bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTVegetated 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTAlteromonassp. 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.