Péan M. , Forano E. , Delort A.-M. 
Published: March 2005

ABSTRACT

Wheat straw degradation by
Fibrobacter succinogenes
was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with
13
C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of
13
C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of
F. succinogenes
S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.
Enumeration and Diversity of Campylobacters and Bacteriophages Isolated during the Rearing Cycles of Free-Range and Organic Chickens
Authors: El-Shibiny A. , Connerton P. L. , Connerton I. F. 
Published: March 2005

ABSTRACT

Campylobacters and
Campylobacter
-specific bacteriophages were isolated and enumerated during the rearing cycle of free-range (56 days) and organic chickens (73 days) at 3-day intervals from hatching until slaughter. In both flocks
Campylobacter jejuni
was the initial colonizer but
Campylobacter coli
was detected more frequently from 5 weeks of age. The diversity of the
Campylobacter
isolates was examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA and antimicrobial resistance typing. Bacteriophages were isolated from 51% (19 of 37 birds) of
Campylobacter
-positive organic birds (log
10
2.5 to log
10
5.7 PFU/g of cecal contents). The bacteriophages were all typical group III
Campylobacter
bacteriophages in terms of genomic size but could be characterized in terms of their host range and placed into five different groups. In contrast to the organic birds, anti-
Campylobacter
activity (bacteriocin-like) was observed in 26% (10 of 38 birds) of
Campylobacter
-positive free-range birds, and only one bacteriophage was isolated. Appearance of either bacteriophages or anti-
Campylobacter
activity was associated with changes in the levels of colonization and the predominant genotypes and species isolated. The frequency and potential influence of naturally occurring bacteriophages and/or inhibitory substances on the diversity and fluctuations of populations of campylobacters have not previously been reported in either free-range or organic chickens.
Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA
Authors: Botero Lina M. , D'Imperio Seth , Burr Mark , McDermott Timothy R. , Young Mark , Hassett Daniel J. 
Published: March 2005

ABSTRACT

We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing
Bacteria
and
Eukarya
taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and
Bacteria
-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the
Firmicutes
and the α-
Proteobacteria
. Unexpectedly, there were no
Archaea
clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and
Archaea
-biased primers and unmodified RNA demonstrated the presence of novel
Archaea
in the soil. Experiments with pure cultures of
Sulfolobus solfataricus
and
Halobacterium halobium
revealed that some
Archaea
rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.
Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in
Listeria innocua
, Identified by Screening for Pediocin AcH Resistance
Authors: Xue Junfeng , Hunter Ian , Steinmetz Tori , Peters Adam , Ray Bibek , Miller Kurt W. 
Published: March 2005

ABSTRACT

To identify genes that are important for class IIa bacteriocin interaction and resistance in
Listeria
species, transposon Tn
917
knockout libraries were constructed for
Listeria innocua
strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 μg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the
mpt
operon (
mptA
/
mptC
/
mptD
) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EII
t
Man
, which serves as a docking protein for class IIa bacteriocins. The transcription of the
mpt
operon is known to be positively controlled by σ
54
factor and ManR (a σ
54
-associated activator). Transcripts for lin0142 and
mpt
were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of
mpt
mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of
manR
and
rpoN
(σ
54
) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three
mpt
regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the
mpt
operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.
Diversity of Proteolytic
Clostridium botulinum
Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach
Authors: Nevas Mari , Lindström Miia , Hielm Sebastian , Björkroth K. Johanna , Peck Michael W. , Korkeala Hannu 
Published: March 2005

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic
Clostridium botulinum
(
C. botulinum
group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic
C. botulinum
strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic
C. botulinum
species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic
C. botulinum
types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic
C. botulinum
types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.
Identification of a New
Borrelia
Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic
Authors: Gil Horacio , Barral Marta , Escudero Raquel , García-Pérez Ana L. , Anda Pedro 
Published: March 2005

ABSTRACT

The role of small mammals as reservoir hosts for
Borrelia burgdorferi
was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by
Ixodes ricinus
nymphs was found in the small mammal populations studied that correlated with the near-absence of
B. burgdorferi
sensu lato in 184 animals tested and with the lack of transmission of
B. burgdorferi
sensu lato to
I. ricinus
larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable
B. burgdorferi
sensu lato repertoire (
B. burgdorferi
sensu stricto,
Borrelia garinii
,
Borrelia valaisiana
, and
Borrelia afzelii
). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other
Borrelia
species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to
B. burgdorferi
sensu lato was found in the animal sera, suggesting cross-reactivity between
B. burgdorferi
sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for
B. burgdorferi
sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.
Patterns of Antimicrobial Resistance Observed in
Escherichia coli
Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water
Authors: Sayah Raida S. , Kaneene John B. , Johnson Yvette , Miller RoseAnn 
Published: March 2005

ABSTRACT

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286
Escherichia coli
strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of
E. coli
were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general,
E. coli
isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant
E. coli
were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.
Presence of Noroviruses and Other Enteric Viruses in Sewage and Surface Waters in The Netherlands
Authors: Lodder W. J. , de Roda Husman A. M. 
Published: March 2005

ABSTRACT

Since virus concentrations in drinking waters are generally below the detection limit, the infectious risk from drinking water consumption requires assessment from the virus concentrations in source waters and removal efficiency of treatment processes. In this study, we estimated from reverse transcription-PCR on 10-fold serially diluted RNA that noroviruses, the most prevalent waterborne gastroenteritis agents, were present at 4 (0.2 to 38) to 4,900 (303 to 4.6 × 10
4
) PCR-detectable units (PDU) per liter of river water (ranges are given in parentheses). These virus concentrations are still high compared with 896 to 7,499 PDU/liter of treated sewage and 5,111 to 850,000 PDU/liter in raw sewage. Sequencing analyses designated human norovirus GGII.4 Lordsdale as the most prevalent strain in the sampling period 1998 to 1999 in both sewage and surface waters. Other GGII strains were also very abundant, indicating that the majority of the virus contamination was derived from urban sewage, although very divergent strains and one animal strain were also detected in the surface and sewage waters. Rotaviruses were also detected in two large rivers (the Maas and the Waal) at 57 to 5,386 PDU/liter. The high virus concentrations determined by PCR may in part be explained by the detection of virus RNA instead of infectious particles. Indeed, reoviruses and enteroviruses that can be cultured were present at much lower levels, of 0.3 to 1 and 2 to 10 PFU/liter, respectively. Assuming 1% of the noroviruses and rotaviruses to be infectious, a much higher disease burden than for other viruses can be expected, not only because of the higher levels but also because of these viruses' higher infectivity and attack rates.
Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness
Authors: Schloss Patrick D. , Handelsman Jo 
Published: March 2005

ABSTRACT

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (
P
> 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.