bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal
Authors: McMahon Katherine D. , Dojka Michael A. , Pace Norman R. , Jenkins David , Keasling Jay D. 
Published: September 2002

ABSTRACT

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in
Rhodocyclus
-like β-
Proteobacteria
known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative
ppk
genes from a pure culture of
Rhodocyclus tenuis
and from organisms in the sludge. Four novel
ppk
homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with
R. tenuis
PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I
ppk
mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg
2+
, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.
Excretion of Human β-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant
Escherichia coli
Authors: Jeong Ki Jun , Lee Sang Yup 
Published: September 2002

ABSTRACT

Escherichia coli
BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by
E. coli
. The
ompF
gene of
E. coli
BL21(DE3) was first knocked out by using the
red
operon of bacteriophage λ to construct
E. coli
MBEL-BL101. For the excretion of human β-endorphin as a model protein, the β-endorphin gene was fused to the C terminus of the
E. coli ompF
gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-β-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-β-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield β-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of β-endorphin was obtained.
Culture-Independent Microbial Community Analysis Reveals that Inulin in the Diet Primarily Affects Previously Unknown Bacteria in the Mouse Cecum
Authors: Apajalahti Juha H. A. , Kettunen Hannele , Kettunen Anu , Holben William E. , Nurminen Päivi H. , Rautonen Nina , Mutanen Marja 
Published: September 2002

ABSTRACT
Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.
Bcmfs1
, a Novel Major Facilitator Superfamily Transporter from
Botrytis cinerea
, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides
Authors: Hayashi Keisuke , Schoonbeek Henk-jan , De Waard Maarten A. 
Published: September 2002

ABSTRACT

Bcmfs1
, a novel major facilitator superfamily gene from
Botrytis cinerea
, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant
Camptotheca acuminata
and the plant pathogenic fungus
Cercospora kikuchii,
respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of
Bcmfs1
and the ATP-binding cassette (ABC) transporter gene
BcatrD
was more sensitive to DMI fungicides than a single-replacement mutant of
BcatrD
, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with
Bcmfs1
expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of
B. cinerea
against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.
Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA
Authors: Nogales Balbina , Timmis Kenneth N. , Nedwell David B. , Osborn A. Mark 
Published: September 2002

ABSTRACT

The expression of five denitrification genes coding for two nitrate reductases (
narG
and
napA
), two nitrite reductases (
nirS
and
nirK
), and nitrous oxide reductase (
nosZ
) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only
nirS
and
nosZ
mRNAs were detected;
nirS
was detected directly as an RT-PCR amplification product, and
nosZ
was detected following Southern blot hybridization. This indicated that active expression of at least the
nirS
and
nosZ
genes was occurring in the sediments at the time of sampling. Amplified
nirS
RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified
nirS
gene sequences from isolates obtained from the same sediments. A high diversity of
nirS
sequences was observed. Most of the cloned
nirS
sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.
Flow Cytometry Analysis of Changes in the DNA Content of the Polychlorinated Biphenyl Degrader
Comamonas testosteroni
TK102: Effect of Metabolites on Cell-Cell Separation
Authors: Hiraoka Yoshinori , Yamada Tohru , Tone Keiko , Futaesaku Yutaka , Kimbara Kazuhide 
Published: September 2002

ABSTRACT

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium
Comamonas testosteroni
TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four
bph
genes,
bphA1
,
bphB
,
bphC
, and
bphD
, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the
bphB
gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.
Mimosine, the Allelochemical from the Leguminous Tree
Leucaena leucocephala
, Selectively Enhances Cell Proliferation in Dinoflagellates
Authors: Yeung Patrick K. K. , Wong Francis T. W. , Wong Joseph T. Y. 
Published: September 2002

ABSTRACT

Mimosine, the allelochemical from the leguminous tree
Leucaena leucocephala
, is toxic to most terrestrial animals and plants. We report here that while mimosine inhibits major phytoplankton groups, it enhances cell proliferation in dinoflagellates. On addition to coastal seawater samples, mimosine is able to confer a growth advantage to dinoflagellates. The use of mimosine will promote the isolation and culture of this group of phytoplankton.
Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light
Authors: Gerba Charles P. , Gramos Dawn M. , Nwachuku Nena 
Published: September 2002

ABSTRACT

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm
2
, respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.
Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons
Authors: Morasch Barbara , Richnow Hans H. , Schink Bernhard , Vieth Andrea , Meckenstock Rainer U. 
Published: September 2002

ABSTRACT

13
C/
12
C and D/H stable isotope fractionation during aerobic degradation was determined for
Pseudomonas putida
strain mt-2,
Pseudomonas putida
strain F1,
Ralstonia pickettii
strain PKO1, and
Pseudomonas putida
strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.
Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B
1
, B
2
, and B
3
by Fifteen Ex-Type Strains of
Fusarium
Species
Authors: Fotso Joseph , Leslie John F. , Smith J. Scott 
Published: September 2002

ABSTRACT

Fifteen
Fusarium
species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B
1
, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B
2
(360 μg/kg) was produced by two species, fumonisin B
3
was not detected in any of the 15 species examined, and
Fusarium bulbicola
produced none of the six mycotoxins that we analyzed.
Significance of Enhanced Morphological Detection of
Cryptosporidium
sp. Oocysts in Water Concentrates Determined by Using 4′,6′-Diamidino-2-Phenylindole and Immunofluorescence Microscopy
Authors: Smith H.V. , Campbell B. M. , Paton C. A. , Nichols R. A. B. 
Published: September 2002

ABSTRACT

Of 2,361 water concentrates analyzed for the presence of
Cryptosporidium
spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-
Cryptosporidium
sp. monoclonal antibody and the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.
Identical nuclei in coherent pion production at intermediate energies
Authors: Deutchman P A 
Published: September 2002

The formalism for the calculation of constructive, coherent production of pions through the collision of intermediate-energy nuclei now includes for the first time the exchange symmetry due to identical nuclei both in the initial two-body state and in the final three-body state. Of the eight terms that contribute to the pion-energy distributions, four of the amplitudes are equal in pairs, effectively leaving four amplitudes with direct terms in the initial state and direct and exchange terms in the final state. Of these remaining amplitudes, the final-state exchange terms are negligible as far as the calculation is concerned. This holds for pion-energy distributions over the incident energies from 100 MeV/nucleon to 2 GeV/nucleon for pions fixed in the forward direction (θπ = 0°) and the projectile and target, respectively, fixed in the fore and aft directions. This work is also generalized to include schematic solutions for the cases of identical nuclei in the initial state only, identical nuclei in the final state only, and the case of no identical nuclei at all. PACS Nos.: 24.10Cn, 24.30Cz, 25.70-z, 25.80-e
Potential curves and rovibrational energies for electronic states of the molecular ion KCs+
Authors: Korek M , Allouche A R , Abdul Al S N 
Published: September 2002

The KCs+ molecular ion potential curves are investigated over a wide range of internuclear distance for electronic states described in a 2Λ(+) representation (neglecting the spin-orbit effect) as well as in an Ω(+) representation (including the spin-orbit effect). This calculation has been done in a one active electron approach by using an ab initio method based on non-empirical pseudopotentials with core-valence effect taken into account through parameterized l-dependent polarization potentials. Gaussian basis sets have been used for both atoms, and spin-orbit effects have been taken into account through a semiempirical spin-orbit pseudopotential. The canonical functions approach is used to do a rovibrational study by calculating the eigenvalues Ev, the rotational constant Bv, the centrifugal distortion constants Dv (up to 106 vibrational levels), and the spectroscopic constants are deduced for six bound states. The permanent and transition dipole moment functions have been derived for transitions between the bound states 2 Σ+ and 2Π as well as the oscillator strengths for the transitions v = 0, 10, 20, and Δ v = 0, 1, 2, ..., 6. To the best of our knowledge neither theoretical nor experimental data are available in the literature for the molecular ion KCs+. Extensive tables of Ev, Bv, Dv, and the energy values versus internuclear distance are displayed at the following address: http://hplasim2.univ-lyon1.fr/allouche/kcsplus.html. PACS Nos.: 33.15Dj, 33.20wr
The intensity dependence of the free-free transition in a bichromatic laser field
Authors: Zhang Sh T , Chen J , Shu-min L 
Published: September 2002

In this paper, we investigate the intensity dependence of a freefree transition in the presence of a bichromatic laser field. Based on the first Born approximation, we calculate the differential cross section and total differential cross section versus the field intensity. This dependence relates to the momentum of the incident electron, the ratio of the two frequencies, and the phase difference between the two components of the bichromatic laser field. While the two intensity components are comparable, this dependence is the most striking. The results also show that the intensity dependence relates to the geometrical configurations between the direction of polarization of the laser fields and the incident electron. PACS Nos.: 34.80Bm, 32.80Qk
Isotopic quantum effects in the structure of liquid ethanol
Authors: Tomberli B , Benmore C J , Neuefeind J , Egelstaff P A 
Published: September 2002

High-energy electromagnetic-radiation-scattering techniques have been used to measure the structural differences between five isotopic samples of ethanol (CH3CH2OH, CD3CD2OD, CD3CH2OH, CH3CH2OD, and CH3CD2OH) at room temperature and ambient pressure. The differences in the X-ray structure factors between several pairs of isotopes, ΔSX(Q), are shown to have maximum amplitudes that are on the order of a few percent compared to the total structure factor for CH3CH2OH. Our uncertainties are an order of magnitude smaller than those of early gamma-ray measurements on methanol (C.J. Benmore and P.A. Egelstaff. J. Phys. Condens. Matter, 8, 9429 (1996)). These studies have shown that isotopic structural differences in room-temperature ethanol vary as a function of substitution site and are in qualitative agreement with similar differences found in liquid methanol. PACS Nos.: 61.20-p, 61.25E, 61.10-E
Reasoning about probabilistic parallel programs
Authors: Rao Josyula R. 
Published: October 2002

The use of randomization in the design and analysis of algorithms promises simple and efficient algorithms to difficult problems, some of which may not have a deterministic solution. This gain in simplicity, efficiency, and solvability results in a trade-off of the traditional notion of absolute correctness of algorithms for a more quantitative notion: correctness with a probability between 0 and 1. The addition of the notion of parallelism to the already unintuitive idea of randomization makes reasoning about probabilistic parallel programs all the more tortuous and difficult.
In this paper we address the problem of specifying and deriving properties of probabilistic parallel programs that either hold deterministically or with probability 1. We present a proof methodology based on existing proof systems for probabilistic algorithms, the theory of the predicate transformer, and the theory of UNITY. Although the proofs of probabilistic programs are slippery at best, we show that such programs can be derived with the same rigor and elegance that we have seen in the derivation of sequential and parallel programs. By applying this methodology to derive probabilistic programs, we hope to develop tools and techniques that would make randomization a useful paradigm in algorithm design.
On the Physical Nature of Time
Authors: Harrison Edward 
Published: October 2002

AbstractIt is argued that certain characteristics of time, normally ascribed to the observer, are actually properties of the natural world, and hence the natural world (physical reality) is more complex than normally supposed. Everything extends in time. Events stretch away into the recent past as recalled in our memories and recorded in daily newspapers, and into the distant past as recounted by historians, paleontologists, and geologists. Events also stretch away into the near future as anticipated in our plans and foretold by weather forecasters, and into the distant future as predicted by climatologists, geologists, and astronomers. Everything also occurs in time. Events happen either quickly or slowly. The glacier takes centuries to reach the sea and the lightning stroke zig-zags to earth faster than the eye can see. Events occupy time and also occur in time; and their occupation and occurrence in time are not the same thing. The extension of time and the transience of time are different aspects of time that in thought, language, and science remain unreconciled. Here I discuss how these two fundamental aspects of time can be reconciled and included in the physical world description. I take the view that both are equally important properties of the natural world and speculate on how the physical world picture would change by their joint incorporation. A theory of "conjugate time" is tentatively proposed.