bluuebunny/crossref_metadata_2025 huggingface dataset:
Three Enzymes for Trehalose Synthesis in
Bradyrhizobium
Cultured Bacteria and in Bacteroids from Soybean Nodules
Authors: Streeter J. G. , Gomez M. L.
Published: June 2006

α,α-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured
Bradyrhizobium japonicum
and
B. elkanii
were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by
Bradyrhizobium
species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.
Identification of the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of
Lactococcus lactis
Authors: Ganesan Balasubramanian , Dobrowolski Piotr , Weimer Bart C.
Published: June 2006

Nutrient starvation and nonculturability in bacteria lead to changes in metabolism not found during the logarithmic phase. Substrates alternate to those used during growth are metabolized in these physiological states, yielding secondary metabolites. In firmicutes and actinobacteria, amino acid catabolic pathways are induced during starvation and nonculturability. Examination of lactococci showed that the population entered a nonculturable state after carbohydrate depletion and was incapable of growth on solid media; however, the cells gained the ability to produce branched-chain fatty acids from amino acids. Gene expression profiling and in silico pathway analysis coupled with nuclear magnetic resonance spectroscopy were used to delineate the leucine catabolic pathway. Lactococci produced acetic and propionic acid during logarithmic growth and starvation. At the onset of nonculturability, 2-methylbutyric acid was produced via hydroxymethyl-glutaryl-coenzyme A (CoA) and acetyl-CoA, along with ATP and oxidation/reduction precursors. Gene expression profiling and genome sequence analysis showed that lactococci contained redundant genes for branched-chain fatty acid production that were regulated by an unknown mechanism linked to carbon metabolism. This work demonstrated the ability of a firmicute to induce new metabolic capabilities in the nonculturable state for producing energy and intermediates needed for transcription and translation. Phylogenetic analyses showed that homologues of these enzymes and their functional motifs were widespread across the domains of life.
The
PKS4
Gene of
Fusarium graminearum
Is Essential for Zearalenone Production
Authors: Lysøe Erik , Klemsdal Sonja S. , Bone Karen R. , Frandsen Rasmus J. N. , Johansen Thomas , Thrane Ulf , Giese Henriette
Published: June 2006

Zearalenones are produced by several
Fusarium
species and can cause reproductive problems in animals. Some aurofusarin mutants of
Fusarium pseudograminearum
produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the
Fusarium graminearum
database was carried out for these mutants.
PKS4
was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An
Agrobacterium tumefaciens-
mediated transformation protocol was used to replace the central part of the
PKS4
gene with a
hygB
resistance gene through double homologous recombination in an
F. graminearum
strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of
PKS4
. High-performance liquid chromatography analysis showed that the
PKS4
replacement mutant did not produce ZON. Thus,
PKS4
encodes an enzyme required for the production of ZON in
F. graminearum
. Barley root infection studies revealed no alteration in the pathogenicity of the
PKS4
mutant compared to the pathogenicity of the wild type. The expression of
PKS13
, which is located in the same cluster as
PKS4
, decreased dramatically in the mutant, while transcription of
PKS4
was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of
PKS4
and that the
PKS4
-encoded protein or its product stimulates expression of
PKS13
. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.
Inactivation ofMycobacterium aviumsubsp.paratuberculosisin Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures
Authors: López-Pedemonte Tomás , Sevilla Iker , Garrido Joseba M. , Aduriz Gorka , Guamis Buenaventura , Juste Ramón A. , Roig-Sagués Artur X.
Published: June 2006

Two strains ofMycobacterium aviumsubsp.paratuberculosis(3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions ofM. aviumsubsp.paratuberculosisincreased with pressure level. Significant differences were also found betweenM. aviumsubsp.paratuberculosisstrains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.
Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR
Authors: Wolffs Petra F. G , Glencross Kari , Thibaudeau Romain , Griffiths Mansel W.
Published: June 2006

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the
invA
gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the
Salmonella
cells. After this, the
Salmonella
cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10
2
CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.
Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from
Sphingopyxis terrae
, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants
Authors: Ohta Takeshi , Kawabata Takeshi , Nishikawa Ken , Tani Akio , Kimbara Kazuhide , Kawai Fusako
Published: June 2006

Polyethylene glycol dehydrogenase (PEGDH) from
Sphingopyxis terrae
(formerly
Sphingomonas terrae
) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from
Aspergillus niger
and
Penicillium amagasakiense
. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q
10
was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.
Comparison of Methods of Extracting
Salmonella enterica
Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control
Authors: Klerks M. M. , van Bruggen A. H. C. , Zijlstra C. , Donnikov M.
Published: June 2006

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of
Salmonella enterica
serovar Enteritidis from soil, manure, and compost and uses an
Escherichia coli
strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of
S. enterica
serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10
3
to 1.8 × 10
3
CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent
S. enterica
-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.
Quantifying the Significance of Phage Attack on Starter Cultures: a Mechanistic Model for Population Dynamics of Phage and Their Hosts Isolated from Fermenting Sauerkraut
Authors: Mudgal P. , Breidt F. , Lubkin S. R. , Sandeep K. P.
Published: June 2006

We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two
Leuconostoc mesenteroides
strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (
P
0
) and multiplicity of infection (MOI) were low (
P
0
< 1 × 10
3
PFU/ml; MOI < 10
−4
) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations.
Mycobacterium aviumsubsp.paratuberculosisin Lake Catchments, in River Water Abstracted for Domestic Use, and in Effluent from Domestic Sewage Treatment Works: Diverse Opportunities for Environmental Cycling and Human Exposure
Authors: Pickup R. W. , Rhodes G. , Bull T. J. , Arnott S. , Sidi-Boumedine K. , Hurley M. , Hermon-Taylor J.
Published: June 2006

Mycobacterium aviumsubsp.paratuberculosisfrom infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests forM. aviumsubsp.paratuberculosisin the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive forM. aviumsubsp.paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.
In Vitro Fermentation of Breast Milk Oligosaccharides by
Bifidobacterium infantis
and
Lactobacillus gasseri
Authors: Ward Robert E. , Niñonuevo Milady , Mills David A. , Lebrilla Carlito B. , German J. Bruce
Published: June 2006

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study,
Bifidobacterium infantis
was shown to ferment purified HMO as a sole carbon source, while another gut commensal,
Lactobacillus gasseri
, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.
Reduction of Acid Tolerance by Tetracycline in
Escherichia coli
Expressing
tetA
(C) Is Reversed by Cations
Authors: Hung Kai F. , Byrd Jeffrey J. , Bose Jeffrey L. , Kaspar Charles W.
Published: June 2006

When tetracycline was present,
tetA
(C) reduced acid tolerance, suppressed
rpoS
expression, and increased the concentration of total soluble proteins in stationary-phase
Escherichia coli
. The suppression of acid tolerance was reversed by 85 mM sodium, potassium, magnesium, and calcium ions but not by 85 mM sucrose. Implications for using TetA(C) are discussed.
Use of Copper Cast Alloys To Control
Escherichia coli
O157 Cross-Contamination during Food Processing
Authors: Noyce J. O. , Michels H. , Keevil C. W.
Published: June 2006

The most notable method of infection from
Escherichia coli
O157 (
E. coli
O157) is through contaminated food products, usually ground beef. The objective of this study was to evaluate seven cast copper alloys (61 to 95% Cu) for their ability to reduce the viability of
E. coli
O157, mixed with or without ground beef juice, and to compare these results to those for stainless steel.
E. coli
O157 (NCTC 12900) (2 × 10
7
CFU) mixed with extracted beef juice (25%) was inoculated onto coupons of each copper cast alloy or stainless steel and incubated at either 22°C or 4°C for up to 6 h.
E. coli
O157 viability was determined by plate counts in addition to staining in situ with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. Without beef extract, three alloys completely killed the inoculum during the 6-h exposure at 22°C. At 4°C, only the high-copper alloys (>85%) significantly reduced the numbers of O157. With beef juice, only one alloy (95% Cu) completely killed the inoculum at 22°C. For stainless steel, no significant reduction in cell numbers occurred. At 4°C, only alloys C83300 (93% Cu) and C87300 (95% Cu) significantly reduced the numbers of
E. coli
O157, with 1.5- and 5-log kills, respectively. Reducing the inoculum to 10
3
CFU resulted in a complete kill for all seven cast copper alloys in 20 min or less at 22°C. These results clearly demonstrate the antimicrobial properties of cast copper alloys with regard to
E. coli
O157, and consequently these alloys have the potential to aid in food safety.
Effect of Growth in Biofilms on Chlorine Susceptibility of
Mycobacterium avium
and
Mycobacterium intracellulare
Authors: Steed Keesha A. , Falkinham Joseph O.
Published: June 2006

Mycobacterium avium
and
Mycobacterium intracellulare
were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured.
M. avium
and
M. intracellulare
readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of
M. avium
and
M. intracellulare
cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 μg chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both
M. avium
and
M. intracellulare
and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.
Shifting the pH Profile of
Aspergillus niger
PhyA Phytase To Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive
Authors: Kim Taewan , Mullaney Edward J. , Porres Jesus M. , Roneker Karl R. , Crowe Sarah , Rice Sarah , Ko Taegu , Ullah Abul H. J. , Daly Catherine B. , Welch Ross , Lei Xin Gen
Published: June 2006

Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from
Aspergillus niger
PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in
Pichia pastoris
yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (
P
< 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering.
Prevalence of
Chlamydophila psittaci
in Fecal Droppings from Feral Pigeons in Amsterdam, The Netherlands
Authors: Heddema Edou R. , ter Sluis Sietske , Buys Jan A. , Vandenbroucke-Grauls Christina M. J. E. , van Wijnen Joop H. , Visser Caroline E.
Published: June 2006

In many cities, the feral rock dove is an abundant bird species that can harbor
Chlamydophila psittaci
. We determined the prevalence and genotype of
C. psittaci
in fresh fecal samples from feral pigeons in Amsterdam, The Netherlands. The prevalence was 7.9% overall (26/331; 95% confidence interval, 5 to 11). Ten genotyped PCR-positive samples were all genotype B.
Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and
vapA
Genotyping of
Rhodococcus equi
Authors: Rodríguez-Lázaro David , Lewis Deborah A. , Ocampo-Sosa Alain A. , Fogarty Ursula , Makrai László , Navas Jesús , Scortti Mariela , Hernández Marta , Vázquez-Boland José A.
Published: June 2006

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete
Rhodococcus equi
, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene
choE
, universally conserved in
R. equi
. The
choE
Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene
vapA
, carried by most horse isolates but infrequently found in isolates from other sources. The
choE
-IAC and
vapA
assays were 100% sensitive and specific as determined using 178
R. equi
isolates, 77 nontarget bacteria, and a panel of 60
R. equi
isolates with known
vapA
+
and
vapA
-negative (including
vapB
+
) plasmid genotypes. The
vapA
+
frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The
choE
-IAC Q-PCR could detect up to one genome equivalent using
R. equi
DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to ≈10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency
E
of >0.94. The
vapA
assay had similar performance but appeared unsuitable for accurate (
vapA
+
)
R. equi
quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of
R. equi
and (optional)
vapA
+
“horse-pathogenic” genotype determination.
Pure and Mixed Genetic Lines of
Saccharomyces bayanus
and
Saccharomyces pastorianus
and Their Contribution to the Lager Brewing Strain Genome
Authors: Rainieri Sandra , Kodama Yukiko , Kaneko Yoshinobu , Mikata Kozaburo , Nakao Yoshihiro , Ashikari Toshihiko
Published: June 2006

The yeast species
Saccharomyces bayanus
and
Saccharomyces pastorianus
are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified “pure” strains containing a single type of genome and “hybrid” strains that contained portions of the genomes from the “pure” lines, as well as alleles termed “Lager” that represent a third genome commonly associated with lager brewing strains. The two pure lines represent
S. uvarum
and
S. bayanus
, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i)
S. cerevisiae
/
S. bayanus
/Lager, (ii)
S. bayanus
/
S. uvarum
/Lager, and (iii)
S. cerevisiae
/
S. bayanus
/
S. uvarum
/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.
Estimating High-Affinity Methanotrophic Bacterial Biomass, Growth, and Turnover in Soil by Phospholipid Fatty Acid
13
C Labeling
Authors: Maxfield P. J. , Hornibrook E. R. C. , Evershed R. P.
Published: June 2006

A time series phospholipid fatty acid (PLFA)
13
C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH
4
concentrations using synthetic air containing 2 parts per million of volume of
13
CH
4
. Flowthrough chambers maintained a stable CH
4
concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of
13
C label into the microbial biomass. Incorporation of the
13
C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The δ
13
C values of individual PLFAs showed that
13
C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of
13
C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant
13
C-labeled PLFA was 18:1ω7c, with 16:1ω5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 × 10
6
cells g
−1
of soil (dry weight). While recycling of
13
C label from the methanotrophic biomass must occur, it is a slower process than initial
13
CH
4
incorporation, with only about 5 to 10% of
13
C-labeled PLFAs reflecting this process. Thus,
13
C-labeled PLFA distributions determined at any time point during
13
CH
4
incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum
13
C labeling for methanotrophic biomass determinations.
Prevalence of
Escherichia coli
O157 in Saskatchewan Cattle: Characterization of Isolates by Using Random Amplified Polymorphic DNA PCR, Antibiotic Resistance Profiles, and Pathogenicity Determinants
Authors: Vidovic Sinisa , Korber Darren R.
Published: June 2006

The prevalence of
Escherichia coli
O157 associated with feedlot cattle in Saskatchewan was determined in a 10-month longitudinal study (3 feedlots) and a point prevalence study (20 feedlots). The prevalence of
E. coli
O157 at the three different sites in the horizontal study varied from 2.5 to 45%. The point prevalence of
E. coli
O157 among Saskatchewan cattle from 20 different feedlots ranged from 0% to a high of 57%. A statistically significant (
P
= 0.003) positive correlation was determined to exist between the density of cattle and the
E. coli
O157 prevalence rate. A significant correlation (
P
= 0.006) was also found between the
E. coli
O157 percent prevalence and the number of cattle housed/capacity ratio. All 194
E. coli
O157 isolates obtained were highly virulent, and random amplified polymorphic DNA PCR analysis revealed that the isolates grouped into 39 different
E. coli
O157 subtypes, most of which were indigenous to specific feedlots. Two of the most predominant subtypes were detected in 11 different feedlots and formed distinct clusters in two geographic regions in the province. Antimicrobial susceptibility testing of the
E. coli
O157 isolates revealed that 10 were multidrug resistant and that 73 and 5 were resistant to sulfisoxazole and tetracycline, respectively.
Atypical
Helicobacter canadensis
Strains Associated with Swine
Authors: Inglis G. Douglas , McConville Malcolm , de Jong Anno
Published: June 2006

Forty-two
Helicobacter
isolates were isolated from swine feces in The Netherlands and Denmark. All 12 isolates sequenced (16S rRNA gene) formed a robust clade with
Helicobacter canadensis
(∼99% similarity). Species-specific PCR indicated that all of the isolates were
H. canadensis
isolates. Although the appearance of the porcine isolates was similar to the appearance of
H. canadensis
, only one of these isolates was able to hydrolyze indoxyl acetate, a cardinal characteristic of this taxon. Examination of the 23S rRNA and
hsp60
genes revealed high levels of similarity between the porcine isolates and
H. canadensis
. However, amplified fragment length polymorphism genomic typing showed that isolates recovered from swine feces were genetically distinct from
H. canadensis
strains obtained from humans and geese.
Microbial Diversity in Water and Sediment of Lake Chaka, an Athalassohaline Lake in Northwestern China
Authors: Jiang Hongchen , Dong Hailiang , Zhang Gengxin , Yu Bingsong , Chapman Leah R. , Fields Matthew W.
Published: June 2006

We employed culture-dependent and -independent techniques to study microbial diversity in Lake Chaka, a unique hypersaline lake (32.5% salinity) in northwest China. It is situated at 3,214 m above sea level in a dry climate. The average water depth is 2 to 3 cm. Halophilic isolates were obtained from the lake water, and halotolerant isolates were obtained from the shallow sediment. The isolates exhibited resistance to UV and gamma radiation. Microbial abundance in the sediments ranged from 108cells/g at the water-sediment interface to 107cells/g at a sediment depth of 42 cm. A major change in the bacterial community composition was observed across the interface. In the lake water, clone sequences affiliated with theBacteroideteswere the most abundant, whereas in the sediments, sequences related to low G+C gram-positive bacteria were predominant. A similar change was also present in the archaeal community. While all archaeal clone sequences in the lake water belonged to theHalobacteriales, the majority of the sequences in the sediments were related to those previously obtained from methanogenic soils and sediments. The observed changes in the microbial community structure across the water-sediment interface were correlated with a decrease in salinity from the lake water (32.5%) to the sediments (approximately 4%). Across the interface, the redox state also changed from oxic to anoxic and may also have contributed to the observed shift in the microbial community.
Analysis of Quantitative Interactions between Two Species of Arbuscular Mycorrhizal Fungi,Glomus mosseaeandG. intraradices, by Real-Time PCR
Authors: Alkan Noam , Gadkar Vijay , Yarden Oded , Kapulnik Yoram
Published: June 2006

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs, known to play an important role in ecological processes. Conventional light microscopy is the most common method used to detect their presence in planta, but this method fails to discern the presence of multiple AMF species and is not quantitative. These two factors are critically important in ecological studies, where the symbiotic contribution of each isolate needs to be defined. This paper describes the use of quantitative real-time PCR (qRT-PCR) as a detection system to address this issue. We used twoGlomusspp., namely,G. intraradicesandG. mosseae, to show that it is possible to study the interactions between these two isolates during the cocolonization of a single root system. Three different physiological studies were set up to assess how the interactions affected the occupancy of these fungi intraradically on a temporal basis. These treatments included saline and phosphorus stress, spatial distribution in the root zone, and preference for a particular host. qRT-PCR could prove a valuable tool in the area of AMF field ecology, where such data are critically important for defining the role of each species in the community structure.
Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines
Authors: Cebollero Eduardo , Gonzalez Ramon
Published: June 2006

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeastSaccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.
Evaluation of a Macrofoam Swab Protocol for the Recovery of
Bacillus anthracis
Spores from a Steel Surface
Authors: Hodges L. R. , Rose L. J. , Peterson A. , Noble-Wang J. , Arduino M. J.
Published: June 2006

A protocol to recover
Bacillus anthracis
spores from a steel surface using macrofoam swabs was evaluated for its accuracy, precision, reproducibility, and limit of detection. Macrofoam swabs recovered 31.7 to 49.1% of spores from 10-cm
2
steel surfaces with a ≤32.7% coefficient of variation in sampling precision and reproducibility for inocula of ≥38 spores.
Evidence for an Intermediate Colony Morphology of
Vibrio vulnificus
Authors: Rosche Thomas M. , Smith Ben , Oliver James D.
Published: June 2006

Vibrio vulnificus
causes both food-borne disease and wound infections. Most
V. vulnificus
strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. Under standard growth conditions, CPS expression is lost at a relatively high frequency (10
−3
to 10
−4
), resulting in a switch from an opaque (Op, CPS
+
) colony morphology to a translucent (Tr, CPS
−
) colony morphology. The
wzb
gene, which encodes a phosphatase required for CPS expression, has been proposed to be involved in this switch through a site-specific deletion of the entire gene. In an examination of five strains, we found that the frequency of
wzb
deletion in Tr colonies varies by strain and therefore does not account for all the Tr colonies that are seen. In addition, we have identified a third, intermediate (Int) colony morphotype, in which the colonies appear less opaque but are not fully translucent. PCR studies have demonstrated that Int colonies still contain the
wzb
gene, while reverse transcriptase PCR studies have shown that although Int strains retain expression of
wzb
, in some cases the transcription of
wzb
is reduced. Int strains switch to a true Tr (
wzb
negative) morphotype at a very high frequency (nearly 100%) under certain conditions. Finally, Int colonies, which in some cases can easily be mistaken for Tr colonies, have been observed to occasionally revert to Op, while Tr colonies containing a
wzb
deletion presumably are unable to revert to the encapsulated form.
Cellulose Acetate Containing Toluidine Blue and Rose Bengal Is an Effective Antimicrobial Coating when Exposed to White Light
Authors: Decraene Valérie , Pratten Jonathan , Wilson Michael
Published: June 2006

Simple methods of reducing the microbial load on surfaces in hospitals are needed to reduce the risk of hospital-associated infections. Here we report on the ability of a cellulose acetate coating containing the photosensitizers toluidine blue and rose bengal to kill microbes (
Staphylococcus aureus
,
Escherichia coli
,
Clostridium difficile
, a bacteriophage, and
Candida albicans
) on its surface when illuminated with white light.
Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation
Authors: Ní Chadhain Sinéad M. , Norman R. Sean , Pesce Karen V. , Kukor Jerome J. , Zylstra Gerben J.
Published: June 2006

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.
Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms
Authors: Burmølle Mette , Webb Jeremy S. , Rao Dhana , Hansen Lars H. , Sørensen Søren J. , Kjelleberg Staffan
Published: June 2006

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga
Ulva australis
, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates,
Microbacterium phyllosphaerae
,
Shewanella japonica
,
Dokdonia donghaensis
, and
Acinetobacter lwoffii
, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing
Pseudoalteromonas tunicata
, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.
Construction of a Large Signature-Tagged Mini-Tn
5
Transposon Library and Its Application to Mutagenesis of
Sinorhizobium meliloti
Authors: Pobigaylo Nataliya , Wetter Danijel , Szymczak Silke , Schiller Ulf , Kurtz Stefan , Meyer Folker , Nattkemper Tim W. , Becker Anke
Published: June 2006

Sinorhizobium meliloti
genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged
S. meliloti
mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.
Completeness of cause of injury coding in healthcare administrative databases in the United States, 2001
Authors: Coben J H , Steiner C A , Barrett M , Merrill C T , Adamson D
Published: June 2006

Objectives:To determine the completeness of external cause of injury coding (E-coding) within healthcare administrative databases in the United States and to identify factors that contribute to variations in E-code reporting across states.

Design:Cross sectional analysis of the 2001 Healthcare Cost and Utilization Project (HCUP), including 33 State Inpatient Databases (SID), a Nationwide Inpatient Sample (NIS), and nine State Emergency Department Databases (SEDD). To assess state reporting practices, structured telephone interviews were conducted with the data organizations that participate in HCUP.

Results:The percent of injury records with an injury E-code was 86% in HCUP’s nationally representative database, the NIS. For the 33 states represented in the SID, completeness averaged 87%, with more than half of the states reporting E-codes on at least 90% of injuries. In the nine states also represented in the SEDD, completeness averaged 93%. Twenty two states had mandates for E-code reporting, but only eight had provisions for enforcing the mandates. These eight states had the highest rates of E-code completeness.

Conclusions:E-code reporting in administrative databases is relatively complete, but there is significant variation in completeness across the states. States with mandates for the collection of E-codes and with a mechanism to enforce those mandates had the highest rates of E-code reporting. Nine statewide ED data systems demonstrate consistently high E-coding completeness.
Trends in childhood injury mortality in Canada, 1979–2002
Authors: Pan S Y , Ugnat A-M , Semenciw R , Desmeules M , Mao Y , MacLeod M
Published: June 2006

Objective:To examine national trends in mortality rates for injuries among Canadian children younger than 15 years in 1979–2002.

Methods:Data on injury deaths were obtained from the Canadian Vital Statistics system at Statistics Canada. Injuries were classified using the codes for external cause of injury and poisoning (E-codes) by intent and by mechanism. Mortality rates were age adjusted to the 1990 world standard population. Negative binomial regression was used to estimate the secular trends.

Results:Annual mortality rates for total and unintentional injuries declined substantially (from 23.8 and 21.7 in 1979 to 7.2 and 5.8 in 2002, respectively), whereas suicide deaths among children aged 10–14 showed an increasing trend. All Canadian provinces and territories showed a decreasing trend in mortality rates of total injuries. Motor vehicle related injuries were the most common cause of injury deaths (accounted for an average of 36.4% of total injury deaths), followed by suffocation (14.3%), drowning (13.5%), and burning (11.1%); however, suffocation was the leading cause for infants. The number of potential years of life lost due to injury before age 75 decreased from 89 343 in 1979 to 27 948 in 2002 for children aged 0–14 years.

Conclusions:During the period 1979–2002, there were dramatic decreases in childhood mortality for total injuries and unintentional injuries as well as various degrees of reduction for all causes of injury except suffocation in children aged 10–14 years and drowning in infants. The reason for the reduction in injury mortality might be multifactoral.
Depressive symptoms as a risk factor for unintentional injury: a cohort study in a rural county
Authors: Tiesman H M , Peek-Asa C , Whitten P , Sprince N L , Stromquist A , Zwerling C
Published: June 2006

Objective:The authors used data from a population based prospective cohort study to determine if depressive symptoms predicted incidence of unintentional injury.

Methods:The Keokuk County Rural Health Study, based in Iowa, is a prospective cohort study of health status that includes injury outcomes. Depressive symptoms were measured using the 11-item Center for Epidemiologic Studies Depression Scale at the beginning of the study on 1493 participants. Quarterly follow up phone calls were made to measure injury incidence.

Results:471 injuries were reported for an overall injury rate of 9.8 per 100 person-years. Crude injury rates were significantly higher for those with depressive symptoms (p = 0.0003). Those with depressive symptoms had a 41% increased risk for injury after controlling for antidepressant medication use, gender, prior injury, income, and sleepiness (RR = 1.41, 95% CI 1.10 to 1.80). Depressive symptoms remained a risk factor for injury regardless of current antidepressant medication use (no medication use, RR = 1.43, 95% CI 1.09 to 1.88; medication use, RR = 1.31, 95% CI 0.76 to 2.26).

Conclusions:Depressive symptoms were found to be risk factors for unintentional injury. Medical practitioners should consider talking about safety with their patients, especially those reporting symptoms of depression, and recognize that an increased risk for injury remains until the depressive symptoms subside.