bluuebunny/crossref_metadata_2025_split huggingface dataset:
Culture-Independent Analysis of Fecal Enterobacteria in Environmental Samples by Single-Cell mRNA Profiling
Authors: Chen Han , Ponniah Gomathinayagam , Salonen Nancy , Blum Paul 
Published: August 2004

ABSTRACT

A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of
fis
and
dps
mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.
Benzoic Acid, a Weak Organic Acid Food Preservative, Exerts Specific Effects on Intracellular Membrane Trafficking Pathways in
Saccharomyces cerevisiae
Authors: Hazan Reut , Levine Alexandra , Abeliovich Hagai 
Published: August 2004

ABSTRACT

Microbial spoilage of food causes losses of up to 40% of all food grown for human consumption worldwide. Yeast growth is a major factor in the spoilage of foods and beverages that are characterized by a high sugar content, low pH, and low water activity, and it is a significant economic problem. While growth of spoilage yeasts such as
Zygosaccharomyces bailii
and
Saccharomyces cerevisiae
can usually be retarded by weak organic acid preservatives, the inhibition often requires levels of preservative that are near or greater than the legal limits. We identified a novel synergistic effect of the chemical preservative benzoic acid and nitrogen starvation: while exposure of
S. cerevisiae
to either benzoic acid or nitrogen starvation is cytostatic under our conditions, the combination of the two treatments is cytocidal and can therefore be used beneficially in food preservation. In yeast, as in all eukaryotic organisms, survival under nitrogen starvation conditions requires a cellular response called macroautophagy. During macroautophagy, cytosolic material is sequestered by intracellular membranes. This material is then targeted for lysosomal degradation and recycled into molecular building blocks, such as amino acids and nucleotides. Macroautophagy is thought to allow cellular physiology to continue in the absence of external resources. Our analyses of the effects of benzoic acid on intracellular membrane trafficking revealed that there was specific inhibition of macroautophagy. The data suggest that the synergism between nitrogen starvation and benzoic acid is the result of inhibition of macroautophagy by benzoic acid and that a mechanistic understanding of this inhibition should be beneficial in the development of novel food preservation technologies.
Ecology and Transmission ofListeria monocytogenesInfecting Ruminants and in the Farm Environment
Authors: Nightingale K. K. , Schukken Y. H. , Nightingale C. R. , Fortes E. D. , Ho A. J. , Her Z. , Grohn Y. T. , McDonough P. L. , Wiedmann M. 
Published: August 2004

ABSTRACTA case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology ofListeria monocytogeneson farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured forL. monocytogenes. While the overall prevalence ofL. monocytogenesin cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P< 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n= 17) and farm (n= 414) isolates differentiated 51 ribotypes.L. monocytogenesribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute toL. monocytogenesdispersal into the farm environment. Ribotype DUP-1038B was significantly (P< 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P< 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission ofL. monocytogenesdiffer between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal ofL. monocytogenesinto the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence ofL. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv)L. monocytogenessubtypes may differ in their abilities to infect animals and to survive in farm environments.
Identification ofCarnobacteriumSpecies by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR
Authors: Rachman Cinta , Kabadjova Petia , Valcheva Rosica , Prévost Hervé , Dousset Xavier 
Published: August 2004

ABSTRACTThe genusCarnobacteriumis currently divided into the following eight species:Carnobacterium piscicola,C. divergens,C. gallinarum,C. mobile,C. funditum,C. alterfunditum,C. inhibens, andC. viridans. An identification tool for the rapid differentiation of these eightCarnobacteriumspecies was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for allCarnobacteriumspecies. The L-ISR sequence revealed the presence of two tRNA genes, tRNAAlaand tRNAIle, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of theCarnobacteriumspecies from other phylogenetically related lactic acid bacteria.
Bactericidal Activity of Glycinecin A, a Bacteriocin Derived from
Xanthomonas campestris
pv. glycines, on Phytopathogenic
Xanthomonas campestris
pv. vesicatoria Cells
Authors: Pham Huy Thang , Riu Key Zoung , Jang Kong Man , Cho Somi K. , Cho Moonjae 
Published: August 2004

ABSTRACT

The ability of glycinecin A, a bacteriocin derived from
Xanthomonas campestris
pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group (S. G. Heu et al., Appl. Environ. Microbiol.
67:
4105-4110, 2001). In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from
X. campestris
pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.
Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based
Escherichia coli
Strain
Authors: Ferrer Manuel , Chernikova Tatyana N. , Timmis Kenneth N. , Golyshin Peter N. 
Published: August 2004

ABSTRACT

A new principle for expression of heat-sensitive recombinant proteins in
Escherichia coli
at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium,
Oleispira antarctica
RB8
T
, that allow
E. coli
to grow at high rates at 4°C (maximum growth rate, 0.28 h
−1
) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing
E. coli
strain grown at 37°C (32,380 versus 190 μmol min
−1
g
−1
). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based
E. coli
strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common
E. coli
strain (7.5°C).
Polydextrose, Lactitol, and Fructo-Oligosaccharide Fermentation by Colonic Bacteria in a Three-Stage Continuous Culture System
Authors: Probert Hollie M. , Apajalahti Juha H. A. , Rautonen Nina , Stowell Julian , Gibson Glenn R. 
Published: August 2004

ABSTRACT

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (
P
= 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where
Bifidobacterium infantis
was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (
P
= 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (
P
= 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (
P
= 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.
Analysis of Structural and Physiological Profiles To Assess the Effects of Cu on Biofilm Microbial Communities
Authors: Massieux B. , Boivin M. E. Y. , van den Ende F. P. , Langenskiöld J. , Marvan P. , Barranguet C. , Admiraal W. , Laanbroek H. J. , Zwart G. 
Published: August 2004

ABSTRACT
We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 μM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.
Inactivation of Caliciviruses
Authors: Duizer Erwin , Bijkerk Paul , Rockx Barry , de Groot Astrid , Twisk Fleur , Koopmans Marion 
Published: August 2004

ABSTRACT

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100°C. UV-B radiation from 0 to 150 mJ/cm
2
caused dose-dependent inactivation, with a 3
D
(
D
= 1 log
10
) reduction in infectivity at 34 mJ/cm
2
for both viruses. Inactivation by 70% ethanol was inefficient, with only 3
D
reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.
Oxidation of Methyl
tert
-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and
n
-Octane-Grown
Pseudomonas putida
GPo1
Authors: Smith Christy A. , Hyman Michael R. 
Published: August 2004

ABSTRACT

The alkane hydroxylase enzyme system in
Pseudomonas putida
GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl
tert
-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of
tert
-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized
tert
-amyl methyl ether but did not appear to oxidize either TBA, ethyl
tert
-butyl ether, or
tert
-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of
P. putida
GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all
n
-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting
n
-alkanes (propane and
n
-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in
n
-octane-grown cells. Kinetic studies with strain GPo1 grown on
n
-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in
n
-octane-grown cells because of the unusually high

K
s

value (20 to 40 mM) for MTBE.
Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in
Streptococcus gordonii
Authors: Lee Chiang W. , Lee Song F. , Halperin Scott A. 
Published: August 2004

ABSTRACT

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in
Streptococcus gordonii
DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant
S. gordonii
recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant
S. gordonii
expressing double copies of DTA (SpaP-DTA
2
) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response.
S. gordonii
SpaP-DTA
2
was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.
Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR
Authors: Kostal Jan , Yang Rosanna , Wu Cindy H. , Mulchandani Ashok , Chen Wilfred 
Published: August 2004

ABSTRACT

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in
Escherichia coli
in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd
2+
or Zn
2+
accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.
Characterization of a Galactokinase-Positive Recombinant Strain of
Streptococcus thermophilus
Authors: Vaillancourt Katy , LeMay Jean-Dominique , Lamoureux Maryse , Frenette Michel , Moineau Sylvain , Vadeboncoeur Christian 
Published: August 2004

ABSTRACT

The lactic acid bacterium
Streptococcus thermophilus
is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species
Streptococcus salivarius
,
S. thermophilus
is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of
S. thermophilus
to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal
−
strain
S. thermophilus
SMQ-301 with
S. salivarius galK
, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing
S. salivarius galK
, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of
S. salivarius galK
and (ii) transcription of the plasmid-borne
galK
gene did not require GalR, a transcriptional regulator of the
gal
and
lac
operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike
S. salivarius
, the ability to metabolize galactose was not sufficient for
S. thermophilus
to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal
+
strain grew and produced acid more rapidly than the Gal
−
wild-type strain.
Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of
Bombyx mori
, That Binds to Cry1A Toxins
Authors: Hossain Delwar M. , Shitomi Yasuyuki , Moriyama Kenta , Higuchi Masahiro , Hayakawa Tohru , Mitsui Toshiaki , Sato Ryoichi , Hori Hidetaka 
Published: August 2004

ABSTRACT

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of
Bombyx mori
. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and

K
d

constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.