bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rectal Administration of
Escherichia coli
O157:H7: Novel Model for Colonization of Ruminants
Authors: Sheng Haiqing , Davis Margaret A. , Knecht Hannah J. , Hovde Carolyn J. 
Published: August 2004

ABSTRACT

Escherichia coli
O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with
E. coli
O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of
E. coli
O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods.
E. coli
O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (∼10
10
CFU) and low (∼10
7
CFU) oral doses of
E. coli
O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of
E. coli
O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of
E. coli
O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of
E. coli
O157:H7 colonization in cattle.
Biodegradation of Chloromethane by
Pseudomonas aeruginosa
Strain NB1 under Nitrate-Reducing and Aerobic Conditions
Authors: Freedman David L. , Swamy Meghna , Bell Nathan C. , Verce Mathew F. 
Published: August 2004

ABSTRACT

Pseudomonas aeruginosa
strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM
−1
under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO
3
−
per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N
2
per mol of NO
3
−
). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O
2
per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (
k
) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS
−1
day
−1
, the maximum specific growth rate (μ
max
) was 0.0506 day
−1
, and the Monod half-saturation coefficient (

K
s

) was 0.067 (±0.004) μM. Under aerobic conditions, the values for
k
, μ

max

, and

K
s

were 10.7 (±0.11) μmol of CM mg of TSS
−1
day
−1
, 0.145 day
−1
, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.
Expression of a Heterologous Manganese Superoxide Dismutase Gene in Intestinal Lactobacilli Provides Protection against Hydrogen Peroxide Toxicity
Authors: Bruno-Bárcena Jose M. , Andrus Jason M. , Libby Stephen L. , Klaenhammer Todd R. , Hassan Hosni M. 
Published: August 2004

ABSTRACT

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in
Lactobacillus
spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (
sodA
) was cloned from
Streptococcus thermophilus
AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the
sodA
gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an
Escherichia coli
strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in
Lactobacillus gasseri
NCK334,
Lactobacillus johnsonii
NCK89
, Lactobacillus acidophilus
NCK56, and
Lactobacillus reuteri
NCK932. Molecular and biochemical analyses confirmed the presence of the gene (
sodA
) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for
L. gasseri
,
L. johnsonii
,
L. acidophilus
, and
L. reuteri
, respectively. The expression of
S. thermophilus
MnSOD in
L. gasseri
and
L. acidophilus
provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O
2
·−
and preventing the redox cycling of iron. To our best knowledge, this is the first report of a
sodA
from
S. thermophilus
being expressed in other lactic acid bacteria.
New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Authors: Stevenson Bradley S. , Eichorst Stephanie A. , Wertz John T. , Schmidt Thomas M. , Breznak John A. 
Published: August 2004

ABSTRACT

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions
Acidobacteria
and
Verrucomicrobia
from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O
2
[vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO
2
(5% [vol/vol]). Significantly more
Acidobacteria
were found on isolation plates that had been incubated with 5% CO
2
. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.
Enhanced Mineralization of [U-
14
C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of
Trifolium pratense
Authors: Shaw Liz J. , Burns Richard G. 
Published: August 2004

ABSTRACT

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-
14
C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of
Lolium perenne
and
Trifolium pratense
. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (
tfdA
) were investigated. The rhizosphere effect on [
14
C]2,4-D mineralization (50 μg g
−1
) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (
P
< 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day
T. pratense
soil but not for 116-day
T. pratense
rhizosphere soil or for
L. perenne
rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g
−1
) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-
Proteobacteria tfdA
-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in
T. pratense
rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.
Characterization of Two
Bacillus thuringiensis
Genes Identified by In Vivo Screening of Virulence Factors
Authors: Fedhila Sinda , Guillemet Elisabeth , Nel Patricia , Lereclus Didier 
Published: August 2004

ABSTRACT

Bacillus thuringiensis
vegetative cells are known to be highly pathogenic when injected into the hemocoel of susceptible insect larvae. This pathogenicity is due to the capacity of
B. thuringiensis
to cause septicemia in the host. We screened a
B. thuringiensis
mini-Tn
10
insertion library for loss of virulence against
Bombyx mori
larvae on injection into the hemocoel. Three clones with attenuated virulence were isolated, corresponding to two different mini-Tn
10
insertions mapping to the
yqgB
/
yqfZ
locus. Single disruptions of the
yqgB
and
yqfZ
genes did not affect virulence against
B. mori
. In contrast, the inactivation of both genes simultaneously reproduced the effect of the mini-Tn
10
insertion and resulted in a significant delay to infection. The double Δ
yqgB
Δ
yqfZ
mutant was also nonmotile, and its growth was affected at 25°C. We analyzed
lacZ
transcriptional fusions and detected promoter activity upstream from
yqgB
at 25 and 37°C. Overall, our findings suggest that the
yqgB
and
yqfZ
genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host.
Biogeography, Evolution, and Diversity of Epibionts in Phototrophic Consortia
Authors: Glaeser Jens , Overmann Jörg 
Published: August 2004

ABSTRACTMotile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bacteria surround a flagellated colorless β-proteobacterium in the center. To date, seven different morphological types of such consortia have been described. In addition, two immotile associations involving green sulfur bacteria are known. By employing a culture-independent approach, different types of phototrophic consortia were mechanically isolated by micromanipulation from 14 freshwater environments, and partial 16S rRNA gene sequences of the green sulfur bacterial epibionts were determined. In the majority of the lakes investigated, different types of phototrophic consortia were found to co-occur. In all cases, phototrophic consortia with the same morphology from the same habitat contained only a single epibiont phylotype. However, morphologically indistinguishable phototrophic consortia collected from different lakes contained different epibionts. Overall, 19 different types of epibionts were detected in the present study. Whereas the epibionts within one geographic region were very similar (Dice coefficient, 0.582), only two types of epibionts were found to occur on both the European and North American continents (Dice coefficient, 0.190). None of the epibiont 16S rRNA gene sequences have been detected so far in free-living green sulfur bacteria, suggesting that the interaction between epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate interaction. Based on our phylogenetic analysis, the epibiont sequences are not monophyletic. Thus, the ability to form symbiotic associations either arose independently from different ancestors or was present in a common ancestor prior to the radiation of green sulfur bacteria and the transition to the free-living state in independent lineages. The present study thus demonstrates that there is great diversity and nonrandom geographical distribution of phototrophic consortia in the natural environment.
Ecological Significance of Microdiversity: Identical 16S rRNA Gene Sequences Can Be Found in Bacteria with Highly Divergent Genomes and Ecophysiologies
Authors: Jaspers Elke , Overmann Jörg 
Published: August 2004

ABSTRACT

A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of
Brevundimonas alba
were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the
B. alba
strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.
Biodegradation of Chlorpyrifos by
Enterobacter
Strain B-14 and Its Use in Bioremediation of Contaminated Soils
Authors: Singh Brajesh K. , Walker Allan , Morgan J. Alun W. , Wright Denis J. 
Published: August 2004

ABSTRACT

Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods. The isolates were indistinguishable, and one (strain B-14) was selected for further analysis. This strain showed greatest similarity to members of the order
Enterobacteriales
and was closest to members of the
Enterobacter asburiae
group. The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus. Studies with ring or uniformly labeled [
14
C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy. The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity. Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos. The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains. Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based. Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (
opd
). The addition of strain B-14 (10
6
cells g
−1
) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg
−1
resulted in a higher degradation rate than was observed in noninoculated soils. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.
Molecular Identification of the Catabolic Vinyl Chloride Reductase from
Dehalococcoides
sp. Strain VS and Its Environmental Distribution
Authors: Müller Jochen A. , Rosner Bettina M. , von Abendroth Gregory , Meshulam-Simon Galit , McCarty Perry L. , Spormann Alfred M. 
Published: August 2004

ABSTRACT

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring
Dehalococcoides
sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (
vcrA
) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The
vcrA
gene was found to be cotranscribed with
vcrB
, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and
vcrC
, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The
vcrAB
genes were subsequently found to be present and expressed in other cultures containing VC-respiring
Dehalococcoides
organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the
vcrA
gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.
Persistence ofMycobacterium paratuberculosisduring Manufacture and Ripening of Cheddar Cheese
Authors: Donaghy J. A. , Totton N. L. , Rowe M. T. 
Published: August 2004

ABSTRACTModel Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 104to 105CFU/ml) and low (101to 102CFU/ml) inocula of three differentMycobacterium paratuberculosisstrains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival ofM. paratuberculosiscells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed inM. paratuberculosisnumbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease inM. paratuberculosisCFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log10) ofM. paratuberculosiswere present in 1-day cheeses, the organism was culturable after the 27-week ripening period. TheDvalues calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination,M. paratuberculosiswas only culturable from 27-week-old cheese spiked with strain 806PSS.M. paratuberculosiswas recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initialM. paratuberculosisload was recovered in the culture-positive whey fractions at either the high or low initial inoculum.
Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate
Authors: North Nadia N. , Dollhopf Sherry L. , Petrie Lainie , Istok Jonathan D. , Balkwill David L. , Kostka Joel E. 
Published: August 2004

ABSTRACTPrevious studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the α, β, δ, and γ subdivisions of the classProteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing δ-Proteobacteriaincreased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed thatGeobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the δ-Proteobacteriasubdivision, includingAnaeromyxobacter dehalogenans-related andGeobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.
Biphenyl and Benzoate Metabolism in a Genomic Context: Outlining Genome-Wide Metabolic Networks in
Burkholderia xenovorans
LB400
Authors: Denef V. J. , Park J. , Tsoi T. V. , Rouillard J.-M. , Zhang H. , Wibbenmeyer J. A. , Verstraete W. , Gulari E. , Hashsham S. A. , Tiedje J. M. 
Published: August 2004

ABSTRACT

We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of
Burkholderia xenovorans
LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was up-regulated in benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C
1
metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.