bluuebunny/crossref_metadata_2025 huggingface dataset:
Controlled clinical and psychometric studies on the relation between periodontitis and depressive mood
Authors: Saletu Alexander , Pirker‐Frühauf Helene , Saletu Franziska , Linzmayer Leopold , Anderer Peter , Matejka Michael
Published: November 2005

Background: Depressive mood is considered a risk factor for the development of periodontitis.

Objectives: Investigation of the relationship between periodontitis and psychopathology utilizing psychometry (both observer‐ and self‐rating scales).

Methods: Forty periodontitis patients were compared with 41 age‐ and sex‐matched controls. The percentage of smokers was similar in both groups (30%versus 24.4%). Dental variables included probing depth, clinical attachment loss (CAL), radiographic loss of attachment, papillary bleeding index (PBI) and approximal plaque index (API). Psychometry comprised the Hamilton Depression Scale, the Zung Self‐Rating Depression and Anxiety Scales, the von Zerssen Well‐being and Complaint Scales, the Epworth Sleepiness Scale, the Pittsburgh Sleep Quality Index, the Quality‐of‐Life Index, crystallized intelligence and the Freiburg Personality Inventory (FPI).

Results: Multifactorial analysis of variance demonstrated increased depression and anxiety scores, reduced well‐being, increased somatic complaints, deteriorated quality of life and introversion in periodontitis. Partial correlation analyses between psychometric measures and dental variables revealed positive correlations of periodontal disease severity/CAL with the depression/anxiety, subjective well‐being and complaints scores, and a negative correlation with quality of life. The API was negatively correlated with social orientation, and the CAL was positively correlated with somatic complaints and introversion in the FPI.

Conclusion: Our clinical‐psychometric studies confirm depressive mood as a relevant pathogenetic factor for periodontitis.
Structural and functional features of human muscle–tendon unit
Authors: Finni T.
Published: November 2005

This paper reviews the architectural details and the in vivo behavior of the human muscle–tendon unit with the focus on the triceps surae and quadriceps femoris muscles. Recent advances in experimental techniques allow in vivo measurements of muscle–tendon architecture and function. In particular, the use of ultrasonography for measurement of tendon and muscle has expanded our knowledge in the last decade. Furthermore, the nuclear magnetic resonance imaging is opening up new insights not only for three‐dimensional anatomical information but also for examining musculo‐skeletal motion in vivo. While these two completely non‐invasive methods provide kinematic data, in vivo force measurements still require somewhat invasive procedures and are scarce. Thus, muscle forces are frequently calculated using both simple and complex models. These models can give us suggestions for further experimental work. There is a need to examine the experimental data ranging from single‐fiber experiments to the muscle function in human movement in order to understand the muscle–tendon function in vivo fully. Furthermore, appreciation of the structure–function relationships may help us to understand the entity of muscle–tendon function both from the perspective of mechanical behavior and neural control.
Relationship between dietary‐induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo
Authors: Lesniewska V. , Rowland I. , Laerke H. N. , Grant G. , Naughton P. J.
Published: November 2005

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16‐month‐old Spraque–Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non‐operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro‐ and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 ± 0.2 to 4.4 ± 0.2 cm min−1 by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.
The interaction of central command and the exercise pressor reflex in mediating baroreflex resetting during exercise in humans
Authors: Gallagher Kevin M. , Fadel Paul J. , Smith Scott A. , Strømstad Morten , Ide Kojiro , Secher Niels H. , Raven Peter B.
Published: November 2005

Central command and the exercise pressor reflex can independently reset the carotid baroreflex (CBR) during exercise. The present investigation assessed the interactive relationship between these two neural mechanisms in mediating baroreflex resetting during exercise. Six men performed static leg exercise at 20% maximal voluntary contraction under four conditions: control, no perturbation; neuromuscular blockade (NMB) induced by administration of the neuromuscular blocking agent Norcuron (central command activation); MAST, application of medical antishock trousers inflated to 100 mmHg (exercise pressor reflex activation); and Combo, NMB plus MAST (concomitant central command and exercise pressor reflex activation). With regard to CBR control of heart rate (HR), both NMB and Combo conditions resulted in a further resetting of the carotid–cardiac stimulus–response curve compared to control conditions, suggesting that CBR–HR resetting is predominately mediated by central command. In contrast, it appears that CBR control of blood pressure can be mediated by signals from either central command or the exercise pressor reflex, since both NMB and MAST conditions equally augmented the resetting of the carotid–vasomotor stimulus–response curve. With regard to the regulation of both HR and blood pressure, the extent of CBR resetting was greater during the Combo condition than during overactivation of either central command or the exercise pressor reflex alone. Therefore, we suggest that central command and the exercise pressor reflex interact such that signals from one input facilitate signals from the other, resulting in an enhanced resetting of the baroreflex during exercise.
Crystallization and chemical structures with annealing in ZrO2 gate insulators studied by photoemission spectroscopy and x-ray absorption spectroscopy
Authors: Okabayashi J. , Toyoda S. , Kumigashira H. , Oshima M. , Usuda K. , Niwa M. , Liu G. L.
Published: November 2005

We have performed photoemission spectroscopy and x-ray absorption spectroscopy (XAS) to investigate the chemical states and the crystallization from the amorphous structure by annealing of the ZrO2 gate insulators on Si. Angular-dependent core-level photoemission spectra revealed the chemical states including the interfacial layers. Annealing-temperature dependence in valence-band spectra and XAS revealed the relationship between crystallization and the changes in spectral line shapes although core-level photoemission spectra are not sensitive to the crystallization. Valence-band spectra are split into double peak structures and the line shapes of O K-edge x-ray absorption spectra become sharp by the annealing at 800°C corresponding to the crystallization temperature of amorphous ZrO2 films. It suggests that the valence-band spectra and XAS can be utilized to characterize the crystallization features in the gate insulators.
Ammonium chloride complex formation during downstream microwave ammonia plasma treatment of parylene-C
Authors: Pruden K. G. , Beaudoin S. P.
Published: November 2005

In this work, parylene-C is exposed to the effluent from a microwave ammonia plasma with a goal of producing primary amine groups on the parylene-C. These amine groups are desired as sites for the attachment of various biomolecules that will influence the biocompatibility of the parylene-C. Ammonia plasma treatment is an effective approach for creating amine species on polymers. In this work, attenuated total reflectance infrared spectroscopy studies showed that no primary amine groups resulted from this treatment of parylene-C. Instead, reactive nitrogen-bearing radicals from the plasma appear to have been complexed by chlorine in the polymer. The formation of these complexes scavenged nitrogen-bearing radicals from the plasma and prevented the formation of nitrogenous species, such as the desired primary amines, on the parylene-C. These results are consistent with results of ammonia plasma treatment of other chlorinated polymers and suggest that alternative approaches are required to create nitrogen-bearing species on parylene-C.
Dry-etch of As2S3 thin films for optical waveguide fabrication
Authors: Li Weitang , Ruan Yinlan , Luther-Davies Barry , Rode Andrei , Boswell Rod
Published: November 2005

Plasma etching to As2S3 thin films for optical waveguide fabrication has been studied using a helicon plasma etcher. The etching effects using the processing gases or gas mixtures of O2, Ar, and CF4 were compared. It was found that the O2 plasma had no chemical etching effect to the As2S3, but it could oxidize the surface of the As2S3. The Ar plasma provided a strong ion sputtering effect to the films. The CF4 plasma exhibited a too strong chemical etch to the As2S3, leading to serious undercutting and very rough sidewalls of the waveguides. Ar and O2 gases were compared as the additives to dilute the CF4 processing gas. The etch rate of the As2S3 was reduced dramatically from over 2000nm∕min to a few hundred nm/min when the pure CF4 gas was heavily diluted with 70% Ar or O2 gas. The undercutting and sidewall roughness of the etched waveguides were also decreased greatly when above dilution was made, which was associated with an enormous weakening of the isotropic chemical etch induced by neutral reactants in the plasma. In addition, the O2 showed a better dilution effect than the Ar in reducing the etch rate of the As2S3; and the O2∕CF4 plasma also enabled a much lower erosion rate to Al mask layers than the Ar∕CF4 plasma at similar plasma conditions. The As2S3 waveguides with near vertical and very smooth sidewalls were obtained using an optimized O2∕CF4 plasma. Moreover, the etching behaviors and mechanisms were explained base on the etching results, and on the characteristics of the applied plasma diagnosed using Langmuir probe and optical spectroscopy techniques.
Low-energy sputtering yields of tungsten and tantalum
Authors: Doerner R. P.
Published: November 2005

The sputtering yields of tungsten and tantalum due to low-energy (30–125eV) xenon ion bombardment in a plasma environment are measured both using weight loss and spectroscopic techniques. The yields of both tungsten and tantalum are practically identical and show a reduction of 3 when compared to the molybdenum yield at 125eV and a reduction of a factor of 10 when compared to the molybdenum yield at 50eV.
Map Plasticity in Somatosensory Cortex
Authors: Feldman Daniel E. , Brecht Michael
Published: November 2005

Sensory maps in neocortex are adaptively altered to reflect recent experience and learning. In somatosensory cortex, distinct patterns of sensory use or disuse elicit multiple, functionally distinct forms of map plasticity. Diverse approaches—genetics, synaptic and in vivo physiology, optical imaging, and ultrastructural analysis—suggest a distributed model in which plasticity occurs at multiple sites in the cortical circuit with multiple cellular/synaptic mechanisms and multiple likely learning rules for plasticity. This view contrasts with the classical model in which the map plasticity reflects a single Hebbian process acting at a small set of cortical synapses.
Surface Microflora of Four Smear-Ripened Cheeses
Authors: Mounier Jérôme , Gelsomino Roberto , Goerges Stefanie , Vancanneyt Marc , Vandemeulebroecke Katrien , Hoste Bart , Scherer Siegfried , Swings Jean , Fitzgerald Gerald F. , Cogan Timothy M.
Published: November 2005

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and
Debaryomyces hansenii
was the dominant species in the four cheeses.
Yarrowia lipolytica
was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species,
Corynebacterium casei
,
Corynebacterium variabile
,
Arthrobacter arilaitensis
,
Arthrobacter
sp.,
Microbacterium gubbeenense
,
Agrococcus
sp. nov.,
Brevibacterium linens
,
Staphylococcus epidermidis
,
Staphylococcus equorum
,
Staphylococcus saprophyticus
,
Micrococcus luteus
,
Halomonas venusta
,
Vibrio
sp., and
Bacillus
sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria,
C. casei
and
A. arilaitensis
, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.
Characterization of Enzymatic Antioxidants in the Lichen
Ramalina lacera
and Their Response to Rehydration
Authors: Weissman Lior , Garty Jacob , Hochman Ayala
Published: November 2005

Lichens are slow-growing associations of fungi and green algae or cyanobacteria. This symbiotic association forms a common thallus that does not possess roots or a waxy cuticle and depends mainly on atmospheric input of mineral nutrients. The lifestyle of most lichens is composed of alternating periods of desiccation with low metabolic activity and hydration that induces increase in their metabolism. We have previously shown that rehydration of the naturally desiccated lichen
Ramalina lacera
resulted in a rapid increase in photosynthesis and was accompanied by a burst of intracellular production of reactive oxygen species and nitric oxide, as well as a transient decrease in water-soluble antioxidant capacity. We report here on enzymatic antioxidants of
R. lacera
and their response to rehydration. Native gel electrophoresis of crude extracts of
R. lacera
stained for superoxide dismutase (SOD) activity revealed four Fe-SOD and four Mn-SOD electromorphs that are synthesized by the alga, a Cu/Zn-SOD and a Mn-SOD that are the product of the fungus, and two catalases synthesized one by the fungus and the other by the algae. In addition, we detected glutathione reductase and glucose-6-phosphate dehydrogenase activities in crude extracts of
R. lacera
. Rehydration of the thalli resulted in a decrease in SOD activity of all forms, and a transient decrease in total catalase activity, as well as a decrease in the antioxidant auxiliary enzymes glutathione reductase and glucose-6-phosphate dehydrogenase.
Differentiation of Spores of
Bacillus subtilis
Grown in Different Media by Elemental Characterization Using Time-of-Flight Secondary Ion Mass Spectrometry
Authors: Cliff John B. , Jarman Kristin H. , Valentine Nancy B. , Golledge Steven L. , Gaspar Daniel J. , Wunschel David S. , Wahl Karen L.
Published: November 2005

We demonstrate the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in a forensics application to distinguish
Bacillus subtilis
spores grown in various media based on the elemental signatures of the spores. Triplicate cultures grown in each of four different media were analyzed to obtain TOF-SIMS signatures comprised of 16 elemental intensities. Analysis of variance was unable to distinguish growth medium types based on
40
Ca-normalized signatures of any single normalized element. Principal component analysis proved successful in separating the spores into groups consistent with the media in which they were prepared. Confusion matrices constructed using nearest-neighbor classification of the PCA scores confirmed the predictive utility of TOF-SIMS elemental signatures in identifying sporulation medium. Theoretical calculations based on the number and density of spores in an analysis area indicate an analytical sample size of about 1 ng, making this technique an attractive method for bioforensics applications.
Importance of Organosulfur Utilization for Survival ofPseudomonas putidain Soil and Rhizosphere
Authors: Mirleau Pascal , Wogelius Roy , Smith Andrew , Kertesz Michael A.
Published: November 2005

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied inPseudomonas putida, a common rhizosphere inhabitant. Survival ofP. putidastrains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness ofP. putidaS-313 was compared with that of isogenic strains with mutations in thesftRandasfAgenes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in thessulocus (required in vitro for the desulfurization of both sulfonates and sulfate esters).asfAorsftRmutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival ofsftRandssumutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.
Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines
Authors: Labrenz Matthias , Jost Günter , Pohl Christa , Beckmann Sabrina , Martens-Habbena Willm , Jürgens Klaus
Published: November 2005

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO
2
dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO
2
dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO
2
dark fixation rates, ranging from 0.7 to 1.4 μmol liter
−1
day
−1
, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO
2
dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter
−1
CO
2
day
−1
. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter
−1
NO
3
−
day
−1
. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic
Thiomicrospira psychrophila
and
Maorithyas hadalis
gill symbiont (both
Gammaproteobacteria
) and to an uncultured nitrate-reducing
Helicobacteraceae
bacterium (
Epsilonproteobacteria
). Our data indicate that this
Helicobacteraceae
bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.
Bacteria Associated with Spores of the Arbuscular Mycorrhizal Fungi
Glomus geosporum
and
Glomus constrictum
Authors: Roesti David , Ineichen Kurt , Braissant Olivier , Redecker Dirk , Wiemken Andres , Aragno Michel
Published: November 2005

Spores of the arbuscular mycorrhizal fungi (AMF)
Glomus geosporum
and
Glomus constrictum
were harvested from single-spore-derived pot cultures with either
Plantago lanceolata
or
Hieracium pilosella
as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both
G. geosporum
and
G. constrictum
spores were affiliated with taxonomic groups known to degrade biopolymers (
Cellvibrio
,
Chondromyces
,
Flexibacter
,
Lysobacter
, and
Pseudomonas
). Scanning electron microscopy of
G. geosporum
spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.
Molecular Epidemiology of Antimicrobial-Resistant CommensalEscherichia coliStrains in a Cohort of Newborn Calves
Authors: Hoyle Deborah V. , Yates Catherine M. , Chase-Topping Margo E. , Turner Esther J. , Davies Sarah E. , Low J. Chris , Gunn George J. , Woolhouse Mark E. J. , Amyes Sebastian G. B.
Published: November 2005

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Ampr) and nalidixic acid-resistant (Nalr) commensalEscherichia colistrains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amprpopulation showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nalrpopulation appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amprgenotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amprgenotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amprisolates carrying sulfamethoxazole resistance increased significantly over the study period (P< 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.
Antimicrobial Resistance and Virulence Genes of
Escherichia coli
Isolates from Swine in Ontario
Authors: Boerlin Patrick , Travis Rebeccah , Gyles Carlton L. , Reid-Smith Richard , Heather Lim Nicol Janecko , Nicholson Vivian , McEwen Scott A. , Friendship Robert , Archambault Marie
Published: November 2005

A total of 318
Escherichia coli
isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine
E. coli
. Antimicrobial resistance frequency among
E. coli
isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic
E. coli
(ETEC). Resistance to quinolones was detected only in enterotoxigenic
E. coli
(≤3%). The presence of
sul3
was demonstrated for the first time in Canada in porcine
E. coli
isolates. Associations were observed among
tetA
,
sul1
,
aadA
, and
aac(3)IV
and among
tetB
,
sul2
, and
strA
/
strB
, with a strong negative association between
tetA
and
tetB
. The
paa
and
sepA
genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between
tetA
,
estA
,
paa
, and
sepA
. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine
E. coli
isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal
E. coli
isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.
Rapid Identification and Enumeration of
Saccharomyces cerevisiae
Cells in Wine by Real-Time PCR
Authors: Martorell P. , Querol A. , Fernández-Espinar M. T.
Published: November 2005

Despite the beneficial role of
Saccharomyces cerevisiae
in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for
S. cerevisiae
using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated
S. cerevisiae
from its sibling species
Saccharomyces bayanus
,
Saccharomyces pastorianus
, and
Saccharomyces paradoxus
. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of
S. cerevisiae
directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify
S. cerevisiae
in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 10
5
to 3.8 CFU/ml in sweet wine and from 5 × 10
6
to 50 CFU/ml in red wine.
Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance
Authors: Yu Zhongtang , Michel Frederick C. , Hansen Glenn , Wittum Thomas , Morrison Mark
Published: November 2005

We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes:
tet
(A) and
tet
(C);
tet
(G); and
tet
genes encoding ribosomal protection proteins, including
tet
(M),
tet
(O),
tetB
(P),
tet
(Q),
tet
(S),
tet
(T), and
tet
(W). The assays were validated using known numbers of sample-derived
tet
gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log
tet
gene copies. New
tet
gene variants were also identified from cloned
tet
amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three
tet
gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of
tet
genes than the swine manures. The composts of swine manures had substantially reduced
tet
gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on
tet
gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of
tet
genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of
tet
genes, which should be useful for ecological studies of antibiotic resistance.
A Method Adapting Microarray Technology for Signature-Tagged Mutagenesis of
Desulfovibrio desulfuricans
G20 and
Shewanella oneidensis
MR-1 in Anaerobic Sediment Survival Experiments
Authors: Groh Jennifer L. , Luo Qingwei , Ballard Jimmy D. , Krumholz Lee R.
Published: November 2005

Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn
10
transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized
Desulfovibrio desulfuricans
G20 in addition to
Shewanella oneidensis
MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.
Metabolic Engineering of
Corynebacterium glutamicum
for
l
-Serine Production
Authors: Peters-Wendisch Petra , Stolz Michael , Etterich Helga , Kennerknecht Nicole , Sahm Hermann , Eggeling Lothar
Published: November 2005

Although
l
-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via
l
-serine formation, previous attempts to obtain a strain producing
l
-serine from glucose have not been successful. We functionally identified the genes
serC
and
serB
from
Corynebacterium glutamicum
, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic
serA
gene,
serA
Δ
197
, encoding an
l
-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of
l
-serine, as did the overexpression of these genes in a strain with the
l
-serine dehydratase gene
sdaA
deleted. However, reduced expression of the serine hydroxymethyltransferase gene
glyA
, in combination with the overexpression of
serA
Δ
197
,
serC
, and
serB
, resulted in a transient accumulation of up to 16 mM
l
-serine in the culture medium. When
sdaA
was also deleted, the resulting strain,
C. glutamicum
Δ
sdaA
::pK18mob
glyA
′(pEC-T18mob2
serA
Δ197
CB
), accumulated up to 86 mM
l
-serine with a maximal specific productivity of 1.2 mmol h
−1
 g (dry weight)
−1
. This illustrates a high rate of
l
-serine formation and also utilization in the
C. glutamicum
wild type. Therefore, metabolic engineering of
l
-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism.
16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught
Anopheles gambiae
Sensu Lato and
A. funestus
Mosquitoes Reveals New Species Related to Known Insect Symbionts
Authors: Lindh Jenny M. , Terenius Olle , Faye Ingrid
Published: November 2005

Field-collected mosquitoes of the two main malaria vectors in Africa,
Anopheles gambiae
sensu lato and
Anopheles funestus
, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate,
Nocardia corynebacterioides
, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of
Anaplasma
species from mosquitoes and a new mosquito-
Spiroplasma
association. The isolates will be further investigated for their suitability for a paratransgenic
Anopheles
mosquito.
Recognition of Individual Genes in Diverse Microorganisms by Cycling Primed In Situ Amplification
Authors: Kenzaka Takehiko , Tamaki Shigeru , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: November 2005

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene inEscherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene inAeromonasspp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of therpoDgenes ofAeromonas sobriaandA. hydrophilain the water samples varied between 2.1 × 103and 9.0 × 103cells ml−1and between undetectable and 5.1 × 102cells ml−1, respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.
Methylotrophic Metabolism Is Advantageous for
Methylobacterium extorquens
during Colonization of
Medicago truncatula
under Competitive Conditions
Authors: Sy Abdoulaye , Timmers Antonius C. J. , Knief Claudia , Vorholt Julia A.
Published: November 2005

Facultative methylotrophic bacteria of the genus
Methylobacterium
are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume
Medicago truncatula
. Competition experiments with
Methylobacterium extorquens
wild-type strain AM1 and methylotrophy mutants revealed that the
ability to use methanol as a carbon and energy source provides a
selective advantage during colonization of
M. truncatula
.
Differences in the fitness of mutants defective in different stages of
methylotrophic metabolism were found; whereas approximately 25% of the
mutant incapable of oxidizing methanol to formaldehyde (deficient in
methanol dehydrogenase) was recovered, 10% or less of the mutants
incapable of oxidizing formaldehyde to CO
2
(defective in
biosynthesis of the cofactor tetrahydromethanopterin) was
recovered. Interestingly, impaired fitness of the mutant
strains compared with the wild type was found on leaves and roots.
Single-inoculation experiments showed, however, that mutants with
defects in methylotrophy were capable of plant colonization at the
wild-type level, indicating that methanol is not the only carbon source
that is accessible to
Methylobacterium
while it is associated
with plants. Fluorescence microscopy with a green fluorescent
protein-labeled derivative of
M. extorquens
AM1 revealed that
the majority of the bacterial cells on leaves were on the surface and
that the cells were most abundant on the lower, abaxial side. However,
bacterial cells were also found in the intercellular spaces inside the
leaves, especially in the epidermal cell layer and immediately
underneath this
layer.
Anaerobic Microbial Communities in Lake Pavin, a Unique Meromictic Lake in France
Authors: Lehours Anne-C. , Bardot Corinne , Thenot Aurelie , Debroas Didier , Fonty Gerard
Published: November 2005

The
Bacteria
and
Archaea
from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of
Bacteria
and
Archaea
and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing
Archaea
involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and
Archaea
detected with probe ARCH915 represented the largest microbial group in the water column, with a mean
Archaea/Eubacteria
ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the
Archaea
community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.
Proof that
Burkholderia
Strains Form Effective Symbioses with Legumes: a Study of Novel
Mimosa
-Nodulating Strains from South America
Authors: Chen Wen-Ming , de Faria Sergio M. , Straliotto Rosângela , Pitard Rosa M. , Simões-Araùjo Jean L. , Chou Jui-Hsing , Chou Yi-Ju , Barrios Edmundo , Prescott Alan R. , Elliott Geoffrey N. , Sprent Janet I. , Young J. Peter W. , James Euan K.
Published: November 2005

Twenty
Mimosa
-nodulating bacterial strains from Brazil and Venezuela, together with eight reference
Mimosa
-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia,
Burkholderia caribensis
,
Burkholderia phymatum
, and
Burkholderia tuberum
. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus
Burkholderia
; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes
nodA
, the NodD-dependent regulatory consensus sequences (
nod
box), and
nifH
. The
nodA
and
nifH
sequences were very close to each other and to those of
B. phymatum
STM815,
B. caribensis
TJ182, and
Cupriavidus taiwanensis
LMG19424 but were relatively distant from those of
B. tuberum
STM678. In addition to nodulating their original hosts, all five strains could also nodulate other
Mimosa
spp., and all produced nodules on
Mimosa pudica
that had nitrogenase (acetylene reduction) activities and structures typical of effective N
2
-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N
2
-fixing nodules on their original hosts,
Mimosa bimucronata
(Br3461) and
Mimosa pigra
(MAP3-5), and hence this confirms strongly that
Burkholderia
strains can form effective symbioses with legumes.
Construction of an Alpha Toxin Gene Knockout Mutant of
Clostridium perfringens
Type A by Use of a Mobile Group II Intron
Authors: Chen Yue , McClane Bruce A. , Fisher Derek J. , Rood Julian I. , Gupta Phalguni
Published: November 2005

In developing
Clostridium perfringens
as a safe vaccine vector, the alpha toxin gene (
plc
) in the bacterial chromosome must be permanently inactivated. Disrupting genes in
C. perfringens
by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the
plc
gene in
C. perfringens
ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the
plc
mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other
C. perfringens
mutants.
Factors Influencing the Ability of
Listeria monocytogenes
To Pass through a Membrane Filter by Active Infiltration
Authors: Nakazawa Kentaro , Hasegawa Hiroyuki , Nakagawa Yoji , Terao Michinori , Matsuyama Tohey
Published: November 2005

Listeria monocytogenes
infiltrated the reticulate structure of a membrane filter and passed through a filter with pore sizes of 0.45 μm and 0.2 μm in 6 to 24 h and 5 to 6 days, respectively. Flagellar motility and expansive pressure generated by the growing bacterial population were indicated as the driving forces of infiltration.
Impact of Bacterial NO
3
−
Transport on Sediment Biogeochemistry
Authors: Sayama Mikio , Risgaard-Petersen Nils , Nielsen Lars Peter , Fossing Henrik , Christensen Peter Bondo
Published: November 2005

Experiments demonstrated that
Beggiatoa
could induce a H
2
S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO
3
−
reduction from denitrification to dissimilatory NO
3
−
reduction to ammonium. pH, O
2
, and H
2
S profiles indicated that the bacteria oxidized H
2
S with NO
3
−
and transported S
0
to the sediment surface for aerobic oxidation.
Bactericidal Actions of a Silver Ion Solution on
Escherichia coli
, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis
Authors: Yamanaka Mikihiro , Hara Keita , Kudo Jun
Published: November 2005

Bactericidal actions of the silver ion on
Escherichia coli
as a model microorganism were studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). EFTEM observations demonstrated that the silver ion readily infiltrates the interior of
E. coli
, contrary to the early hypothesis that it resides initially in the cell membrane area. Furthermore, 2-DE and MALDI-TOF MS indicated that the expression of a ribosomal subunit protein as well as that of some other enzymes and proteins is affected by the silver ion. The present results demonstrate for the first time that one of the major bactericidal functions of the silver ion is its interaction with the ribosome and the ensuing inhibition in expression of the enzymes and proteins essential to ATP production.
Discrimination between
Francisella tularensis
and
Francisella
-Like Endosymbionts when Screening Ticks by PCR
Authors: Kugeler Kiersten J. , Gurfield Nikos , Creek Jean G. , Mahoney Kerry S. , Versage Jessica L. , Petersen Jeannine M.
Published: November 2005

The presence of
Francisella
-like endosymbionts in tick species known to transmit tularemia poses a potential diagnostic problem for laboratories that screen tick samples by PCR for
Francisella tularensis.
Tick samples initially considered positive for
F. tularensis
based on standard 16S rRNA gene PCR were found to be positive only for
Francisella-
like endosymbionts using a multitarget
F. tularensis
TaqMan assay (IS
Ftu2
,
tul4
, and
iglC
) and 16S rRNA gene sequencing. Specificity of PCR-based diagnostics for
F. tularensis
should be carefully evaluated with appropriate specimen types prior to diagnostic use.
Creating New Genes by Plasmid Recombination in
Escherichia coli
and
Bacillus subtilis
Authors: Gomez Ana , Galic Tatjana , Mariet Jean-François , Matic Ivan , Radman Miroslav , Petit Marie-Agnès
Published: November 2005

Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that
Bacillus subtilis
efficiently recombines sequences with 4% divergence, and
Escherichia coli mutS
is more appropriate for sequences with 22% divergence.