bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protease Inhibitors Fail To Prevent Pore Formation by the Activated
Bacillus thuringiensis
Toxin Cry1Aa in Insect Brush Border Membrane Vesicles
Authors: Kirouac Martin , Vachon Vincent , Quievy Delphine , Schwartz Jean-Louis , Laprade Raynald 
Published: January 2006

ABSTRACT

To investigate whether membrane proteases are involved in the activity of
Bacillus thuringiensis
insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with
Manduca sexta
midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors,
o
-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.
Prevalence, Persistence, and Molecular Characterization of Glycopeptide-Resistant Enterococci in Norwegian Poultry and Poultry Farmers 3 to 8 Years after the Ban on Avoparcin
Authors: Sørum M. , Johnsen P. J. , Aasnes B. , Rosvoll T. , Kruse H. , Sundsfjord A. , Simonsen G. S. 
Published: January 2006

ABSTRACT

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant
Enterococcus faecium
(GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn
1546
-plasmid junction fragment in 93.9% of
E. faecium
isolates. A putative postsegregation killing (PSK) system linked to Tn
1546
was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (
n
= 10) and -resistant (
n
= 10)
E. faecium
isolates from humans (
n
= 10) and poultry (
n
= 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated
vanA
-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.
Stable Concentrated Emulsions of the 1-Monoglyceride of Capric Acid (Monocaprin) with Microbicidal Activities against the Food-Borne Bacteria
Campylobacter jejuni
,
Salmonella
spp., and
Escherichia coli
Authors: Thormar Halldor , Hilmarsson Hilmar , Bergsson Gudmundur 
Published: January 2006

ABSTRACT

Of 11 fatty acids and monoglycerides tested against
Campylobacter jejuni
, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against
C. jejuni
upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log
10
reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of
Campylobacter
isolates from humans and poultry and also killed strains of
Campylobacter coli
and
Campylobacter lari
, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log
10
reduction in viable bacterial counts of
Salmonella
spp. and
Escherichia coli
in 10 min.
C. jejuni
was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to
Campylobacter
-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.
Sequence Analysis of Two Cryptic Plasmids from
Bifidobacterium longum
DJO10A and Construction of a Shuttle Cloning Vector
Authors: Lee Ju-Hoon , O’Sullivan Daniel J. 
Published: January 2006

ABSTRACT

Bifidobacterium longum
DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other
B. longum
plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused
rep
gene and an RCR-type
ori
consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a
Rhodococcus rhodochrous
plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in
B. longum
DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of
Rhodococcus rhodochrous
(67%) and significantly higher than that of
B. longum
(60.1%), it may have been acquired through horizontal gene transfer from a
Rhodococcus
species, as both genera are members of the
Actinomycetes
and are intestinal inhabitants. An
Escherichia coli-B. longum
shuttle cloning vector was constructed from pDOJH10S and the
E. coli ori
region of p15A, a
lacZ
gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into
E. coli
and
B. longum
. It could not be introduced into lactic acid bacteria (
Lactococcus
and
Lactobacillus
), showing it was not very promiscuous. It was stably maintained in
B. longum
in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.
Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in
Saccharomyces cerevisiae
Authors: Zhang Bo , Carlson Ross , Srienc Friedrich 
Published: January 2006

ABSTRACT

Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of
Saccharomyces cerevisiae
by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical β-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C
6
to C
14
) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from
Pseudomonas oleovorans
. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the
Ralstonia eutropha
scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that
S. cerevisiae
expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.
Effect of Temperature and Light on Growth of and Photosynthesis bySynechococcusIsolates Typical of Those Predominating in the Octopus Spring Microbial Mat Community of Yellowstone National Park
Authors: Allewalt Jessica P. , Bateson Mary M. , Revsbech Niels Peter , Slack Kimberly , Ward David M. 
Published: January 2006

ABSTRACTPrevious molecular analysis of the Octopus Spring cyanobacterial mat revealed numerous genetically distinct 16S rRNA sequences from predominantSynechococcuspopulations distantly related to the readily cultivated unicellular cyanobacteriumSynechococcus lividus. Patterns in genotype distribution relative to temperature and light conditions suggested that the organisms contributing these 16S rRNA sequences may fill distinct ecological niches. To test this hypothesis,Synechococcusisolates were cultivated using a dilution and filtration approach and then shown to be genetically relevant to natural mat populations by comparisons of similarities of 16S rRNA genes and 16S-23S internal transcribed spacer (ITS) regions. Most isolates were identical or nearly identical at both loci to predominant mat genotypes; others showed 1- to 2-nucleotide differences at the 16S rRNA locus and even greater difference in ITS sequences. Isolates with predominant mat genotypes had distinct temperature ranges and optima for growth that were consistent with their distributions in the mat. Isolates with genotypes not previously detected or known to be predominant in the mat exhibited temperature ranges and optima that were not representative of predominant mat populations and also grew more slowly. Temperature effects on photosynthesis did not reflect temperature relations for growth. However, the isolate with the highest temperature optimum and upper limit was capable of performing photosynthesis at a higher temperature than other isolates. Growth rate and photosynthetic responses provided evidence for light acclimation but evidence of, at best, only subtle light adaptation.
Diversity of Microorganisms in Fe-As-Rich Acid Mine Drainage Waters of Carnoulès, France
Authors: Bruneel O. , Duran R. , Casiot C. , Elbaz-Poulichet F. , Personné J.-C. 
Published: January 2006

ABSTRACT

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg · liter
−1
), iron (750 to 2,700 mg · liter
−1
), and sulfate (2,000 to 7,500 mg · liter
−1
). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with
Gallionella ferruginea
, a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.
Persistence of
Mycobacterium avium
subsp.
paratuberculosis
and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure
Authors: Grewal Sukhbir K. , Rajeev Sreekumari , Sreevatsan Srinand , Michel Frederick C. 
Published: January 2006

ABSTRACT

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as
Escherichia coli
O157:H7,
Listeria monocytogenes
,
Salmonella
spp., and
Mycobacterium avium
subsp.
paratuberculosis
(
Mycobacterium paratuberculosis
). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated
M. paratuberculosis
, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with
M. paratuberculosis
at 10
6
CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of
M. paratuberculosis
was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO
2
, while no significant change in carbon content was observed in the liquid slurry.
Escherichia coli
,
Salmonella
, and
Listeria
were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days.
M. paratuberculosis
was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56,
M. paratuberculosis
was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However,
M. paratuberculosis
DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.
Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms
Authors: Chénier M. R. , Beaumier D. , Fortin N. , Roy R. , Driscoll B. T. , Lawrence J. R. , Greer C. W. 
Published: January 2006

ABSTRACTBiofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. BothnirSandnirKdenitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, andnirSandnirKgenes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence ofnirSandnirKgenes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P≤ 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.
Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in
Burkholderia xenovorans
LB400
Authors: Denef V. J. , Klappenbach J. A. , Patrauchan M. A. , Florizone C. , Rodrigues J. L. M. , Tsoi T. V. , Verstraete W. , Eltis L. D. , Tiedje J. M. 
Published: January 2006

ABSTRACT

Transcriptomic and proteomic analyses of
Burkholderia xenovorans
LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol
ortho
cleavage (
ben
-
cat
) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (

box
C

) and the megaplasmid (

box
M

). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the
ben-cat
pathway (the Δ
benABCD
::
kan
mutant), the

box
C

pathway (the Δ

boxAB
C

::
kan
mutant), and both pathways (the Δ
benABCD
Δ

boxAB
C

::
kan
mutant). All three mutants oxidized benzoate in resting-cell assays. However, the Δ
benABCD
::
kan
and Δ
benABCD
Δ

boxAB
C

::
kan
mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the Δ
benABCD
::
kan
mutant growing on benzoate expressed both
box
pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of
benABCD
abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the
box
pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.
Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases
Authors: van Beilen Jan B. , Funhoff Enrico G. , van Loon Alexander , Just Andrea , Kaysser Leo , Bouza Manuel , Holtackers René , Röthlisberger Martina , Li Zhi , Witholt Bernard 
Published: January 2006

ABSTRACT

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed
Pseudomonas putida
GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.