bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of DuplicatemsaGenes in the Salmonid PathogenRenibacteriumsalmoninarum
Authors: Rhodes Linda D. , Coady Alison M. , Strom Mark S.
Published: April 2009

Renibacterium salmoninarumis a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout.R.salmoninarumhas two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies ofmsaare expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region frommsa1ormsa2were constructed and introduced intoR.salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that bothmsa1andmsa2are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination inR.salmoninarum.
Transport ofCryptosporidium parvumOocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency
Authors: Atwill Edward R. , Hou Lingling , Karle Betsy M. , Harter Thomas , Tate Kenneth W. , Dahlgren Randy A.
Published: April 2009

Vegetated buffer strips were evaluated for their ability to remove waterborneCryptosporidium parvumfrom surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log10reductions for spikedC. parvumoocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm3. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborneC. parvum(1- to 2-log10reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log10reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% ofC. parvumoocysts from agricultural runoff generated during events involving mild to moderate precipitation.
Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon
Pyrococcus abyssi
Authors: Lucas Soizick , Toffin Laurent , Zivanovic Yvan , Charlier Daniel , Moussard Hélène , Forterre Patrick , Prieur Daniel , Erauso Gaël
Published: April 2009

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea,
Pyrococcus
spp
.
, is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon
Pyrococcus abyssi
. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from
P. abyssi
strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for
Pyrococcus
spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from
P. abyssi
strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the
pyrE
and/or
pyrF
genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two
pyrE
mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the
pyrE
gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote
Sulfolobus acidocaldarius
, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform
P. abyssi
GE9
pyrE
mutants to prototrophy, though with low frequency (10
2
to 10
3
transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both
Escherichia coli
and
P. abyssi
.
Widespread
N
-Acetyl-
d
-Glucosamine Uptake among Pelagic Marine Bacteria and Its Ecological Implications
Authors: Riemann Lasse , Azam Farooq
Published: April 2009

Dissolved free and combined
N
-acetyl-
d
-glucosamine (NAG) is among the largest pools of amino sugars in the ocean. NAG is a main structural component in chitin and a substantial constituent of bacterial peptidoglycan and lipopolysaccharides. We studied the distribution and kinetics of NAG uptake by the phosphoenolpyruvate:NAG phosphotransferase systems (PTS) in marine bacterial isolates and natural bacterial assemblages in near-shore waters. Of 78 bacterial isolates examined, 60 took up
3
H-NAG, while 18 showed no uptake. No systematic pattern in NAG uptake capability relative to phylogenetic affiliation was found, except that all isolates within
Vibrionaceae
took up NAG. Among 12 isolates, some showed large differences in the relationship between polymer hydrolysis (measured as chitobiase activity) and uptake of the NAG, the hydrolysis product. Pool turnover time and estimated maximum ambient concentration of dissolved NAG in samples off Scripps Pier (La Jolla, Calif.) were 5.9 ± 3.0 days (
n
= 10) and 5.2 ± 0.9 nM (
n
= 3), respectively. Carbohydrate competition experiments indicated that glucose, glucosamine, mannose, and fructose were taken up by the same system as NAG. Sensitivity to the antibiotic and NAG structural analog streptozotocin (STZ) was developed into a culture-independent approach, which demonstrated that approximately one-third of bacteria in natural marine assemblages that were synthesizing DNA took up NAG. Isolates possessing a NAG PTS system were found to be predominantly facultative anaerobes. These results suggest the hypothesis that a substantial fraction of bacteria in natural pelagic assemblages are facultative anaerobes. The adaptive value of fermentative metabolism in the pelagic environment is potentially significant, e.g., to bacteria colonizing microenvironments such as marine snow that may experience periodic O
2
-limitation.
A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium,Alteromonassp. Strain O-7
Authors: Miyamoto Katsushiro , Nukui Eiji , Hirose Mariko , Nagai Fumi , Sato Takaji , Inamori Yoshihiko , Tsujibo Hiroshi
Published: April 2009

Alteromonassp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned inEscherichia coli. The open reading frame ofmprIIIencoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated thatmprIIIwas induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant ofAlteromonassp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.
Presence of a Single Genotype of the Newly Described Species
Mycobacterium immunogenum
in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis
Authors: Wallace, Richard J. , Zhang Yansheng , Wilson Rebecca W. , Mann Linda , Rossmoore Harold
Published: April 2009

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species
Mycobacterium immunogenum
. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as
M. immunogenum
and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes
Dra
I and
Xba
I, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of
M. immunogenum
, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.
MtrB Is Required for Proper Incorporation of the Cytochromes OmcA and OmcB into the Outer Membrane of
Shewanella putrefaciens
MR-1
Authors: Myers Charles R. , Myers Judith M.
Published: April 2009

When grown under anaerobic conditions,
Shewanella putrefaciens
MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO
2
) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg
2+
, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an
mtrF
gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.
Salmonellae in Avian Wildlife in Norway from 1969 to 2000
Authors: Refsum Thorbjørn , Handeland Kjell , Baggesen Dorte Lau , Holstad Gudmund , Kapperud Georg
Published: April 2009

Postmortem records of wild-living birds in Norway with laboratory-confirmed findings of salmonella infection were summarized for the period from 1969 to 2000.
Salmonella
spp. were isolated from 470 birds belonging to 26 species. The salmonella-positive birds included 441 small passerines, 15 gulls, 5 waterfowl, 4 birds of prey, 3 doves, and 2 crows. The bullfinch (
Pyrrhula pyrrhula
) was by far the most frequently recorded species (54% of the cases).
Salmonella enterica
serover Typhimurium was recovered from all cases except from one hooded crow (
Corvus corone
), which yielded serovar Paratyphi-B var. Java. Variant O:4,12 comprised 96% (451 cases) of all serovar Typhimurium isolates, including all the passerines, while variant O:4,5,12 accounted for the remaining 4% (18 cases). The occurrence of salmonellae in small passerines showed a distinct seasonality, with a peak in February and March. Plasmid profile analysis of 346 isolates of serovar Typhimurium O:4,12 detected six profiles, of which two comprised 66 and 28% of the isolates, respectively. Phage typing of 52 randomly selected isolates of serovar Typhimurium O:4,12 from passerines detected four types: DT 40 (54%), U277 (35%), DT 99 (6%), and DT 110 (4%).
Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening
Authors: Benech R.-O. , Kheadr E. E. , Lacroix C. , Fliss I.
Published: April 2009

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against
Listeria innocua, Lactococcus
spp., and
Lactobacillus casei
subsp.
casei
and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing
Lactococcus lactis
subsp.
lactis
biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and
L. casei
subsp.
casei
as secondary flora, and ripened at 7°C for 6 months. In some trials,
L. innocua
was added to cheese milk at 10
5
to 10
6
CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in
L. casei
subsp.
casei
and
L. innocua
, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.
Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in
Escherichia coli
Authors: March J. C. , Eiteman M. A. , Altman E.
Published: April 2009

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in
Escherichia coli
containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, β-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.
Bacteria Belonging to the Genus
Cycloclasticus
Play a Primary Role in the Degradation of Aromatic Hydrocarbons Released in a Marine Environment
Authors: Kasai Yuki , Kishira Hideo , Harayama Shigeaki
Published: April 2009

To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus
Cycloclasticus
became predominant in the enrichment cultures. The
Cycloclasticus
strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of
Cycloclasticus
strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of
Cycloclasticus
cells associated with the grains was on the order of 10
3
cells/g of grains before crude oil was added to the tanks and increased to 3 × 10
6
cells/g of grains after crude oil was added. The number increased further after 14 days to 10
8
cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 × 10
6
cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of
Cycloclasticus
cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus
Cycloclasticus
play an important role in the degradation of petroleum PAHs in a marine environment.
Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical
Vibrio vulnificus
Strains in Artificial Seawater
Authors: Fischer-Le Saux Marion , Hervio-Heath Dominique , Loaec Solen , Colwell Rita R. , Pommepuy Monique
Published: April 2009

The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of
Vibrio vulnificus.
For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of
V. vulnificus
and targeted the cytotoxin-hemolysin virulence gene
vvhA.
Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4°C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of
vvhA
mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC
V. vulnificus.
Moreover,
vvhA
transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.
Metabolic Engineering of Acetaldehyde Production by
Streptococcus thermophilus
Authors: Chaves A. C. S. D. , Fernandez M. , Lerayer A. L. S. , Mierau I. , Kleerebezem M. , Hugenholtz J.
Published: April 2009

The process of acetaldehyde formation by the yogurt bacterium
Streptococcus thermophilus
is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the
glyA
gene. In
S. thermophilus
, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type
S. thermophilus
strains were screened for acetaldehyde production in the presence and absence of
l
-threonine. Supplementation of the growth medium with
l
-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a
glyA
mutant was constructed by gene disruption. Inactivation of
glyA
resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an
S. thermophilus
strain was constructed in which the
glyA
gene was cloned under the control of a strong promoter (P
LacA
). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in
S. thermophilus
, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with
S. thermophilus
as starter culture.
Use of the
alr
Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria
Authors: Bron Peter A. , Benchimol Marcos G. , Lambert Jolanda , Palumbo Emmanuelle , Deghorain Marie , Delcour Jean , de Vos Willem M. , Kleerebezem Michiel , Hols Pascal
Published: April 2009

Both
Lactococcus lactis
and
Lactobacillus plantarum
contain a single
alr
gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of
d
-alanine and
l
-alanine. The
alr
genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an
alr
deletion (Δ
alr
) showed auxotrophy for
d
-alanine, plasmids carrying a heterologous
alr
were constructed and could be selected, since they complemented
d
-alanine auxotrophy in the
L. plantarum
Δ
alr
and
L. lactis
Δ
alr
strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous
alr
genes could be stably maintained in wild-type strains of
L. plantarum
and
L. lactis
by selection for resistance to
d
-cycloserine, a competitive inhibitor of Alr (600 and 200 μg/ml, respectively). In addition, a plasmid carrying the
L. plantarum alr
gene under control of the regulated
nisA
promoter was constructed to demonstrate that
d
-cycloserine resistance of
L. lactis
is linearly correlated to the
alr
expression level. Finally, the
L. lactis alr
gene controlled by the
nisA
promoter, together with the nisin-regulatory genes
nisRK,
were integrated into the chromosome of
L. plantarum
Δ
alr.
The resulting strain could grow in the absence of
d
-alanine only when expression of the
alr
gene was induced with nisin.
Engineering of Polyploid
Saccharomyces cerevisiae
for Secretion of Large Amounts of Fungal Glucoamylase
Authors: Ekino Keisuke , Hayashi Hiroyuki , Moriyama Masahiro , Matsuda Minoru , Goto Masatoshi , Yoshino Sadazo , Furukawa Kensuke
Published: April 2009

We engineered
Saccharomyces cerevisiae
cells that produce large amounts of fungal glucoamylase (GAI) from
Aspergillus awamori
var.
kawachi
. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.
In Vitro and In Vivo Invasiveness of Different Pulsed-Field Gel Electrophoresis Types of
Listeria monocytogenes
Authors: Larsen Charlotte Nexmann , Nørrung Birgit , Sommer Helle Mølgaard , Jakobsen Mogens
Published: April 2009

The virulence of different pulsed-field gel electrophoresis (PFGE) types of
Listeria monocytogenes
was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of
L. monocytogenes
commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10
9
L. monocytogenes
cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.
Altered Glycosylation of 63- and 68-Kilodalton Microvillar Proteins in
Heliothis virescens
Correlates with Reduced Cry1 Toxin Binding, Decreased Pore Formation, and Increased Resistance to
Bacillus thuringiensis
Cry1 Toxins
Authors: Jurat-Fuentes Juan Luis , Gould Fred L. , Adang Michael J.
Published: April 2009

The binding and pore formation abilities of Cry1A and Cry1Fa
Bacillus thuringiensis
toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of
Heliothis virescens
.
125
I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and
125
I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the
N
-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of
H. virescens
and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.
Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton
Authors: Pernthaler Annelie , Pernthaler Jakob , Schattenhofer Martha , Amann Rudolf
Published: April 2009

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 μm) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the
Roseobacter
lineage remained constant, but the fraction of BrdU-incorporating
Roseobacter
sp. cells doubled, from 24 to 42%. In
Alteromonas
sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.
Sensitive In Situ Monitoring of a Recombinant Bioluminescent
Yersinia enterocolitica
Reporter Mutant in Real Time on Camembert Cheese
Authors: Maoz Ariel , Mayr Ralf , Bresolin Geraldine , Neuhaus Klaus , Francis Kevin P. , Scherer Siegfried
Published: April 2009

Bioluminescent mutants of
Yersinia enterocolitica
were generated by transposon mutagenesis using a promoterless, complete
lux
operon (
luxCDABE
) derived from
Photorhabdus luminescens
, and their production of light in the cheese environment was monitored. Mutant B94, which had the
lux
cassette inserted into an open reading frame of unknown function was used for direct monitoring of
Y
.
enterocolitica
cells on cheeses stored at 10°C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm
2
. Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the
lux
operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a “real-product” status, and at a low temperature.
Mercury Methylation by
Desulfovibrio desulfuricans
ND132 in the Presence of Polysulfides
Authors: Jay Jenny Ayla , Murray Karen J. , Gilmour Cynthia C. , Mason Robert P. , Morel François M. M. , Roberts A. Lynn , Hemond Harold F.
Published: April 2009

The extracellular speciation of mercury may control bacterial uptake and methylation. Mercury-polysulfide complexes have recently been shown to be prevalent in sulfidic waters containing zero-valent sulfur. Despite substantial increases in total dissolved mercury concentration, methylation rates in cultures of
Desulfovibrio desulfuricans
ND132 equilibrated with cinnabar did not increase in the presence of polysulfides, as expected due to the large size and charged nature of most of the complexes. In natural waters not at saturation with cinnabar, mercury-polysulfide complexes would be expected to shift the speciation of mercury from HgS
0
(aq)
toward charged complexes, thereby decreasing methylation rates.
Large-Scale Production of Coenzyme F
420
-5,6 by Using
Mycobacterium smegmatis
Authors: Isabelle Dale , Simpson D. Randall , Daniels Lacy
Published: April 2009

Production of coenzyme F
420
and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth,
Mycobacterium smegmatis
was the best source for F
420
-5,6.
M
.
smegmatis
produced 1 to 3 μmol of intracellular F
420
per liter of culture, which was more than the 0.85 to 1.0 μmol of F
420
-2 per liter usually obtained with
Methanobacterium thermoautotrophicum
and ∼10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F
420
than with previous methods.
Transcript Analysis of Genes Encoding a Family 61 Endoglucanase and a Putative Membrane-Anchored Family 9 Glycosyl Hydrolase from
Phanerochaete chrysosporium
Authors: Wymelenberg Amber Vanden , Denman Stuart , Dietrich Diane , Bassett Jennifer , Yu Xiaochun , Atalla Rajai , Predki Paul , Rudsander Ulla , Teeri Tuula T. , Cullen Daniel
Published: April 2009

Phanerochaete chrysosporium
cellulase genes were cloned and characterized. The
cel61A
product was structurally similar to fungal endoglucanases of glycoside hydrolase family 61, whereas the
cel9A
product revealed similarities to
Thermobifida fusca
Cel9A (E4), an enzyme with both endo- and exocellulase characteristics. The fungal Cel9A is apparently a membrane-bound protein, which is very unusual for microbial cellulases. Transcript levels of both genes were substantially higher in cellulose-grown cultures than in glucose-grown cultures. These results show that
P. chrysosporium
possesses a wide array of conventional and unconventional cellulase genes.
High-Frequency Rugose Exopolysaccharide Production by
Vibrio cholerae
Authors: Ali Afsar , Rashid Mohammed H. , Karaolis David K. R.
Published: April 2009

Vibrio cholerae
can shift to a “rugose” phenotype, thereby producing copious exopolysaccharide (EPS), which promotes its environmental survival and persistence. We report conditions that promote high-frequency rugose EPS production (HFRP), whereby cells switch at high frequency (up to 80%) to rugose EPS production. HFRP appeared to be more common in clinical strains, as HFRP was found in 6 of 19 clinical strains (32%) (including classical, El Tor, and non-O1 strains) but in only 1 of 16 environmental strains (6%). Differences were found between strains in rugose colony morphology, conditions promoting HFRP, the frequency of rugose-to-smooth (R-S) cell reversion, and biofilm formation. We propose that rugose EPS and HFRP provide an evolutionary and adaptive advantage to specific epidemic
V. cholerae
strains for increased persistence in the environment.
Application of Carbon Source Utilization Patterns To Measure the Metabolic Similarity of Complex Dental Plaque Biofilm Microcosms
Authors: Anderson Sally A. , Sissons Christopher H. , Coleman Megan J. , Wong Lisa
Published: April 2009

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H
2
-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (
Streptococcus mutans
plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.
Serum as a Factor Influencing Adhesion ofEnterococcus faecalisto Glass and Silicone
Authors: Gallardo-Moreno Amparo M. , González-Martín M. Luisa , Pérez-Giraldo Ciro , Bruque José M. , Gómez-García Antonio C.
Published: April 2009

The purpose of this work was to analyze the effect of serum on the physicochemical surface properties and adhesion to glass and silicone ofEnterococcus faecalisATCC 29212 at 37°C. As is presented using thermodynamics analysis, serum minimizes the interaction of cells with water, which correlates well with the increase in hydrophobicity and in bacterial adhesion to glass and silicone.
Influence of Environmental Conditions on Methanogenic Compositions in Anaerobic Biogas Reactors
Authors: Karakashev Dimitar , Batstone Damien J. , Angelidaki Irini
Published: April 2009

The influence of environmental parameters on the diversity of methanogenic communities in 15 full-scale biogas plants operating under different conditions with either manure or sludge as feedstock was studied. Fluorescence in situ hybridization was used to identify dominant methanogenic members of the
Archaea
in the reactor samples; enriched and pure cultures were used to support the in situ identification. Dominance could be identified by a positive response by more than 90% of the total members of the
Archaea
to a specific group- or order-level probe. There was a clear dichotomy between the manure digesters and the sludge digesters. The manure digesters contained high levels of ammonia and of volatile fatty acids (VFA) and were dominated by members of the
Methanosarcinaceae
, while the sludge digesters contained low levels of ammonia and of VFA and were dominated by members of the
Methanosaetaceae
. The methanogenic diversity was greater in reactors operating under mesophilic temperatures. The impact of the original inoculum used for the reactor start-up was also investigated by assessment of the present population in the reactor. The inoculum population appeared to have no influence on the eventual population.
Intramolecular base-catalyzed reactions involving interaction between benzene nitro groups and ortho carbon chains
Authors: Hanusek Jiří , Macháček Vladimír
Published: April 2009

The review is focused on the understanding of processes involving chemical interaction between benzene nitro group and ortho carbon chain containing heteroatom (N, O, S) adjacent to the ring. In most cases these compounds undergo base-catalyzed cyclization to give heteroaromatic N-oxides that can be subsequently transformed to related heterocycles under the same conditions. However, in some cases, depending on substitution of the benzene ring, side chain or the base used, the formation of other compounds – both heterocyclic and non-heterocyclic such as nitroso and azoxy compounds, spiro Meisenheimer adducts – is observed. Review with 31 references.
Synthesis, structural characterisation and electrochemistry of bis[(diphenylphosphino)ferrocene]diruthenium complexes [Ru2(μ-RCO2)2(CO)4(FcPPh2)2] (R = H and Me)
Authors: Štěpnička Petr , Císařová Ivana
Published: April 2009

Polymeric carboxylatoruthenium complexes [Ru2(μ-RCO2)2(CO)4]n (1, R = H; 2, R = Me) react with (diphenylphosphino)ferrocene (FcPPh2) to give the corresponding discrete diruthenium complexes [Ru2(μ-RCO2)2(CO)4(FcPPh2)2] (3, R = H; 4, R = Me) in good yields. Compounds 3 and 4 were characterised by spectroscopic (NMR, IR and MS) and voltammetric methods. The crystal structure of 4 was determined by single-crystal X-ray diffraction.
A DYNAMIC SEATING SYSTEM FOR CHILDREN WITH CEREBRAL PALSY
Authors: Hahn Michael E. , Simkins Sheri L. , Gardner Jacob K. , Kaushik Gaurav
Published: April 2009

The study's aim was to determine the initial effects of a dynamic seating system as a therapeutic intervention in children with cerebral palsy. A two-factor, repeated-measures design was used. Twelve children with neuromuscular dysfunction (mean age 6.0, SD 2.7 years) were included in the study, randomly assigned to an experimental or a control group. At study initiation the experimental group received a wheelchair with dynamic seating components that allows limited range of motion in the hip and knee, and the control group received a static setting wheelchair. Participants were evaluated for range of motion, muscle spasticity (Modified Ashworth Scale), motor function (Gross Motor Function Measure), and level of disability (Pediatric Evaluation of Disability Inventory) at study initiation, 3-months, and 6-months post intervention. Both groups improved in motor function over time, particularly in the categories of Sitting and Crawl/Kneel. Measures of disability improved in both groups for the categories of self-care, mobility, and social function. A larger, more homogeneous sample would likely show significant group differences in measures of muscle spasticity, gross motor function and disability.
ANATOMIC AND CLINICAL STUDY OF A NEUROVASCULAR CAUSE OF LATERAL EPICONDYLITIS
Authors: Du Xin-Ru , Zhao Ling-Xiu
Published: April 2009

Because of few anatomic reports investigating the mechanism of lateral epicondylitis (tennis elbow), we performed cadaveric and clinical studies to investigate the involvement of neurovascular bundles passing through the common extensor origin. We dissected and observed under a light microscope tissue samples of neurovascular bundles passing through the common extensor tendon from 40 upper left and right limbs from cadavers. Tissue samples were prepared by hematoxylin & eosin and Weil's myelin staining. We also investigated the records of 20 patients who had been treated for lateral epicondylitis between 1991 and 2004. From cadavers, we found 60 bundles in the common extensor tendon, each 0.5 to 1.0 mm in diameter, with more bundles in the right than left limbs. Twenty-four of these bundles passed over the vertex of the lateral epicondyle of the humerus, and most of the bundles contained only one artery each. The bundles mostly originated from the radial recurrent artery, passing through the aponeurosis of the extensor corpi radialis brevis, but in some cases originated from the radial collateral artery, passing through the aponeurosis of the triceps brachii muscle. The bundles had a membranous covering when passing through the aponeurosis and produced a hiatus. Histological analysis of resected common extensor tendon tissue, 1 cm in diameter, of patients showed hyaline degeneration and fibrosis formation infiltration. Neurovascular bundles passed through the common extensor tendon in nine cases; six cases showed pulsing bleeding. After a mean follow-up of two years (6–48 months), 16 cases showed excellent results, two showed good results and two showed reliefs. Lateral epicondylitis could be caused by damage to neurovascular bundles when they pass through the common extensor origin; one cause of pain is the neurovascular bundle being compressed when passing through the common extensor tendon, secondary to the pathologic degeneration of the origin of the common extensor tendon.
SIX MONTH PHYSIOTHERAPY ASSESSMENTS ON THE PATIENTS EXPOSED TO THE TWO DIFFERENT OPERATIONS FOR THEIR LUMBAR DISC HERNIAS
Authors: Yildirim Yücel , Kara Bilge , Erbayraktar Serhat
Published: April 2009

Purpose: To compare the patients operated on for lumbar disc hernia with two different methods in view of pain, disability, physical performance, physical activity and aerobic capacity. Methods: Forty-one patients were included in the study (group I, nucleoplasty – n = 14), (group II, lumbar discectomy – n = 27). Visual Analog Scale (VAS) was performed for pain; Oswestry Disability Index for lumbar disability level; total rolling time, lie/sit, sit and stand, 50 foot walking and five-minute walk tests for physical performance measurements; the Compendium of Physical Activities (CPA) scale for activity level measurement, and VO2max measurements for aerobic capacity. Results: In the second month's controls, changes of pain and disability levels were insignificant in both groups, even though pain in nucleoplasty group and disability in discectomy group were found to be better. In performance measurements, group II showed better results in the second postoperative month, whereas group I had better results in the sixth postoperative month. The levels of physical activity in group I, and of aerobic capacity in group II increased significantly. Conclusion: Different physiotherapy approaches are necessary to obtain good results in terms of pain intensity, disability, physical performance, physical activity and aerobic capacity, since these parameters are found to be worse during the sixth month postoperative in patients who had undergone lumbar discectomy.
LOCAL SYMPLECTIC INVARIANTS FOR CURVES
Authors: KAMRAN NIKY , OLVER PETER , TENENBLAT KETI
Published: April 2009

We consider curves in ℝ2n endowed with the standard symplectic structure. We introduce the concept of symplectic arc length for curves. We construct an adapted symplectic Frenet frame and we define 2n - 1 local differential invariants that we call symplectic curvatures of the curve. We prove that up to a rigid symplectic motion of ℝ2n, there exists a unique curve with prescribed symplectic curvatures. We characterize the curves in ℝ4 with constant symplectic curvatures.
CONTACT STRUCTURES, σ-CONFOLIATIONS AND CONTAMINATIONS IN 3-MANIFOLDS
Authors: OERTEL ULRICH , ŚWIATKOWSKI JACEK
Published: April 2009

We propose in this paper a method for studying contact structures in 3-manifolds by means of branched surfaces. We explain what it means for a contact structure to be carried by a branched surface embedded in a 3-manifold. To make the transition from contact structures to branched surfaces, we first define auxiliary objects called σ-confoliations and pure contaminations, both generalizing contact structures. We study various deformations of these objects and show that the σ-confoliations and pure contaminations obtained by suitably modifying a contact structure remember the contact structure up to isotopy.

After defining tightness for all pure contaminations in a natural way, generalizing the definition of tightness for contact structures, we obtain some conditions on (the embedding of) a branched surface in a 3-manifold sufficient to guarantee that any pure contamination carried by the branched surface is tight. We also find conditions sufficient to prove that a branched surface carries only overtwisted (non-tight) contact structures.

Our long-term goal in developing these methods is twofold: Not only do we want to study tight contact structures and pure contaminations, but we also wish to use them as tools for studying 3-manifold topology.
INEQUALITIES ASSOCIATED WITH DILATIONS
Authors: OZAWA TOHRU , SASAKI HIRONOBU
Published: April 2009

Some properties of distributions f satisfying x · ∇ f ∈ Lp (ℝn), 1 ≤ p < ∞, are studied. The operator x · ∇ is the generator of a semi-group of dilations. We first give Sobolev type inequalities with respect to the operator x · ∇. Using the inequalities, we also show that if [Formula: see text], x · ∇ f ∈ Lp (ℝn) and |x|n/p|f(x)| vanishes at infinity, then f belongs to Lp (ℝn). One of the Sobolev type inequalities is shown to be equivalent to the Hardy inequality in L2 (ℝn).