bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Real-Time PCR Assay for Detection and Quantitation of
Mycobacterium avium
subsp.
paratuberculosis
DNA in Artificially Contaminated Milk
Authors: O'Mahony Jim , Hill Colin
Published: August 2004

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS
900
which allowed rapid detection of
Mycobacterium avium
subsp.
paratuberculosis
DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg
2+
concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 10
6
to 3 × 10
1
copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating
M. avium
subsp.
paratuberculosis
DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify
M. avium
subsp.
paratuberculosis
in spiked milk from heavily and mildly contaminated samples.
Novel Haloperoxidase from the Agaric Basidiomycete
Agrocybe aegerita
Oxidizes Aryl Alcohols and Aldehydes
Authors: Ullrich René , Nüske Jörg , Scheibner Katrin , Spantzel Jörg , Hofrichter Martin
Published: August 2004

Agrocybe aegerita
, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS).
A. aegerita
peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H
2
O
2
(2 mM) for optimum activity. The apparent
K

m

values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H
2
O
2
were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete
Caldariomyces fumago
. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.
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

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

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

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

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

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

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

Motile 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

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

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

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

Model 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

Previous 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

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.
Explorative Multivariate Analyses of 16S rRNA Gene Data from Microbial Communities in Modified-Atmosphere-Packed Salmon and Coalfish
Authors: Rudi Knut , Maugesten Tove , Hannevik Sigrun E. , Nissen Hilde
Published: August 2004

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with
Photobacterium phosphoreum
was observed.
Brochothrix
spp. and
Carnobacterium
spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve
Carnobacterium
isolates were identified as either
Carnobacterium piscicola
(five isolates) or
Carnobacterium divergens
(seven isolates), while the eight
Brochothrix
isolates were identified as
Brochothrix thermosphacta
by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that
C. piscicola
had distinct substrate requirements, while the requirements of
B. thermosphacta
and
C. piscicola
were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.
Strategy for Cloning Large Gene Assemblages as Illustrated Using the Phenylacetate and Polyhydroxyalkanoate Gene Clusters
Authors: García Belén , Olivera Elías R. , Sandoval Ángel , Arias-Barrau Elsa , Arias Sagrario , Naharro Germán , Luengo José M.
Published: August 2004

We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in
Pseudomonas putida
U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs).
Nisin-Controlled Production of Pediocin PA-1 and Colicin V in Nisin- and Non-Nisin-Producing
Lactococcus lactis
Strains
Authors: Horn Nikki , Fernández Antonio , Dodd Helen M. , Gasson Michael J. , Rodríguez Juan M.
Published: August 2004

The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible
nisA
promoter and the lactococcin A-dedicated secretion genes (
lcnCD
) into
Lactococcus lactis
strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.
Nisin-Producing
Lactococcus lactis
Strains Isolated from Human Milk
Authors: Beasley Shea S. , Saris Per E. J.
Published: August 2004

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing
Lactococcus lactis
bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.
Molecular basis of transcriptional silencing in budding yeast
Authors: Chen Lingyi , Widom Jonathan
Published: August 2004

Transcriptional silencing is a phenomenon in which the transcription of genes by RNA polymerase II or III is repressed, dependent on the chromosomal location of a gene. Transcriptional silencing normally occurs in highly condensed heterochromatin regions of the genome, suggesting that heterochromatin might repress transcription by restricting the ability of sequence-specific gene activator proteins to access their DNA target sites. However, recent studies show that heterochromatin structure is inherently dynamic, and that sequence-specific regulatory proteins are able to bind to their target sites in heterochromatin. The molecular basis of transcriptional silencing is plainly more complicated than simple steric exclusion. New ideas and experiments are needed.Key words: transcriptional silencing, heterochromatin, accessibility.
Gene regulation by Sp1 and Sp3
Authors: Li Lin , He Shihua , Sun Jian-Min , Davie James R
Published: August 2004

The Sp family of transcription factors is united by a particular combination of three conserved Cys2His2 zinc fingers that form the sequence-specific DNA-binding domain. Within the Sp family of transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. They can bind and act through GC boxes to regulate gene expression of multiple target genes. Although Sp1 and Sp3 have similar structures and high homology in their DNA binding domains, in vitro and in vivo studies reveal that these transcription factors have strikingly different functions. Sp1 and Sp3 are able to enhance or repress promoter activity. Regulation of the transcriptional activity of Sp1 and Sp3 occurs largely at the post-translational level. In this review, we focus on the roles of Sp1 and Sp3 in the regulation of gene expression.Key words: Sp1, Sp3, gene regulation, sub-cellular localization.
The FACT chromatin modulator: genetic and structure/function relationships
Authors: Singer Richard A , Johnston Gerald C
Published: August 2004

The chromatin configuration of DNA inhibits access by enzymes such as RNA polymerase II. This inhibition is alleviated by FACT, a conserved transcription elongation factor that has been found to reconfigure nucleosomes to allow transit along the DNA by RNA polymerase II, thus facilitating transcription. FACT also reorganizes nucleosomes after the passage of RNA polymerase II, as indicated by the effects of certain FACT mutations. The larger of the two subunits of FACT is Spt16/Cdc68, while the smaller is termed SSRP1 (vertebrates) or Pob3 (budding yeast). The HMG-box domain at the C terminus of SSRP1 is absent from Pob3; the function of this domain for yeast FACT is supplied by the small HMG-box protein Nhp6. In yeast, this "detachable" HMG domain is a general chromatin component, unlike FACT, which is found only in transcribed regions and associated with RNA polymerase II. The several domains of the larger FACT subunit are also likely to have different functions. Genetic studies suggest that FACT mediates nucleosome reorganization along several pathways, and reinforce the notion that protein unfolding and (or) refolding is involved in FACT activity for transcription.Key words: nucleosomes, transcription, FACT, yeast, genetics.
S100 proteins and their influence on pro-survival pathways in cancer
Authors: Emberley Ethan D , Murphy Leigh C , Watson Peter H
Published: August 2004

The S100 gene family is composed of at least 20 members that share a common structure defined in part by the Ca2+binding EF-hand motif. These genes which are expressed in a discriminate fashion in specific cells and tissues, have been described to have either an intracellular or extracellular function, or both. S100 proteins are implicated in the immune response, differentiation, cytoskeleton dynamics, enzyme activity, Ca2+homeostasis and growth. A potential role for S100 proteins in neoplasia stems from these activities and from the observation that several S100 proteins have altered levels of expression in different stages and types of cancer. While the precise role and importance of S100 proteins in the development and promotion of cancer is poorly understood, it appears that the binding of Ca2+is essential for exposing amino acid residues that are important in forming protein-protein interactions with effector molecules. The identity of some of these effector molecules has also now begun to emerge, and with this the elucidation of the signaling pathways that are modulated by these proteins. Some of these interactions are consistent with the diverse functions noted above. Others suggest that, many S100s may also promote cancer progression through specific roles in cell survival and apoptosis pathways. This review summarizes these findings and their implications.
Assessment of intra-species diversity among strains ofAcinetobacterbaumanniiisolated from sites contaminated with petroleum hydrocarbons
Authors: Sarma Priyangshu Manab , Bhattacharya Dhruva , Krishnan S , Lal Banwari
Published: August 2004

A total of 96 crude oil - degrading bacterial strains were isolated from 5 geographically diverse sites in India that were contaminated with different types of petroleum hydrocarbons. The strains were identified by sequencing the genes that encode for 16S rRNA. Out of the 96 isolates, 25 strains were identified as Acinetobacter baumannii and selected for the study. All of the selected strains could degrade the total petroleum hydrocarbon fractions of crude oil. These 25 strains were biochemically profiled and grouped into 8 phenovars on the basis of multivariate analysis of their substrate utilization profiles. PCR-based DNA fingerprinting was performed using intergenic repetitive DNA sequences, which divided the selected 25 strains into 7 specific genomic clusters. tRNA intergenic spacer length polymorphism was performed to determine the intra-species relatedness among these 25 strains. It delineated the strains into 8 genomic groups. The present study detected specific variants among the A. baumannii strains with differential degradation capacities for different fractions of crude oil. This could play a significant role in in situ bioremediation. The study also revealed the impact of environmental factors that cause intra-species diversity within the selected strains of A. baumannii.Key words: genotypic diversity, phenotypic diversity, crude oil, Acinetobacter baumannii.
Evaluation of the in vitro and in vivo dimorphism ofSporothrix schenckii,Blastomyces dermatitidis, andParacoccidioides brasiliensisisolates after preservation in mineral oil
Authors: Lima Renata Ferretti de , Santos Brito Marcelly Maria dos , Schäffer Guido Manoel Vidal , Lima Osana Cunha de , Borba Cintia de Moraes
Published: August 2004

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 °C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 °C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 °C and their micromorphology showed thin filaments with single hyaline conidia. At 36 °C the colonies did not differ from those grown at 25 °C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 °C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.Key words: pathogenic fungi, in vitro storage, in vivo passage, morphogenesis.
Pyrimidine nucleotide synthesis inPseudomonascitronellolis
Authors: West Thomas P
Published: August 2004

Pyrimidine biosynthesis was active in Pseudomonas citronellolis ATCC 13674 and appeared to be regulated by pyrimidines. When wild-type cells were grown on succinate in the presence of uracil, the de novo enzyme activities were depressed while only four enzyme activities were depressed in the glucose-grown cells. On either carbon source, orotic acid-grown cells had diminished aspartate transcarbamoylase, dihydroorotase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells resulted in de novo enzyme activities, except for transcarbamoyolase activity, that were elevated by more than 5-fold compared to their activities in uracil-grown cells. Since pyrimidine limitation of succinate-grown mutant cells produced less enzyme derepression, catabolite repression appeared to be a factor. At the level of enzyme activity, aspartate transcarbamoylase activity in P. citronellolis was strongly inhibited by all effectors tested. Compared to the regulation of pyrimidine biosynthesis in taxonomically-related species, pyrimidine biosynthesis in P. citronellolis appeared more highly regulated.Key words: pyrimidine biosynthesis, regulation, Pseudomonas citronellolis, auxotroph, aspartate transcarbamoylase, inhibition.
Note types and coding in parid vocalizations. I: The chick-a-dee call of the black-capped chickadee (Poecile atricapillus)
Authors: Charrier Isabelle , Bloomfield Laurie L , Sturdy Christopher B
Published: August 2004

The chick-a-dee call of the black-capped chickadee, Poecile atricapillus (L., 1766), consists of four note types and is used in a wide variety of contexts including mild alarm, contact between mates, and for mobilizing members of winter flocks. Because note-type composition varies with context and because birds need to identify flock mates and individuals by their calls, it is important that birds are able to discriminate between note types and birds. Moreover, previous experiments have shown that black-capped chickadees are able to discriminate their four note types, but the acoustical basis of this process is still unknown. Here, we present the results of a bioacoustic analysis that suggests which acoustic features may be controlling the birds' perception of note types and of individual identity. Several acoustic features show high note type and individual specificity, but frequency and frequency modulation cues (in particular, those of the initial part of the note) appear more likely to be used in these processes. However, only future experiments testing the bird's perceptual abilities will determine which acoustic cues in particular are used in the discrimination of note types and in individual recognition.
Butterfly assemblages along a human disturbance gradient in Ontario, Canada
Authors: Hogsden Kristy L , Hutchinson T C
Published: August 2004

This study relates patterns of butterfly abundance and species richness to position along an urban disturbance gradient in southeastern Ontario, Canada. Observed assemblages along the gradient (N = 15) included butterflies from the Papilionidae, Pieridae, Lycaenidae, Nymphalidae, and Hesperiidae families. Of the total 26 observed species, 15 were noticeably absent from the disturbed sites. Butterfly assemblages had equal or higher number of individuals and species richness at moderately disturbed sites compared with the least disturbed sites. In relation to distribution patterns along the gradient, 28% of butterfly species were classified as disturbance adaptable and 58% as disturbance avoiders. These classifications were correlated with host-plant use and voltinism. Canonical correspondence analysis of local-scale data strongly associated disturbance avoiders with a specific environmental variable (e.g., Everes comyntas (Godart, 1824) with grasslands), whereas disturbance-adaptable species were weakly associated with any variable. One-time disturbances (i.e., mowing) during the survey resulted in pronounced changes in butterfly abundance and species composition at two sites, reducing species richness and total abundance by up to 80%. Species were patchily distributed along the gradient, suggesting that they respond differentially to disturbance in the landscape.
Proximate mechanisms that contribute to female-biased sexual size dimorphism in an anguid lizard
Authors: Rutherford Pamela L
Published: August 2004

Various proximate mechanisms have been proposed to explain sexual size dimorphism (SSD) in vertebrates. Identifying the proximate causation of SSD allows insight into the ultimate reasons why SSD exists. I explored whether differential growth rates and (or) mortality explain SSD in the lizard Elgaria coerulea (Baird and Girard, 1852). I estimated growth parameters for males and females using the logistic-by-weight growth curves and determined survivorship using two complimentary methods: standard life-table calculations and capturerecapture methods. The former calculated age-specific survivorship, whereas the latter tested for differences in survivorship between males and females while considering differences in their recapture rates. I considered age-specific SSD as further evidence of SSD independent of differential mortality. Differences in growth asymptote, not intrinsic growth rate, contribute to SSD in this population. SSD is not due to differential mortality, as there is no difference in survivorship of males and females over 3 years of age. In addition, there is age-specific SSD with females larger than males for individuals 4 years of age and greater. The female-biased SSD may be a result of selection for large body size, although further studies are necessary to identify the ultimate cause of SSD in this species.
IMPLICATIONS OF A LIGHT RADION ON β(λ) AND β(gt) AND A LOWER BOUND ON RADION VEV
Authors: DAS PRASANTA KUMAR , MAHANTA UMA
Published: August 2004

In this paper we determine how the beta funtions of Higgs self-coupling λ and top Yukawa coupling gt to one-loop order are modified by a light stabilized radion in the Randall–Sundrum model. We then use these beta functions to evolve λ from the cutoff <ϕ> down to μ0=115 GeV starting from different boundary values λ(<ϕ>). We find that although for large <ϕ> the value of λ(μ0) does depend on λ(<ϕ>), for small enough <ϕ> the curves merge together. We then determine a lower bound on <ϕ> by demanding that [Formula: see text] in order to be consistent with the LEPII lower bound on the Higgs mass. The lower bound on <ϕ> obtained by us is insensitive to the value of λ(<ϕ>).
RECONCILING THE POSITIVE DAMA ANNUAL MODULATION SIGNAL WITH THE NEGATIVE RESULTS OF THE CDMS II EXPERIMENT
Authors: FOOT R.
Published: August 2004

We examine the recent CDMS II results in the context of the mirror matter interpretation of the DAMA/NaI experiment. We find that the favoured mirror matter interpretation of the DAMA/NaI experiment — a He ′/ H ′-dominated halo with a small O ′ component is fully consistent with the null results reported by CDMS II. While the CDMS II experiment is quite sensitive to a heavy Fe ′ component, and may yet find a positive result, a more decisive test of mirror matter-type dark matter would require a lower threshold experiment using light target elements.
THE USES OF CONNES AND KREIMER'S ALGEBRAIC FORMULATION OF RENORMALIZATION THEORY
Authors: FIGUEROA HÉCTOR , GRACIA-BONDÍA JOSÉ M.
Published: August 2004

We show how, modulo the distinction between the antipode and the "twisted" or "renormalized" antipode, Connes and Kreimer's algebraic paradigm trivializes the proofs of equivalence of the (corrected) Dyson–Salam, Bogoliubov–Parasiuk–Hepp and Zimmermann procedures for renormalizing Feynman amplitudes. We discuss the outlook for a parallel simplification of computations in quantum field theory, stemming from the same algebraic approach.
SEMICLASSICAL STRING SOLUTIONS ON DEFORMED NS5-BRANE BACKGROUNDS AND NEW PLANE WAVE
Authors: ALISHAHIHA MOHSEN , MOSAFFA AMIR E.
Published: August 2004

We study different Penrose limits of supergravity solution of NS5-brane in the presence of RR field. Although in the case of NS5-brane we get a four-dimensional plane wave, in the case with RR field we will get two different plane waves; a four-dimensional and a three-dimensional one. These plane wave solutions are the backgrounds that a particular string solution feels at one-loop approximation. Using the one-loop correction one can identify a particular subsector of LST/deformed LST which is dual to type II string theories on these plane waves.