bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow visualization and pressure distributions in a model of the glottis with a symmetric and oblique divergent angle of 10 degrees
Authors: Shinwari Daoud , Scherer Ronald C. , DeWitt Kenneth J. , Afjeh Abdollah A.
Published: January 2003

Modeling the human larynx can provide insights into the nature of the flow and pressures within the glottis. In this study, the intraglottal pressures and glottal jet flow were studied for a divergent glottis that was symmetric for one case and oblique for another. A Plexiglas model of the larynx (7.5 times life size) with interchangeable vocal folds was used. Each vocal fold had at least 11 pressure taps. The minimal glottal diameter was held constant at 0.04 cm. The glottis had an included divergent angle of 10 degrees. In one case the glottis was symmetric. In the other case, the glottis had an obliquity of 15 degrees. For each geometry, transglottal pressure drops of 3, 5, 10, and 15 cm H2O were used. Pressure distribution results, suggesting significantly different cross-channel pressures at glottal entry for the oblique case, replicate the data in another study by Scherer et al. [J. Acoust. Soc. Am. 109, 1616–1630 (2001b)]. Flow visualization using a LASER sheet and seeded airflow indicated separated flow inside the glottis. Separation points did not appear to change with flow for the symmetric glottis, but for the oblique glottis moved upstream on the divergent glottal wall as flow rate increased. The outgoing glottal jet was skewed off-axis for both the symmetric and oblique cases. The laser sheet showed asymmetric circulating regions in the downstream region. The length of the laminar core of the glottal jet was less than approximately 0.6 cm, and decreased in length as flow increased. The results suggest that the glottal obliquity studied here creates significantly different driving forces on the two sides of the glottis (especially at the entrance to the glottis), and that the skewed glottal jet characteristics need to be taken into consideration for modeling and aeroacoustic purposes.
Comment on “Development of panel loudspeaker system: Design, evaluation and enhancement” [J. Acoust Soc. Am. 106, 2751–2761 (2001)] (L)
Authors: Fahy Frank
Published: January 2003

This letter concerns the paper “Development of panel loudspeaker system: Design, evaluation and enhancement” [M. R. Bai and T. Huang, J. Acoust. Soc. Am. 109, 2751–2761 (2001)]. It is suggested that the radiation field generated by the near vibration field induced by a point force acting on the plate has been neglected. It is pointed out that its relative contribution is crucially dependent upon the mechanical loss factor of the panel, for which no data are presented. The conclusion that the radiated power per unit mean square force is independent of frequency neglects the radiation efficiency factor. Other perceived shortcomings of the paper are noted.
The influence of flight speed on the ranging performance of bats using frequency modulated echolocation pulses
Authors: Boonman Arjan M. , Parsons Stuart , Jones Gareth
Published: January 2003

Many species of bat use ultrasonic frequency modulated (FM) pulses to measure the distance to objects by timing the emission and reception of each pulse. Echolocation is mainly used in flight. Since the flight speed of bats often exceeds 1% of the speed of sound, Doppler effects will lead to compression of the time between emission and reception as well as an elevation of the echo frequencies, resulting in a distortion of the perceived range. This paper describes the consequences of these Doppler effects on the ranging performance of bats using different pulse designs. The consequences of Doppler effects on ranging performance described in this paper assume bats to have a very accurate ranging resolution, which is feasible with a filterbank receiver. By modeling two receiver types, it was first established that the effects of Doppler compression are virtually independent of the receiver type. Then, used a cross-correlation model was used to investigate the effect of flight speed on Doppler tolerance and range–Doppler coupling separately. This paper further shows how pulse duration, bandwidth, function type, and harmonics influence Doppler tolerance and range–Doppler coupling. The influence of each signal parameter is illustrated using calls of several bat species. It is argued that range–Doppler coupling is a significant source of error in bat echolocation, and various strategies bats could employ to deal with this problem, including the use of range rate information are discussed.
A comparison of the fragmentation thresholds and inertial cavitation doses of different ultrasound contrast agents
Authors: Chen Wen-Shiang , Matula Thomas J. , Brayman Andrew A. , Crum Lawrence A.
Published: January 2003

Contrast bubble destruction is important in several new diagnostic and therapeutic applications. The pressure threshold of destruction is determined by the shell material, while the propensity for of the bubbles to undergo inertial cavitation (IC) depends both on the gas and shell properties of the ultrasound contrast agent (UCA). The ultrasonic fragmentation thresholds of three specific UCAs (Optison, Sonazoid, and biSpheres), each with different shell and gas properties, were determined under various acoustic conditions. The acoustic emissions generated by the agents, or their derivatives, characteristic of IC after fragmentation, was also compared, using cumulated broadband-noise emissions (IC “dose”). Albumin-shelled Optison and surfactant-shelled Sonazoid had low fragmentation thresholds (mean=0.13 and 0.15 MPa at 1.1 MHz, 0.48 and 0.58 MPa at 3.5 MHz, respectively), while polymer-shelled biSpheres had a significant higher threshold (mean=0.19 and 0.23 MPa at 1.1 MHz, 0.73 and 0.96 MPa for thin- and thick-shell biSpheres at 3.5 MHz, respectively, p<0.01). At comparable initial concentrations, surfactant-shelled Sonazoid produced a much larger IC dose after shell destruction than did either biSpheres or Optison (p<0.01). Thick-shelled biSpheres had the highest fragmentation threshold and produced the lowest IC dose. More than two and five acoustic cycles, respectively, were necessary for the thin- and thick-shell biSpheres to reach a steady-state fragmentation threshold.
Development and Application of Different Methods for the Detection of
Toxoplasma gondii
in Water
Authors: Kourenti Christina , Heckeroth Anja , Tenter Astrid , Karanis Panagiotis
Published: January 2003

Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of
Toxoplasma gondii
oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated
T. gondii
oocysts (1 × 10
5
and 1 × 10
4
oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various
g
values or by flocculation with two coagulants, Fe
2
(SO
4
)
3
and Al
2
(SO
4
)
3
. The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al
2
(SO
4
)
3
(96.5% ± 21.7%) than by flocculation with Fe
2
(SO
4
)
3
(93.1% ± 8.1%) or by centrifugation at 2,073 ×
g
(82.5% ± 6.8%). For the unsporulated oocysts, flocculation with Fe
2
(SO
4
)
3
was more successful (100.3% ± 26.9%) than flocculation with Al
2
(SO
4
)
3
(90.4% ± 19.1%) or centrifugation at 2,565 ×
g
(97.2% ± 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated
Toxoplasma
oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of
Toxoplasma
oocysts in water.
Isolation and Characterization of a Sulfur-Oxidizing Chemolithotroph Growing on Crude Oil under Anaerobic Conditions
Authors: Kodama Yumiko , Watanabe Kazuya
Published: January 2003

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the ε subclass of the class
Proteobacteria
constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was
Thiomicrospira denitrificans
(90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S
2−
+ 4NO
3
−
→ SO
4
2−
+ 4NO
2
−
(Δ
G
0
= −534 kJ mol
−1
). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.
Oxygen Consumption by Anaerobic
Saccharomyces cerevisiae
under Enological Conditions: Effect on Fermentation Kinetics
Authors: Rosenfeld Eric , Beauvoit Bertrand , Blondin Bruno , Salmon Jean-Michel
Published: January 2003

The anaerobic growth of the yeast
Saccharomyces cerevisiae
normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (
erg1
and
ole1
mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the
ole1
mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.
High pH during Trisodium Phosphate Treatment Causes Membrane Damage and Destruction ofSalmonella entericaSerovar Enteritidis
Authors: Sampathkumar Balamurugan , Khachatourians George G. , Korber Darren R.
Published: January 2003

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria suchSalmonellaspp. remains unclear. Thus,Salmonella entericaserovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 ± 0.2 (mean ± standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.
Genetic Diversity and Spoilage Potentials among
Pseudomonas
spp. Isolated from Fluid Milk Products and Dairy Processing Plants
Authors: Dogan Belgin , Boor Kathryn J.
Published: January 2003

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by
Pseudomonas
spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among
Pseudomonas
spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338
Pseudomonas
spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as
P. fluorescens
and
P. putida
by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among
Pseudomonas
isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating
Pseudomonas
strains with dairy food spoilage potential.
Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution
Authors: Sakaue Ryoichi , Kajiyama Naoki
Published: January 2003

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45°C, whereas the wild-type enzyme was not stable above 37°C. The

K
m

values of FAOX-TE for
d
-fructosyl-
l
-valine and
d
-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.
Novel Approach to the Improvement of Biphenyl and Polychlorinated Biphenyl Degradation Activity: Promoter Implantation by Homologous Recombination
Authors: Ohtsubo Yoshiyuki , Shimura Minoru , Delawary Mina , Kimbara Kazuhide , Takagi Masamichi , Kudo Toshiaki , Ohta Akinori , Nagata Yuji
Published: January 2003

To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the
bph
operon (
bphEGF
[orf4]
A1A2A3CD
[orf1]
A4R
) of
Pseudomonas
sp. strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the
bph
operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.
Role ofctcfromListeria monocytogenesin Osmotolerance
Authors: Gardan Rozenn , Duché Ophélie , Leroy-Sétrin Sabine ,   the European Listeria Genome Consortium, Labadie Jean
Published: January 2003

Listeria monocytogenesis a food-borne pathogen with the ability to grow under conditions of high osmolarity. In a previous study, we reported the identification of 12 proteins showing high induction after salt stress. One of these proteins is highly similar to the general stress protein Ctc ofBacillus subtilis. In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments. To explore the role of thectcgene product in resistance to stresses, we constructed actcinsertional mutant. No difference in growth was observed between the wild-type strain LO28 and thectcmutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress. However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2. Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium. Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of thectcmutant. Altogether, these results suggest that the Ctc protein ofL. monocytogenesis involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium.
Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression
Authors: Tsuruoka Naoki , Nakayama Toru , Ashida Masako , Hemmi Hisashi , Nakao Masahiro , Minakata Hiroyuki , Oyama Hiroshi , Oda Kohei , Nishino Tokuzo
Published: January 2003

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest.Alicyclobacillus sendaiensisstrain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s−1;Km, 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s−1;Km, 214 μM), where the asterisks denote the scissile bonds. The clonedscpAgene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of thescpAgene in theEscherichia colicells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of thescpAgene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.
Coevolution of Bacteriophage PP01 and
Escherichia coli
O157:H7 in Continuous Culture
Authors: Mizoguchi Katsunori , Morita Masatomo , Fischer Curt R. , Yoichi Masatoshi , Tanji Yasunori , Unno Hajime
Published: January 2003

The interaction between
Escherichia coli
O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01,
E. coli
O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h
−1
and by 3 orders of magnitude at a lower dilution rate (0.327 h
−1
). However, the appearance of a series of phage-resistant
E. coli
isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled
E. coli
O157:H7 to escape PP01 infection. One of the escape mutants of
E. coli
O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h
−1
and persisted until the end of the experiment (∼200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and
E. coli
O157:H7 proceeded as a mutual arms race in chemostat continuous culture.
Microbial Characterization of Biofilms in Domestic Drains and the Establishment of Stable Biofilm Microcosms
Authors: McBain Andrew J. , Bartolo Robert G. , Catrenich Carl E. , Charbonneau Duane , Ledder Ruth G. , Rickard Alexander H. , Symmons Sharon A. , Gilbert Peter
Published: January 2003

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log
10
cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log
10
cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan
Colpoda maupasi
, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.
Bacterial Competition for Human Nasal Cavity Colonization: Role of Staphylococcal
agr
Alleles
Authors: Lina Gerard , Boutite Florent , Tristan Anne , Bes Michèle , Etienne Jerome , Vandenesch Francois
Published: January 2003

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal
agr
alleles. The
Staphylococcus aureus
colonization rate correlated negatively with the rate of colonization by
Corynebacterium
spp. and non-
aureus
staphylococci, especially
S. epidermidis
, suggesting that both
Corynebacterium
spp. and
S. epidermidis
antagonize
S. aureus
colonization. Most of the
S. aureus
and
S. epidermidis
isolates were
agr
typed by a PCR method. Only one
S. aureus agr
(

agr
Sa

) allele was detected in each carrier. Multiple logistic regression of the two most prevalent

agr
Sa

alleles (

agr-1
Sa

and

agr-2
Sa

) and the three
S. epidermidis agr
(

agr
Se

) alleles showed a specific influence of the
agr
system. The results of this model did not support conclusions drawn from previous in vitro
agr
-specific cross-inhibition experiments. Our findings suggest that the
agr
alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties—including mediators of bacterial interference—in the strains that bear them.
Degradation of Anthracene by
Mycobacterium
sp. Strain LB501T Proceeds via a Novel Pathway, through
o
-Phthalic Acid
Authors: van Herwijnen René , Springael Dirk , Slot Pieter , Govers Harrie A. J. , Parsons John R.
Published: January 2003

Mycobacterium
sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of
o
-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through
o
-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in
Mycobacterium
sp. LB501T.
Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene
Authors: Mancini Silvia A. , Ulrich Ania C. , Lacrampe-Couloume Georges , Sleep Brent , Edwards Elizabeth A. , Lollar Barbara Sherwood
Published: January 2003

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (ε) for carbon (range of −1.9 to −3.6‰) and hydrogen (range of −29 to −79‰) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.
Differential Growth Response of Colony-Forming α- and γ-Proteobacteria in Dilution Culture and Nutrient Addition Experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat
Authors: Pinhassi Jarone , Berman Tom
Published: January 2003

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of α-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the
Roseobacter
clade, while lake dilution cultures were dominated by bacteria affiliated with the genera
Sphingomonas
and
Caulobacter
. In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing γ-proteobacteria dominated; in the marine experiments primarily different
Vibrio
and
Alteromonas
species appeared, while in the lake water experiments species of the genera
Shewanella
,
Aeromonas
, and
Rheinheimera
grew. These results suggest that major, but different, γ-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by α-proteobacteria in the presence of low nutrient concentrations.
l
-Proline Accumulation and Freeze Tolerance in
Saccharomyces cerevisiae
Are Caused by a Mutation in the
PRO1
Gene Encoding γ-Glutamyl Kinase
Authors: Morita Yuko , Nakamori Shigeru , Takagi Hiroshi
Published: January 2003

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular
l
-proline derived from
l
-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and
l
-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-
l
-proline-utilizing strain, the mutant was found to carry an allele of the wild-type
PRO1
gene encoding γ-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of
PRO1
was shown to enhance the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of
l
-proline synthesis from
l
-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated γ-glutamyl kinase were found to accumulate intracellular
l
-proline and showed a prominent increase in cell viability after freezing at −20°C compared to the viability of cells harboring the wild-type
PRO1
gene. These results suggest that the altered γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-glutamyl phosphate reductase activity, which leads to an increase in
l
-proline production in
Saccharomyces cerevisiae
. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.
Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis
Authors: Temmerman R. , Scheirlinck I. , Huys G. , Swings J.
Published: January 2003

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.
Purification and Characterization of a Prolyl Aminopeptidase from
Debaryomyces hansenii
Authors: Bolumar Tomás , Sanz Yolanda , Aristoy M.-Concepción , Toldrá Fidel
Published: January 2003

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of
Debaryomyces hansenii
CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate,
p
-chloromercuribenzoic acid, and Hg
2+
, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The

K
m

for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about
D. hansenii
proteases and their possible roles in meat fermentation.
Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered
Escherichia coli
without Loss of Specific Isotopic Enrichment
Authors: Sampaio Maria-Manuel , Santos Helena , Boos Winfried
Published: January 2003

We report the construction of an
Escherichia coli
mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-
O
-α-
d
-mannosyl-
d
-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the
manA
gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the
cps
gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (
cpsG
) and mannose-1-phosphate guanylyltransferase (
cpsB
) ensure the formation of GDP-mannose. A second plasmid harbors
msg
, a gene from
Rhodothermus marinus
that encodes mannosylglycerate synthase, which catalyzes the formation of 2-
O
-α-
d
-mannosyl-
d
-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-
O
-α-
d
-mannosyl-
d
-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-α-
d
-mannosyl-
d
-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.
Induction of Sucrose Utilization Genes from
Bifidobacterium lactis
by Sucrose and Raffinose
Authors: Trindade Marla I. , Abratt Valerie R. , Reid Sharon J.
Published: January 2003

The probiotic organism
Bifidobacterium lactis
was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of
B. lactis
was identified by complementation of a gene library in
Escherichia coli
. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The
scrP
gene was expressed constitutively from its own promoter in
E. coli
grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with
scrP
performed by using RNA isolated from
B. lactis
identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In
B. lactis
, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the
scrP
,
scrR
, and
scrT
genes in
B. lactis
indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous
E. coli
indicated that ScrR functions as a positive regulator.
Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge:
13
C Labeling and In Vivo Nuclear Magnetic Resonance
Authors: Lemos Paulo C. , Serafim Luísa S. , Santos Margarida M. , Reis Maria A. M. , Santos Helena
Published: January 2003

In vivo
13
C and
31
P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-
13
C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV
5
, 59%; HV
4
, 5.0%; HV
3
, 1.1%; HV
2
, 3.5%; and HV
1
, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV
4
is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.
Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins
Authors: Worthington Penny , Blum Paul , Perez-Pomares Francisco , Elthon Tom
Published: January 2003

An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon
Sulfolobus solfataricus
and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the
S
.
solfataricus
-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.
Attachment of
Listeria monocytogenes
to Radish Tissue Is Dependent upon Temperature and Flagellar Motility
Authors: Gorski Lisa , Palumbo Jeffrey D. , Mandrell Robert E.
Published: January 2003

Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by
Listeria monocytogenes
. In order to begin to understand the physiology of the organism in a produce habitat, the ability of
L
.
monocytogenes
to attach to freshly cut radish tissue was examined. All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices. A screen was developed to identify Tn
917
-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized. Two of the three mutations were in genes with unknown functions. Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect. The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system. All three mutants were defective in attachment when tested at 30°C; the motility mutant had the most severe phenotype. However, not all of the mutants were defective when tested at other temperatures. These results indicate that
L
.
monocytogenes
may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of
Listeria
fitness in complex environments.
Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of
Cryptosporidium
Oocysts and
Giardia
Cysts in Water
Authors: McCuin Randi M. , Clancy Jennifer L.
Published: January 2003

Collaborative and in-house laboratory trials were conducted to evaluate
Cryptosporidium
oocyst and
Giardia
cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of
Cryptosporidium
oocysts and
Giardia
cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for
Cryptosporidium
oocysts and
Giardia
cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.