bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluidization of Membrane Lipids Enhances the Tolerance of
Saccharomyces cerevisiae
to Freezing and Salt Stress
Authors: Rodríguez-Vargas Sonia , Sánchez-García Alicia , Martínez-Rivas Jose Manuel , Prieto Jose Antonio , Randez-Gil Francisca
Published: December 2006

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but
Saccharomyces cerevisiae
can introduce only a single double bond at the Δ
9
position. We expressed two sunflower (
Helianthus annuus
) oleate Δ
12
desaturases encoded by
FAD2-1
and
FAD2-3
in yeast cells of the wild-type W303-1A strain (
trp1
) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ
9,12
, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the
FAD2
strains, probably due to tryptophan auxotrophy, since the
trp1
(
TRP1
) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp
+
or Trp
−
strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in
S. cerevisiae
, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.
Respiration and Growth of
Shewanella decolorationis
S12 with an Azo Compound as the Sole Electron Acceptor
Authors: Hong Yiguo , Xu Meiying , Guo Jun , Xu Zhicheng , Chen Xingjuan , Sun Guoping
Published: December 2006

The ability of
Shewanella decolorationis
S12 to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory azoreduction was investigated. This microorganism can reduce a variety of azo dyes by use of formate, lactate, pyruvate, or H
2
as the electron donor. Furthermore, strain S12 grew to a maximal density of 3.0 × 10
7
cells per ml after compete reduction of 2.0 mM amaranth in a defined medium. This was accompanied by a stoichiometric consumption of 4.0 mM formate over time when amaranth and formate were supplied as the sole electron acceptor and donor, respectively, suggesting that microbial azoreduction is an electron transport process and that this electron transport can yield energy to support growth. Purified membranous, periplasmic, and cytoplasmic fractions from S12 were analyzed, but only the membranous fraction was capable of reducing azo dyes with formate, lactate, pyruvate, or H
2
as the electron donor. The presence of 5 μM Cu
2+
ions, 200 μM dicumarol, 100 μM stigmatellin, and 100 μM metyrapone inhibited anaerobic azoreduction activity by both whole cells and the purified membrane fraction, showing that dehydrogenases, cytochromes, and menaquinone are essential electron transfer components for azoreduction. These results provide evidence that the microbial anaerobic azoreduction is linked to the electron transport chain and suggest that the dissimilatory azoreduction is a form of microbial anaerobic respiration. These findings not only expand the number of potential electron acceptors known for microbial energy conservation but also elucidate the mechanisms of microbial anaerobic azoreduction.
Bacterial Population Dynamics in Dairy Waste during Aerobic and Anaerobic Treatment and Subsequent Storage
Authors: McGarvey Jeffery A. , Miller William G. , Zhang Ruihong , Ma Yanguo , Mitloehner Frank
Published: December 2006

The objective of this study was to model a typical dairy waste stream, monitor the chemical and bacterial population dynamics that occur during aerobic or anaerobic treatment and subsequent storage in a simulated lagoon, and compare them to those of waste held without treatment in a simulated lagoon. Both aerobic and anaerobic treatment methods followed by storage effectively reduced the levels of total solids (59 to 68%), biological oxygen demand (85 to 90%), and sulfate (56 to 65%), as well as aerobic (83 to 95%), anaerobic (80 to 90%), and coliform (>99%) bacteria. However, only aerobic treatment reduced the levels of ammonia, and anaerobic treatment was more effective at reducing total sulfur and sulfate. The bacterial population structure of waste before and after treatment was monitored using 16S rRNA gene sequence libraries. Both treatments had unique effects on the bacterial population structure of waste. Aerobic treatment resulted in the greatest change in the type of bacteria present, with the levels of eight out of nine phyla being significantly altered. The most notable differences were the >16-fold increase in the phylumProteobacteriaand the approximately 8-fold decrease in the phylumFirmicutes. Anaerobic treatment resulted in fewer alterations, but significant decreases in the phylaActinobacteriaandBacteroidetes, and increases in the phylaPlanctomycetes,Spirochetes, and TM7 were observed.
Vertical Distribution of Denitrification in an Estuarine Sediment: Integrating Sediment Flowthrough Reactor Experiments and Microprofiling via Reactive Transport Modeling
Authors: Laverman Anniet M. , Meile Christof , Van Cappellen Philippe , Wieringa Elze B. A.
Published: December 2006

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N2O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N2O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm−3h−1. The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (± 10) nmol cm−2h−1, which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.
A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of
Listeria monocytogenes
Authors: Ducey Thomas F. , Page Brent , Usgaard Thomas , Borucki Monica K. , Pupedis Kitty , Ward Todd J.
Published: December 2006

Listeria monocytogenes
is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of
L. monocytogenes
are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for
L. monocytogenes
lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65
L. monocytogenes
isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in
inlA
, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.
Effects of Target Length on the Hybridization Efficiency and Specificity of rRNA-Based Oligonucleotide Microarrays
Authors: Liu Wen-Tso , Guo Huiling , Wu Jer-Horng
Published: December 2006

The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of
Escherichia coli
. The targets included unfragmented native rRNA, fragmented rRNA (∼20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies.
Spatial and Temporal Expression of
Lactobacillus plantarum
Genes in the Gastrointestinal Tracts of Mice
Authors: Marco Maria L. , Bongers Roger S. , de Vos Willem M. , Kleerebezem Michiel
Published: December 2006

Lactobacillus plantarum
is a common inhabitant of mammalian gastrointestinal tracts, and
L. plantarum
strain WCFS1 is a human isolate with a known genome sequence.
L. plantarum
WCFS1 survives intestinal passage in an active form, and its transit time and transcriptional activities were monitored in 15 BALB/c mice at 2, 4, 6, 8, and 24 h after being fed a single intragastric dose of this organism. Enumeration of viable cells isolated from fecal material revealed that the majority of the
L. plantarum
inoculum transited the mouse intestine within 4 h after ingestion. Three mice were sacrificed at each time point, and total RNA was isolated from the mouse intestinal compartments (stomach through colon). Quantification of
L. plantarum
16S rRNA by quantitative real-time reverse-transcription-PCR revealed that
L. plantarum
was present at elevated levels in the stomach and small intestine for at least 4 h following ingestion and for over 8 h in the cecum and colon. We also examined the expression of 9
L. plantarum
housekeeping genes and 15
L. plantarum
in vivo-inducible (
ivi
) genes previously identified by recombination-based in vivo expression technology to be induced in the mouse gastrointestinal tract. The relative expression levels of the
ivi
genes increased up to 350-fold in the mouse intestine compared to levels observed for
L. plantarum
WCFS1 cells grown in a rich laboratory medium. Moreover, several genes displayed intestinal compartment-specific (small intestine versus colon) activities. These results confirm that
L. plantarum
displays specific and differential responses at various sites along the mammalian intestine.
Sources of Variation in the Ampicillin-Resistant
Escherichia coli
Concentration in the Feces of Organic Broiler Chickens
Authors: Pleydell E. J. , Brown P. E. , Woodward M. J. , Davies R. H. , French N. P.
Published: December 2006

Currently, there are limited published data for the population dynamics of antimicrobial-resistant commensal bacteria. This study was designed to evaluate both the proportions of the
Escherichia coli
populations that are resistant to ampicillin at the level of the individual chicken on commercial broiler farms and the feasibility of obtaining repeated measures of fecal
E. coli
concentrations. Short-term temporal variation in the concentration of fecal
E. coli
was investigated, and a preliminary assessment was made of potential factors involved in the shedding of high numbers of ampicillin-resistant
E. coli
by growing birds in the absence of the use of antimicrobial drugs. Multilevel linear regression modeling revealed that the largest component of random variation in log-transformed fecal
E. coli
concentrations was seen between sampling occasions for individual birds. The incorporation of fixed effects into the model demonstrated that the older, heavier birds in the study were significantly more likely (
P
= 0.0003) to shed higher numbers of ampicillin-resistant
E. coli
. This association between increasing weight and high shedding was not seen for the total fecal
E. coli
population (
P
= 0.71). This implies that, in the absence of the administration of antimicrobial drugs, the proportion of fecal
E. coli
that was resistant to ampicillin increased as the birds grew. This study has shown that it is possible to collect quantitative microbiological data on broiler farms and that such data could make valuable contributions to risk assessments concerning the transfer of resistant bacteria between animal and human populations.
Longitudinal Study of Finnish
Campylobacter jejuni
and
C. coli
Isolates from Humans, Using Multilocus Sequence Typing, Including Comparison with Epidemiological Data and Isolates from Poultry and Cattle
Authors: Kärenlampi Rauni , Rautelin Hilpi , Schönberg-Norio Daniela , Paulin Lars , Hänninen Marja-Liisa
Published: December 2006

We describe a study on the application of multilocus sequence typing for the analysis of
Campylobacter jejuni
and
C. coli
isolates from human domestically acquired infections in the Helsinki-Uusimaa area of Finland in 1996, 2002, and 2003. In addition, isolates from poultry meat and fecal samples of cattle from the seasonal peak (July to September) in 2003 were included in the study. In total, 361 Finnish
C. jejuni
and
C. coli
strains were typed. Sequence type 45 (ST-45) (45%), ST-21 (21%), and ST-677 (11%) clonal complexes were the most prevalent. The ST-45 and ST-677 complexes were overrepresented in comparison with previous studies. The longitudinal study revealed an association between
C. coli
(ST-828 complex) infection and elderly patients (≥60 years). Analysis of exposure factors, determined by a previous case-control study conducted during the seasonal peak in 2002, revealed that the ST-48 complex was significantly (
P
< 0.05) associated with the tasting or eating of raw minced meat. New and unassigned STs were associated with swimming in natural bodies of water, whereas the ST-677 complex was related to drinking nonchlorinated water from a small water plant or water from natural sources. The ST-45 complex was associated with contact with pet cats and dogs. In 2003, ST-45 occurrence was significantly associated with poultry whereas ST-50 was associated with isolates from humans. In contrast, ST-53, ST-58, ST-61, and ST-883 were significantly associated with isolates from cattle. Further studies are needed to reveal the significance of the observed associations.
Potential of the
Bacillus thuringiensis
Toxin Reservoir for the Control of
Lobesia botrana
(Lepidoptera: Tortricidae), a Major Pest of Grape Plants
Authors: Ruiz de Escudero Iñigo , Estela Anna , Escriche Baltasar , Caballero Primitivo
Published: December 2006

The potential of
Bacillus thuringiensis
Cry proteins to control the grape pest
Lobesia botrana
was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from
B. thuringiensis
, was the protein most toxic to
L. botrana
larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of
B. thuringiensis
sprays or expressed together in transgenic plants.
Comparison of Media for the Isolation of
Enterobacter sakazakii
Authors: Iversen Carol , Forsythe Stephen J.
Published: December 2006

Enterobacter sakazakii
is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some
E. sakazakii
strains do not grow in standard media for
Enterobacteriaceae
and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three
E. sakazakii
enrichment broths—
Enterobacteriaceae
enrichment broth (EE),
E. sakazakii
selective broth (ESSB), and modified lauryl sulfate broth (mLST)—were compared with a novel broth designed for maximum recovery of
E. sakazakii
,
E. sakazakii
enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB.
E. sakazakii
possesses α-glucosidase activity, and a number of selective, chromogenic agars for
E. sakazakii
isolation based on this enzyme have been developed.
E. sakazakii
isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more
E. sakazakii
strains. It was also determined that 2% of
E. sakazakii
strains did not produce yellow pigmentation on tryptone soya agar at 25°C, a characteristic frequently cited in the identification of
E. sakazakii
. The recovery of desiccated
E. sakazakii
(0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of
E. sakazakii
do not support the growth of all currently known
E. sakazakii
phenotypes; therefore, improvements in the proposed methods are desirable.
Isolation of
Salmonella enterica
and Shiga-Toxigenic
Escherichia coli
O157 from Feces of Animals in Public Contact Areas of United States Zoological Parks
Authors: Keen James E. , Durso Lisa M. , Meehan Thomas P.
Published: December 2006

The fecal prevalence of subclinical
Salmonella enterica
and Shiga-toxigenic
Escherichia coli
O157 among animals in human-animal contact exhibits at institutions in the United States accredited by the Association of Zoos and Aquariums was estimated to assess public health risk. The prevalence was less than 0.6% for both zoonotic pathogens among 997 animals sampled at 36 exhibits.
Trigonopsis variabilisd-Amino Acid Oxidase: Control of Protein Quality and Opportunities for Biocatalysis through Production inEscherichia coli
Authors: Dib Iskandar , Stanzer Damir , Nidetzky Bernd
Published: December 2006

Trigonopsis variabilisd-amino acid oxidase accounts for 35% ofEscherichia coliprotein when addedd-methionine suppresses the toxic activity of the recombinant product. PermeabilizedE. colicells are reusable and stabilized enzyme preparations. The purified oxidase lacks the microheterogeneity of the natural enzyme. Oriented immobilization of a chimeric oxidase maintains 80% of the original activity in microparticle-bound enzymes.
Communities of
Archaea
and
Bacteria
in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps, and Evidence of Ammonia-Oxidizing
Crenarchaeota
Authors: Weidler Gerhard W. , Dornmayr-Pfaffenhuemer Marion , Gerbl Friedrich W. , Heinen Wolfgang , Stan-Lotter Helga
Published: December 2006

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the “Franz-Josef-Quelle” in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated
Euryarchaeota
. These findings support recent reports that
Crenarchaeota
are not restricted to high-temperature environments. Most of the bacterial sequences were related to the
Proteobacteria
(α, β, γ, and δ),
Bacteroidetes
, and
Planctomycetes
. One OTU was allied with
Nitrospina
sp. (
δ-Proteobacteria
) and three others grouped with
Nitrospira.
Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since
Crenarchaeota
have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (
amoA
) gene. Sequences were obtained which were related to crenarchaeotic
amoA
genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.
Gene Copy Number Polymorphisms in an Arbuscular Mycorrhizal Fungal Population
Authors: Corradi Nicolas , Croll Daniel , Colard Alexandre , Kuhn Gerrit , Ehinger Martine , Sanders Ian R.
Published: December 2006

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus
Glomus intraradices
by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.
Multiple-Level Regulation of Genes for Protocatechuate Degradation in
Acinetobacter baylyi
Includes Cross-Regulation
Authors: Siehler Simone Yasmin , Dal Süreyya , Fischer Rita , Patz Patricia , Gerischer Ulrike
Published: December 2006

The bacterium
Acinetobacter baylyi
uses the branched β-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the
pca-qui
operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter
pcaIp
, namely protocatechuate-dependent activation of
pca-qui
operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (
Escherichia coli
) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the
pca-qui
operon. The increase of the
pcaU
gene copy number leads to a decrease of the basal expression at
pcaIp
, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of
pcaIp
when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (cross-regulation). The same repression pattern was observed for promoter
pcaUp
. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (
pobAp
upstream of
pobA
,
pcaIp
, and
pcaUp
) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on
pobA
expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of
pobA
expression.
Lytic Activity of Recombinant Bacteriophage φ11 and φ12 Endolysins on Whole Cells and Biofilms of
Staphylococcus aureus
Authors: Sass Peter , Bierbaum Gabriele
Published: December 2006

The recombinant φ11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells, and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the φ12 endolysin was inactive and caused aggregation of the cells.
Identification and Characterization of Conjugative Transposons CTn86 and CTn9343 in
Bacteroides fragilis
Strains
Authors: Buckwold Simy L. , Shoemaker Nadja B. , Sears Cynthia L. , Franco Augusto A.
Published: December 2006

The related genetic elements flanking the
Bacteroides fragilis
pathogenicity island (PAI) in enterotoxigenic
B. fragilis
(ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic
B. fragilis
(NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the
B. fragilis
PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in
B. fragilis
strains and predominantly in division I,
cepA
-positive strains.
Identification of Acetate-Assimilating Microorganisms under Methanogenic Conditions in Anoxic Rice Field Soil by Comparative Stable Isotope Probing of RNA
Authors: Hori Tomoyuki , Noll Matthias , Igarashi Yasuo , Friedrich Michael W. , Conrad Ralf
Published: December 2006

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH
4
and/or CO
2
. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with
13
C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which
13
C-labeled acetate was added at a concentration of 500 μM every 3 days.
13
C-labeled CH
4
and CO
2
were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate
13
C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the
13
C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a
Geobacter
sp. and an
Anaeromyxobacter
sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO
2
was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for
Geobacter
spp. and
Anaeromyxobacter
spp. under these conditions.
Removal of Astrovirus from Water and Sewage Treatment Plants, Evaluated by a Competitive Reverse Transcription-PCR
Authors: Morsy El-Senousy Waled , Guix Susana , Abid Islem , Pintó Rosa M. , Bosch Albert
Published: December 2006

Quantification of human astrovirus genogroups A and B was undertaken with sewage and water samples, collected from the Greater Cairo area in Egypt from November 1998 to October 1999, by a competitive reverse transcription (RT)-PCR with an internal control. The number of RNA copies of genogroup A/liter in quantifiable samples ranged from 3.4 × 10
3
to 5.6 × 10
6
in raw sewage and from 3.4 × 10
3
to 1.1 × 10
4
in treated effluents, while the number of infectious units per liter in these samples as determined by cell culture RT-PCR (CC-RT-PCR U/liter) ranged from 3.3 × 10
1
to 3.3 × 10
3
in raw sewage and was 3.3 × 10
0
in treated effluents. On the other hand, the number of RNA copies/liter in quantifiable genogroup B samples ranged from 1.1 × 10
4
to 8.7 × 10
6
in raw sewage and from 1.1 × 10
3
to 6.2 × 10
5
in treated effluents, while the number of infectious units ranged from 3.3 × 10
1
to 3.3 × 10
5
CC-RT-PCR U/liter in raw sewage and from 3.3 × 10
1
to 3.3 × 10
2
CC-RT-PCR U/liter in treated effluents. These higher numbers of both RNA copies/liter and infectious particles of genogroup B may indicate the emergence of genogroup B in the area. Additionally, genogroup B astrovirus exhibited a higher resistance to removal treatments with regard to the number of RNA copies per ml. When the equipment for real-time approaches is unavailable, a competitive PCR or RT-PCR with an internal control may be employed for virus quantification in validations of the efficiency of virus removal treatments.
Folate Production by Bifidobacteria as a Potential Probiotic Property
Authors: Pompei Anna , Cordisco Lisa , Amaretti Alberto , Zanoni Simona , Matteuzzi Diego , Rossi Maddalena
Published: December 2006

The ability of 76
Bifidobacterium
strains to produce folate was investigated. In order to evaluate folic acid productivity, bifidobacteria were cultivated in the folate-free semisynthetic medium SM7. Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were not a function of species but were distinctive features of individual strains. Six strains among the 17 that grew without folate produced significantly higher concentrations of vitamin (between 41 and 82 ng ml
−1
). The effects of exogenous folate and
p
-aminobenzoic acid (PABA) concentrations on folate production were evaluated. In contrast to most of the other strains, the folate yield of
B. adolescentis
MB 239 was not negatively affected by either PABA or exogenous folic acid. Folate production by
B. adolescentis
MB 239 was studied in the pH range of the colonic environment, and a comparison of folate production on raffinose, lactose, and fructo-oligosaccharides, which belong to three important groups of fermentable intestinal carbon sources, was established. Differences in folate biosynthesis by
B. adolescentis
MB 239 were not observed as a function either of the pH or of the carbon source. Fecal culture experiments demonstrated that the addition of
B. adolescentis
MB 239 may increase the folate concentration in the colonic environment.
Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR
Authors: Fukushima Hiroshi , Katsube Kazunori , Hata Yukiko , Kishi Ryoko , Fujiwara Satomi
Published: December 2006

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (
Salmonella enterica
,
Escherichia coli
,
Yersinia enterocolitica
,
Campylobacter jejuni
,
Vibrio cholerae
O139,
Vibrio parahaemolyticus
O3K6,
Vibrio vulnificus
,
Providencia alcalifaciens
,
Aeromonas hydrophila
,
Bacillus cereus
,
Staphylococcus aureus
, and
Clostridium perfringens
) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10
1
to 10
3
CFU/g using the RTi-qPCR assay, and amounts as small as 10
0
to 10
1
CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10
1
to 10
2
CFU/g of
Salmonella
and
C. jejuni
directly in naturally contaminated chicken and the presence of
S. aureus
directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.
Biotransformation of Hg(II) by Cyanobacteria
Authors: Lefebvre Daniel D. , Kelly David , Budd Kenneth
Published: December 2006

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl
2
in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl
2
. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that
meta
-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria:
Limnothrix planctonica
(Lemm.),
Synechococcus leopoldiensis
(Racib.) Komarek, and
Phormidium limnetica
(Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.
Identification and Quantification of Methanogenic Archaea in Adult Chicken Ceca
Authors: Saengkerdsub Suwat , Anderson Robin C. , Wilkinson Heather H. , Kim Woo-Kyun , Nisbet David J. , Ricke Steven C.
Published: December 2006

By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to
Methanobrevibacter woesei
, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log
10
/g (wet weight), respectively.
Localization of Functional Polypeptides in Bacterial Inclusion Bodies
Authors: García-Fruitós Elena , Arís Anna , Villaverde Antonio
Published: December 2006

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a β-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular β-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.
Conjugative Transfer Facilitates Stable Maintenance of IncP-1 Plasmid pKJK5 in
Escherichia coli
Cells Colonizing the Gastrointestinal Tract of the Germfree Rat
Authors: Bahl Martin Iain , Hansen Lars Hestbjerg , Licht Tine Rask , Sørensen Søren J.
Published: December 2006

Quantitative determination of IncP-1 plasmid loss from
Escherichia coli
cells colonizing the gastrointestinal tracts of germfree rats was achieved by flow cytometry. Results show that the plasmid's ability to conjugate counteracts plasmid loss and is thus an important mechanism for the stable maintenance of IncP-1 plasmids within the gastrointestinal environment.
Increased Insect Virulence in
Beauveria bassiana
Strains Overexpressing an Engineered Chitinase
Authors: Fan Yanhua , Fang Weiguo , Guo Shujuan , Pei Xiaoqiong , Zhang Yongjun , Xiao Yuehua , Li Demou , Jin Kai , Bidochka Michael J. , Pei Yan
Published: December 2006

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as
Beauveria bassiana
, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in
B. bassiana
, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several
B. bassiana
hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm
Bombyx mori
chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bb
chit1
-Bm
ChBD
) was then placed under the control of a fungal constitutive promoter (
gpd
-Bb
chit1
-Bm
ChBD
) and transformed into
B. bassiana
. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (
gpd
-Bb
chit1
) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.
Highly Prevalent
Coxiella
sp. Bacterium in the Tick Vector
Amblyomma americanum
Authors: Jasinskas Algimantas , Zhong Jianmin , Barbour Alan G.
Published: December 2006

Laboratory-reared and field-collected
Amblyomma americanum
ticks were hosts of a
Coxiella
sp. and a
Rickettsia
sp. While the
Coxiella
sp. was detected in 50 of 50 field-collected ticks, the
Rickettsia
sp. was absent from 32% of ticks. The
Coxiella
sp. showed evidence of a reduced genome and may be an obligate endosymbiont.
Evidence that Accumulation of Mutants in a Biofilm Reflects Natural Selection Rather than Stress-Induced Adaptive Mutation
Authors: Banas Jeffrey A. , Miller Justin D. , Fuschino Meghan E. , Hazlett Karsten R. O. , Toyofuku Wendy , Porter Kristen A. , Reutzel Sarah B. , Florczyk Matthew A. , McDonough Kathleen A. , Michalek Suzanne M.
Published: December 2006

The accumulation of mutant genotypes within a biofilm evokes the controversy over whether the biofilm environment induces adaptive mutation or whether the accumulation can be explained by natural selection. A comparison of the virulence of two strains of the dental pathogen
Streptococcus mutans
showed that rats infected with one of the strains accumulated a high proportion (average, 22%) of organisms that had undergone a deletion between two contiguous and highly homologous genes. To determine if the accumulation of deletion mutants was due to selection or to an increased mutation rate, accumulations of deletion mutants within in vitro planktonic and biofilm cultures and within rats inoculated with various proportions of deletion organisms were quantified. We report here that natural selection was the primary force behind the accumulation of the deletion mutants.
Isolation and Identification of a
Paenibacillus polymyxa
Strain That Coproduces a Novel Lantibiotic and Polymyxin
Authors: He Zengguo , Kisla Duygu , Zhang Liwen , Yuan Chunhua , Green-Church Kari B. , Yousef Ahmed E.
Published: December 2006

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as
Paenibacillus polymyxa
and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including
Bacillus
spp.,
Clostridium sporogenes
,
Lactobacillus
spp.,
Lactococcus lactis
,
Leuconostoc mesenteroides
,
Listeria
spp.,
Pediococcus cerevisiae
,
Staphylococcus aureus
, and
Streptococcus agalactiae
. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.
Rhizobium etli
USDA9032 Engineered To Produce a Phenazine Antibiotic Inhibits the Growth of Fungal Pathogens but Is Impaired in Symbiotic Performance
Authors: Krishnan Hari B. , Kang Beom Ryong , Hari Krishnan Ammulu , Kim Kil Yong , Kim Young Cheol
Published: December 2006

Phenazine production was engineered in
Rhizobium etli
USDA9032 by the introduction of the
phz
locus of
Pseudomonas chlororaphis
O6. Phenazine-producing
R. etli
was able to inhibit the growth of
Botrytis cinerea
and
Fusarium oxysporum
in vitro. Black bean inoculated with phenazine-producing
R. etli
produced brownish Fix
−
nodules.
Synthetic Consolidants Attacked by Melanin-Producing Fungi: Case Study of the Biodeterioration of Milan (Italy) Cathedral Marble Treated with Acrylics
Authors: Cappitelli Francesca , Nosanchuk Joshua D. , Casadevall Arturo , Toniolo Lucia , Brusetti Lorenzo , Florio Sofia , Principi Pamela , Borin Sara , Sorlini Claudia
Published: December 2006

Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.