bluuebunny/crossref_metadata_2025 huggingface dataset:
A Viewpoint‐Dependent Approach to Ray Trace Free‐Form Surfaces
Authors: Formella A. , Müller K.
Published: July 2004

For convincing realistic scenes objects with free‐form surfaces are essential. Especially for photorealistic rendering pure polygonal models are often not sufficient. We present a new kind of algorithm to render free‐form surfaces in a rendering system based on ray tracing. We describe a triangular patch as usual by its three points and normal vectors, but base the intersection calculation as well on the viewpoint of the camera (or, in general, on the ray itself). Hence, the shape of the object depends to some extent on the sampling rays. However, the resulting differences of, for instance, the shape of the silhouette to the shape of the corresponding shadow is usually not perceived by the observer of the rendered image. Because we perform a direct computation without a tessellation process, the resulting surface, its shadows, and its reflections appear smooth independent of the distance to the camera. Furthermore, the memory consumption depends only linearly on the number of input triangles. Special features like creases, T‐vertices, and darts are also well supported. The computed uv‐coordinates provide a direct means for texture mapping whose visual appearance improves significantly compared to triangle meshes of the same resolution.
Visualizing Objects with Mirrors
Authors: Hurtado F. , Noy M. , Robert J.‐M. , Sacristán V. , Skiena S.
Published: July 2004

We present an automatic camera and mirrors placement method for visualizing 3D scenes such that complete and nonredundant visibility is guaranteed, as well as highest image resolution. We provide algorithms and geometries for optimal object‐independent mirrors and viewpoint placement for all objects in a specific class. We also briefly consider object‐dependent algorithmic problems, where mirrors and viewpoints are positioned as a function of the object.
Image Interpolation by Pixel‐Level Data‐Dependent Triangulation
Authors: Su Dan , Willis Philip
Published: July 2004

We present a novel image interpolation algorithm. The algorithm can be used in arbitrary resolution enhancement, arbitrary rotation and other applications of still images in continuous space. High‐resolution images are interpolated from the pixel‐level data‐dependent triangulation of lower‐resolution images. It is simpler than other methods and is adaptable to a variety of image manipulations. Experimental results show that the new “mesh image” algorithm is as fast as the bilinear interpolation method. We assess the interpolated images' quality visually and also by the MSE measure which shows our method generates results comparable in quality to slower established methods. We also implement our method in graphics card hardware using OpenGL which leads to real‐time high‐quality image reconstruction. These features give it the potential to be used in gaming and image‐processing applications.
Assessing the role of architecture in conspicuous consumption in the middle minoan I–II periods
Authors: Schoep Ilse
Published: July 2004

Summary.  This paper uses Middle Minoan architecture to explore the degree to which the conceptualization and reconstruction of the First Palaces on Crete have been unduly influenced by the model of the Minoan palace as the centralized political, economic and religious authority. It is generally assumed that this model, first formulated on the basis of the LM II–III palace at Knossos, also serves to explain the First Palaces despite the fact that relatively little attention has hitherto been paid to their external and internal characteristics. Detailed reassessment of the available data strongly suggests that the First Palaces differed from their Late Bronze Age counterparts in several important ways. Particularly striking is the absence of so‐called ‘palatial’ architectural features (e.g. ashlar masonry, Minoan Hall, Lustral Basin, etc.), which hitherto had been thought to form an integral part of the First Palaces. Rather, the earliest evidence for these architectural features seems to be found in elite residences in settlement contexts (e.g. Malia). This observation urges a reassessment not only of the term ‘palatial’ architecture but also of the nature and location of power in Middle Bronze Age Crete and the role played by architecture as a medium of elite conspicuous consumption.
Gabbroic clay sources in Cornwall: a petrographic study of prehistoric pottery and clay samples
Authors: Harrad Lucy
Published: July 2004

Summary.  This analysis of prehistoric pottery and clay samples from Cornwall demonstrates that the clay used to make Cornish gabbroic pottery in prehistory originated around the gabbro rock outcrop in a small area of the Lizard peninsula. The research uses petrographic and chemical analysis to subdivide the prehistoric pottery into six groups. Owing to the unusual geology of the Lizard these groups can be attributed to specific locations. The most abundant pottery fabric, Typical Gabbroic, was made using coarse clay which is mainly found in a 1 km2 area near Zoar. A finer version of this clay, found higher in the soil profile or slightly transported and redeposited, was used to make Fine Gabbroic pottery and an even finer variant called FNS (Fine Non‐Sandy) Gabbroic. We identify for the first time here a Loessic/Gabbroic pottery fabric which can be matched exactly to clay found at Lowland Point. Serpentinitic/Gabbroic pottery was made using clay from the gabbro/serpentinite border zone. Pottery made from the Granitic/Gabbroic fabric did not match any clay from the Lizard, showing that gabbroic clay was sometimes removed and made into pottery elsewhere in Cornwall. The main clay source near Zoar was used for clay extraction throughout the Bronze Age and Iron Age for pottery which was traded all over Cornwall. Other gabbroic clay sources produced pottery only during certain periods and exclusively supplied particular settlements, such as the Loessic/Gabbroic fabric which was found only at Gear and Caer Vallack. The results suggest that pottery was produced by several small‐scale cottage industries, which may have operated on a seasonal, part‐time basis and probably formed only part of a wide range of activities located around the Lizard area.
Chaining and shaming: images of defeat, from Llyn Cerrig Bach to Sarmitzegetusa
Authors: Aldhouse‐Green Miranda
Published: July 2004

Summary.  This paper seeks to address issues relating to physical restraint, disempowerment and the symbolisms of humiliation, particularly within the contexts of warfare and conquest in Iron Age and Roman Britain and Europe, although the enormous topic of ancient slavery per se is beyond the scope of the present study. The enquiry is based upon evidence from iconography, human remains, the physical paraphernalia of restraint and, for the latest Iron Age onwards, the testimony of such ancient authors as Tacitus. The subject is approached from the perspective not only of empirical material but also from that of social and symbolic theory. Furthermore, in seeking to interpret the relevant material culture, I have deemed it useful to draw broad analogies with other periods and contexts, including the iconography of the ancient Nile Valley and aspects of the nineteenth century French penal system. The material under discussion is scrutinized within contexts of ritual practice and performance, together with presentations of degradation and attitudes to foreignness, subjugation, supremacy and inferiority. Accordingly, questions are raised concerning the symbolic meaning of gang‐chains and chain‐gangs, grammars of victory‐imagery (including somatic position, dimorphism and hair‐grasping) and issues associated with shaming the body, whether by means of binding and shackling, violence, head‐shaving or sensory deprivation.
Thioesterase II of
Escherichia coli
Plays an Important Role in 3-Hydroxydecanoic Acid Production
Authors: Zheng Zhong , Gong Qiang , Liu Tao , Deng Ying , Chen Jin-Chun , Chen Guo-Qiang
Published: July 2004

3-Hydroxydecanoic acid (3HD) was produced in
Escherichia coli
by mobilizing (
R
)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the
phaG
gene). By employing an isogenic
tesB
(encoding thioesterase II)-negative knockout
E. coli
strain, CH01, it was found that the expressions of
tesB
and
phaG
can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant
E. coli
harboring
phaG
and
tesB
. This study supports the hypothesis that the physiological role of thioesterase II in
E. coli
is to prevent the abnormal accumulation of intracellular acyl-coenzyme A.
Thermus thermophilus
as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in
Escherichia coli
Authors: Hidalgo Aurelio , Betancor Lorena , Moreno Renata , Zafra Olga , Cava Felipe , Fernández-Lafuente Roberto , Guisán José M. , Berenguer José
Published: July 2004

Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H
2
O
2
-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from
Thermus thermophilus
HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in
Escherichia coli
, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a
Thermus-
specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in
T. thermophilus
under the control of the
Pnar
promoter. This promoter belongs to a respiratory nitrate reductase from of
T. thermophilus
HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in
T. thermophilus
HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain
T. thermophilus
HB27::
nar
was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of
T. thermophilus
as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.
Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids
Authors: Helinck Sandra , Le Bars Dominique , Moreau Daniel , Yvon Mireille
Published: July 2004

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of
Lactobacillus delbrueckii
subsp.
lactis
,
Lactobacillus helveticus,
and
Streptococcus thermophilus
to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only
S. thermophilus
exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only
S. thermophilus
was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than
S. thermophilus,
which mainly produced α-keto acids and a small amount of hydroxy acids and acids.
L. helveticus
mainly produced acids from phenylalanine and leucine, while
L. delbrueckii
subsp.
lactis
produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by
L. delbrueckii
subsp.
lactis
mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in
L. helveticus
and
S. thermophilus
is an α-keto acid dehydrogenase that produces acyl coenzymes A.
Accurate Estimation of Viral Abundance by Epifluorescence Microscopy
Authors: Wen Kevin , Ortmann Alice C. , Suttle Curtis A.
Published: July 2004

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% ± 5.2% (
n
= 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h
−1
, respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%;
n
= 6). Aldehyde fixation of samples followed by storage at 4°C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h
−1
during the first hour to 0.01 h
−1
between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at −20°C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at −86°C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.
The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in
Botrytis cinerea
Authors: Siewers Verena , Smedsgaard Jørn , Tudzynski Paul
Published: July 2004

The phytopathogenic ascomycete
Botrytis cinerea
is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of
B. cinerea
. Targeted inactivation of the gene
bccpr1
encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these,
bcaba1
, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus,
bcaba1
represents the first identified fungal ABA biosynthetic gene.
Disruption of the Subtilase Gene,
albin1
, in
Ophiostoma piliferum
Authors: Hoffman Brad , Breuil Colette
Published: July 2004

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet.
41:
168-175, 2002). In this work we examined the role of the subtilase Albin1 from
Ophiostoma piliferum
. Reamplification of cDNA ends PCR was used to obtain the
albin1
gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an
Agrobacterium tumefaciens
-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus,
albin1
plays an important role in
O. piliferum
's ability to acquire nitrogen from wood proteins.
Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies
Authors: Caballero Santiago , Abad F. Xavier , Loisy Fabienne , Le Guyader Françoise S. , Cohen Jean , Pintó Rosa M. , Bosch Albert
Published: July 2004

Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.
Analysis and Validation of a Predictive Model for Growth and Death ofAeromonas hydrophilaunder Modified Atmospheres at Refrigeration Temperatures
Authors: Pin Carmen , Velasco de Diego Raquel , George Susan , García de Fernando Gonzalo D. , Baranyi József
Published: July 2004

Specific growth and death rates ofAeromonas hydrophilawere measured in laboratory media under various combinations of temperature, pH, and percent CO2and O2in the atmosphere. Predictive models were developed from the data and validated by means of observations obtained from (i) seafood experiments set up for this purpose and (ii) the ComBase database (http://www.combase.cc;http://wyndmoor.arserrc.gov/combase/).Two main reasons were identified for the differences between the predicted and observed growth in food: they were the variability of the growth rates in food and the bias of the model predictions when applied to food environments. A statistical method is presented to quantitatively analyze these differences. The method was also used to extend the interpolation region of the model. In this extension, the concept of generalizedZvalues (C. Pin, G. García de Fernando, J. A. Ordóñez, and J. Baranyi, Food Microbiol.18:539-545, 2001) played an important role. The extension depended partly on the density of the model-generating observations and partly on the accuracy of extrapolated predictions close to the boundary of the interpolation region. The boundary of the growth region of the organism was also estimated by means of experimental results for growth and death rates.
Properties of a Novel Thermostable Glucoamylase from the Hyperthermophilic Archaeon
Sulfolobus solfataricus
in Relation to Starch Processing
Authors: Kim Mi-Sun , Park Jong-Tae , Kim Young-Wan , Lee Hee-Seob , Nyawira Rose , Shin Hyoun-Seung , Park Cheon-Seok , Yoo Sang-Ho , Kim Yong-Ro , Moon Tae-Wha , Park Kwan-Hwa
Published: July 2004

A gene (
ssg
) encoding a putative glucoamylase in a hyperthermophilic archaeon,
Sulfolobus solfataricus
, was cloned and expressed in
Escherichia coli
, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90°C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated β-
d
-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from
S. solfataricus
produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from
S. solfataricus
has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.
A New Black
Aspergillus
Species,
A. vadensis
, Is a Promising Host for Homologous and Heterologous Protein Production
Authors: de Vries Ronald P. , Burgers Kim , van de Vondervoort Peter J. I. , Frisvad Jens C. , Samson Robert A. , Visser Jaap
Published: July 2004

A new species of the group of black aspergilli,
Aspergillus vadensis
, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of
A. tubingensis
feruloyl esterase A (FaeA) was compared upon production in wild-type
A. vadensis
,
A. tubingensis
, and an
A. niger
strain in which the three main protease-encoding genes were disrupted. The production of FaeA in
A. vadensis
resulted in larger amounts of intact protein than production in
A. tubingensis
and was similar to production in an
A. niger
protease disruptant, confirming in vivo the low proteolytic activity of
A. vadensis
. The protoplast formation and transformation efficiencies of
A. vadensis
were much higher than those of
A. niger
. These characteristics make
A. vadensis
a very promising candidate for homologous, and possibly heterologous, protein production.
Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of
Spiroplasma citri
by the Leafhopper
Circulifer haematoceps
Authors: Boutareaud A. , Danet J. L. , Garnier M. , Saillard C.
Published: July 2004

Spiroplasma citri
is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of
S. citri
affected in their transmission must be available. For this purpose, transposon (Tn
4001
) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector
Circulifer haematoceps
to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as
S. citri
wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn
4001
was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the
Sc76
gene restored the wild phenotype, showing that Sc76 protein is involved in
S. citri
transmission by the leafhopper vector
C. haematoceps
.
Abundance of Dioxygenase Genes Similar to
Ralstonia
sp. Strain U2
nagAc
Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments
Authors: Dionisi Hebe M. , Chewning Christopher S. , Morgan Katherine H. , Menn Fu-Min , Easter James P. , Sayler Gary S.
Published: July 2004

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the
Ralstonia
sp. strain U2
nagAc
gene (
nagAc
-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of
nagAc
-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for
nagAc
-like sequences indicated the presence of (4.1 ± 0.7) × 10
3
to (2.9 ± 0.3) × 10
5
copies of
nagAc
-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 10
5
to (5.4 ± 0.4) × 10
7
copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and
nagAc
-like gene copies per microgram of DNA (
r
= 0.89) and per gram of dry weight sediment (
r
= 0.77). These results provide evidence of the ecological significance of organisms carrying
nagAc
-like genes in the biodegradation of naphthalene.
Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community
Authors: Norman R. Sean , Moeller Peter , McDonald Thomas J. , Morris Pamela J.
Published: July 2004

Pseudomonas aeruginosa
is an
n
-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing
P. aeruginosa
and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 μM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C
29
and C
30
hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.
Multivariate Analyses of
Burkholderia
Species in Soil: Effect of Crop and Land Use History
Authors: Salles Joana Falcão , van Veen Johannes Antonius , van Elsas Jan Dirk
Published: July 2004

The assessment of
Burkholderia
diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications. Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus
Burkholderia
. In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles. The diversity of
Burkholderia
spp. in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA). CCA ordination plots showed that previous land use was the main factor affecting the composition of the
Burkholderia
community. Although most variation in the
Burkholderia
community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed. Plant species affected
Burkholderia
community structure to a lesser extent than did land use history. Similarities were observed between
Burkholderia
populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand. Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation. The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with
Burkholderia
species that are commonly isolated from soil, in particular
Burkholderia glathei
,
B. caledonica
,
B. hospita
, and
B. caribiensis
.
Effect of High-Pressure-Induced Ice I-to-Ice III Phase Transitions on Inactivation of
Listeria innocua
in Frozen Suspension
Authors: Luscher C. , Balasa A. , Fröhling A. , Ananta E. , Knorr D.
Published: July 2004

The inactivation of
Listeria innocua
BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices. The state of aggregation of water was taken into account. The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect. The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition. This effect was independent of the applied pressure holding time. Flow cytometric analyses supported the assumption of different mechanisms of inactivation of
L. innocua
in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture). Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food.
Evaluation of a Strategy for
Toxoplasma gondii
Oocyst Detection in Water
Authors: Villena Isabelle , Aubert Dominique , Gomis Philippe , Ferté Hubert , Inglard Jean-Christophe , Denis-Bisiaux Hélène , Dondon Julie-Muriel , Pisano Eric , Ortis Naïma , Pinon Jean-Michel
Published: July 2004

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for
Toxoplasma
oocyst detection as part of an approach to detecting multiple waterborne parasites, including
Giardia
and
Cryptosporidium
spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover
Toxoplasma
oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected
Toxoplasma
DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects
Toxoplasma
oocysts in water and may be suitable as a public health sentinel method.
Effect of Dilution Rate on Metabolic Pathway Shift between Aceticlastic and Nonaceticlastic Methanogenesis in Chemostat Cultivation
Authors: Shigematsu Toru , Tang Yueqin , Kobayashi Tsutomu , Kawaguchi Hiromi , Morimura Shigeru , Kida Kenji
Published: July 2004

Acetate conversion pathways of methanogenic consortia in acetate-fed chemostats at dilution rates of 0.025 and 0.6 day
−1
were investigated by using
13
C-labeled acetates, followed by gas chromatography-mass spectrometry (GC-MS) analysis of the CH
4
and CO
2
produced. Nonaceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens was suggested to occupy a primary pathway (approximately 62 to 90%) in total methanogenesis at the low dilution rate. In contrast, aceticlastic cleavage of acetate by aceticlastic methanogens was suggested to occupy a primary pathway (approximately 95 to 99%) in total methanogenesis at the high dilution rate. Phylogenetic analyses of transcripts of the methyl coenzyme M reductase gene (
mcrA
) confirmed that a significant number of transcripts of the genera
Methanoculleus
(hydrogenotrophic methanogens) and
Methanosarcina
(aceticlastic methanogens) were present in the chemostats at the low and high dilution rates, respectively. The
mcrA
transcripts of the genus
Methanosaeta
(aceticlastic methanogens), which dominated the population in a previous study (T. Shigematsu, Y. Tang, H. Kawaguchi, K. Ninomiya, J. Kijima, T. Kobayashi, S. Morimura, and K. Kida, J. Biosci. Bioeng.
96:
547-558, 2003), were poorly detected at both dilution rates due to the limited coverage of the primers used. These results demonstrated that the dilution rate could cause a shift in the primary pathway of acetate conversion to methane in acetate-fed chemostats.
Mineralization of Individual Congeners of Linear Alkylbenzenesulfonate by Defined Pairs of Heterotrophic Bacteria
Authors: Schleheck David , Knepper Thomas P. , Fischer Karin , Cook Alasdair M.
Published: July 2004

Parvibaculum lavamentivoransDS-1Tutilized the commercial surfactant linear alkylbenzenesulfonate (LAS) (20 congeners with C10to C13side chains) as a carbon and energy source by shortening the side chain, and sulfophenylcarboxylates (SPCs) and similar compounds (e.g., α,β-unsaturated SPCs [SPC-2Hs]) were excreted with quantitative recovery of the sulfophenyl moiety. 2-(4-Sulfophenyl)decane (2-C10-LAS) was converted largely to 3-(4-sulfophenyl)butyrate (3-C4-SPC), as were 2-C12-LAS and 2-C14-LAS; the other products were 5-C6-SPC (SPC+2C) and 3-C4-SPC-2H. 2-C11-LAS was converted largely to 4-C5-SPC with the corresponding SPC+2C and SPC-2H; similarly, 3-C12-LAS yielded 4-C6-SPC with the corresponding SPC+2C and SPC-2H. This pattern of products confirmed that LAS is degraded by ω-oxygenation and chain shortening through β-oxidation. At least nine major SPCs were formed from commercial LAS. The novel isolatesComamonas testosteroniSPB-2 and KF-1 utilized 3-C4-SPC;Delftia acidovoransSPH-1 utilized 4-C6-SPC enantioselectively. The substrate-dependent oxygen uptake of whole cells of strain SPB-2 indicated that there was inducible oxygenation of 3-C4-SPC and of 4-sulfophenol in whole cells of the strains ofC. testosteroniduring growth with 3-C4-SPC or 4-sulfophenol. The degradative pathways apparently involved 4-sulfocatechol and 4-sulfocatechol 1,2-dioxygenase. Strain SPB-2 and strain DS-1Tgrew together in LAS-salts medium, and only seven of the nine major SPCs were recovered. Strain SPB-2 utilized 3-C4-SPC, 3-C5-SPC, and 3-C4-SPC-2H. Strain SPH-1 grew together with strain DS-1Tin LAS-salts medium, and a different set of seven major SPCs was recovered. Strain SPH-1 utilized 4-C6-SPC, 4-C5-SPC, 4-C6-SPC-2H, and 4-C5-SPC-2H. A three-member community consisting of strains DS-1T, SPB-2, and SPH-1 utilized four major SPCs. We inferred that this community mineralized the major SPCs derived from 8 of the 20 LAS congeners.
Light Dependence of [3H]Leucine Incorporation in the Oligotrophic North Pacific Ocean
Authors: Church Matthew J. , Ducklow Hugh W. , Karl David M.
Published: July 2004

The influence of irradiance on bacterial incorporation of [3H]leucine was evaluated at Station ALOHA in the oligotrophic North Pacific subtropical gyre. Six experiments were conducted on three cruises to Station ALOHA to examine how [3H]leucine incorporation varied as a function of irradiance. Two experiments were also conducted to assess the photoautotrophic response to irradiance (based on photosynthetic uptake of [14C]bicarbonate) in both the upper and lower photic zones. Rates of [3H]leucine incorporation responded to irradiance in a photosynthesis-like manner, increasing sharply at low light and then saturating and sometimes declining with increasing light intensity. The influence of irradiance on bacterial growth was evaluated in both the well-lit (5 to 25 m) and dimly lit regions of the upper ocean (75 to 100 m) to determine whether the bacterial response to irradiance differed along the depth-dependent light gradient of the photic zone. [3H]leucine incorporation rates were analyzed with a photosynthesis-irradiance model for a quantitative description of the relationships between [3H]leucine incorporation and irradiance. Maximum rates of [3H]leucine incorporation in the upper photic zone increased 48 to 92% relative to those of dark-incubated samples, with [3H]leucine incorporation saturating at light intensities between 58 and 363 μmol of quanta m−2s−1. Rates of [3H]leucine incorporation in the deep photic zone were photostimulated 53 to 114% and were susceptible to photoinhibition, with rates declining at light intensities of >100 μmol of quanta m−2s−1. The results of these experiments revealed that sunlight directly influences bacterial growth in this open-ocean ecosystem.
Populations Implicated in Anaerobic Reductive Dechlorination of 1,2-Dichloropropane in Highly Enriched Bacterial Communities
Authors: Ritalahti Kirsti M. , Löffler Frank E.
Published: July 2004

1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that
Dehalococcoides
populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of
Dehalococcoides
populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by
Dehalococcoides
species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of
Dehalococcoides
populations.
Effects of Seeding Procedures and Water Quality on Recovery of
Cryptosporidium
Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623
Authors: Francy Donna S. , Simmons Otto D. , Ware Michael W. , Granger Emma J. , Sobsey Mark D. , Schaefer Frank W.
Published: July 2004

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for
Cryptosporidium
oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter.
Cryptosporidium
oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (
r
= 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.
Contribution of SAR11 Bacteria to Dissolved Dimethylsulfoniopropionate and Amino Acid Uptake in the North Atlantic Ocean
Authors: Malmstrom Rex R. , Kiene Ronald P. , Cottrell Matthew T. , Kirchman David L.
Published: July 2004

SAR11 bacteria are abundant in marine environments, often accounting for 35% of total prokaryotes in the surface ocean, but little is known about their involvement in marine biogeochemical cycles. Previous studies reported that SAR11 bacteria are very small and potentially have few ribosomes, indicating that SAR11 bacteria could have low metabolic activities and could play a smaller role in the flux of dissolved organic matter than suggested by their abundance. To determine the ecological activity of SAR11 bacteria, we used a combination of microautoradiography and fluorescence in situ hybridization (Micro-FISH) to measure assimilation of
3
H-amino acids and [
35
S]dimethylsulfoniopropionate (DMSP) by SAR11 bacteria in the coastal North Atlantic Ocean and the Sargasso Sea. We found that SAR11 bacteria were often abundant in surface waters, accounting for 25% of all prokaryotes on average. SAR11 bacteria were typically as large as, if not larger than, other prokaryotes. Additionally, more than half of SAR11 bacteria assimilated dissolved amino acids and DMSP, whereas about 40% of other prokaryotes assimilated these compounds. Due to their high abundance and activity, SAR11 bacteria were responsible for about 50% of amino acid assimilation and 30% of DMSP assimilation in surface waters. The contribution of SAR11 bacteria to amino acid assimilation was greater than would be expected based on their overall abundance, implying that SAR11 bacteria outcompete other prokaryotes for these labile compounds. These data suggest that SAR11 bacteria are highly active and play a significant role in C, N, and S cycling in the ocean.
Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography
Authors: Franciosa Giovanna , Pourshaban Manoocheher , De Luca Alessandro , Buccino Anna , Dallapiccola Bruno , Aureli Paolo
Published: July 2004

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from
Clostridium botulinum
, neurotoxigenic
C. butyricum
type E, and
C. baratii
type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.
Molecular Analysis of Shower Curtain Biofilm Microbes
Authors: Kelley Scott T. , Theisen Ulrike , Angenent Largus T. , St. Amand Allison , Pace Norman R.
Published: July 2004

Households provide environments that encourage the formation of microbial communities, often as biofilms. Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals. One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains. Over time, vinyl shower curtains accumulate films, commonly referred to as “soap scum,” which microscopy reveals are constituted of lush microbial biofilms. To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households. Each of the shower curtain communities was highly complex. No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities. However, the sequences generally represented similar phylogenetic kinds of organisms. Particularly abundant sequences represented members of the α-group of proteobacteria, mainly
Sphingomonas
spp. and
Methylobacterium
spp. Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens. Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits. In addition, the study detected many other kinds of organisms at lower abundances. These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms. Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.