bluuebunny/crossref_metadata_2025 huggingface dataset:
Load History Effects on Fretting Contacts of Isotropic Materials
Authors: Rajeev P. T. , Murthy H. , Farris T. N.
Published: June 2004

The load history that blade/disk contacts in jet engine attachment hardware are subject to can be very complex. Using finite element method (FEM) to track changes in the contact tractions due to changing loads can be computationally very expensive. For two-dimensional plane-strain contact problems with friction involving similar/dissimilar isotropic materials, the contact tractions can be related to the initial gap function and the slip function using coupled Cauchy singular integral equations (SIEs). The effect of load history on the contact tractions is illustrated by presenting results for an example fretting “mission.” For the case of dissimilar isotropic materials the mission results show the effect of the coupling between the shear traction and the contact pressure.
Carbon Dioxide Emission Analysis With Energy Payback Effect
Authors: Utamura Motoaki
Published: June 2004

Analytical model is proposed to account for carbon emission behavior during replacement of power source from fossil to renewable energy in which sustainability of energy supply is stressed. Analyses show that energy payback time (EPT) should be much shorter than the doubling time of manufacturing cycle to secure adequate available energy during as well as after the replacement. Nuclear, small hydropower, and photovoltaic cell are taken as representative candidates and investigated as an option to replace fossil power until mid-century. Nuclear and small hydropower can be promising candidates but photovoltaic cell needs further development efforts to reduce EPT to avoid energy expense after the replacement.
Small-Scale Well-Proven Inherently Safe Nuclear Power Conversion
Authors: Crommelin G. A. K.
Published: June 2004

Over the last few years a number of papers have discussed the progress on studies and thoughts on small-scale nuclear power, especially nuclear power conversion systems aiming at the nonutility markets, such as the stand-alone heat generation, combined heat and power production, stand-alone electricity conversion, and ship propulsion. The design of these installations must fully comply with the philosophies as are common in these markets, where the expression “the engine is a means to an end” applies. So design to cost, design to be operated by non professional energy producers, to be managed by a pool-management system, maintained, repaired and overhauled by replacement, etc. The paper will discuss such a design. So far all papers mentioned have discussed the gas turbine directly coupled to the heat source. However, the helium turbine is considered quite a challenge for the gas turbine industry, so alternatives had to be found. At the moment the possibilities of gas turbines with an indirect heat source (to burn refuse, wood, refinery waste, etc.) are getting much more attention. The paper therefore will discuss how an inherently safe, well proven, nuclear heat source can be coupled by an intermediate heat exchanger to a recuperative, existing but adapted gas turbine.
Microscale Radial-Flow Compressor Impeller Made of Silicon Nitride: Manufacturing and Performance
Authors: Kang Sangkyun , Johnston James P. , Arima Toshiyuki , Matsunaga Minoru , Tsuru Hideaki , Printz Friedrich B.
Published: June 2004

A microscale, high-speed compressor impeller (12 mm diameter, 800,000 rpm) was tested for feasibility in regard to aerodynamic performance. The compressor was designed for application in a first-sized gas turbine generator. To survive high stresses at such high temperatures, the rotor was manufactured as a single turbine/compressor/shaft unit in silicon nitride, by the Mold SDM process. Performance testing was conducted in a cold-flow rig at reduced speed of 420,000 rpm. Results from a CFD code compared favorably to measured data at this speed. Extrapolation from test conditions to full design speed was accomplished by application of CFD applied at both speeds.
In Vivo Immobilization of Fusion Proteins on Bioplastics by the Novel Tag BioF
Authors: Moldes Cristina , García Pedro , García José L. , Prieto María A.
Published: June 2004

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from
Pseudomonas putida
GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-β-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.
Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from
Clonostachys rosea
Authors: Takahashi-Ando Naoko , Ohsato Shuichi , Shibata Takehiko , Hamamoto Hiroshi , Yamaguchi Isamu , Kimura Makoto
Published: June 2004

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by
zhd101
. An enhanced green fluorescent protein (EGFP) gene was fused to
zhd101
(i.e.,
egfp
::
zhd101
) and expressed in
Escherichia coli
. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (
k
cat
at 30°C, 0.51 s
−1
). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the
k
cat
was highest with zearalanone (1.5 s
−1
) and lowest with β-zearalenol (0.075 s
−1
). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used
egfp
::
zhd101
to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of
zhd101
for in vivo detoxification of ZEN. While recombinant
E. coli
and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant
Saccharomyces cerevisiae
gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, β-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade
Lactobacillus
strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol.
68:
3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.
Protein Engineering of Toluene-
o
-Xylene Monooxygenase from
Pseudomonas stutzeri
OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol
Authors: Vardar Gönül , Wood Thomas K.
Published: June 2004

Toluene-
o
-xylene monooxygenase (ToMO) from
Pseudomonas stutzeri
OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example,
Escherichia coli
TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from
o
-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From
o
-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22%
o
-cresol, 44%
m
-cresol, and 34%
p
-cresol, whereas wild-type ToMO formed 32%
o
-cresol, 21%
m
-cresol, and 47%
p
-cresol.
In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host
Escherichia coli
Authors: Gruenewald Stephan , Mootz Henning D. , Stehmeier Per , Stachelhaus Torsten
Published: June 2004

Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide
d-
Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host
Escherichia coli
, and the use of artificial nonribosomal peptide synthetases.
E. coli
provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4′-phosphopantetheine transferase gene
sfp
to ensure heterologous production of fully active holoenzmyes.
d-
Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in
Bacillus brevis
. Coexpression of the corresponding genes in
E. coli
gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to
E. coli
and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of
d
-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.
Evaluation ofNostocStrain ATCC 53789 as a Potential Source of Natural Pesticides
Authors: Biondi Natascia , Piccardi Raffaella , Margheri M. Cristina , Rodolfi Liliana , Smith Geoffrey D. , Tredici Mario R.
Published: June 2004

The cyanobacteriumNostocstrain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillariasp.,Fusarium oxysporumf. sp.melonis,Penicillium expansum,Phytophthora cambivora,P. cinnamomi,Rhizoctonia solani,Rosellinia, sp.,Sclerotinia sclerotiorum, andVerticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity againstS. sclerotiorumwas further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity ofNostocstrain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.
Purification, Characterization, and Sequencing of an Extracellular Cold-Active Aminopeptidase Produced by Marine Psychrophile
Colwellia psychrerythraea
Strain 34H
Authors: Huston Adrienne L. , Methe Barbara , Deming Jody W.
Published: June 2004

The limited database on cold-active extracellular proteases from marine bacteria was expanded by successful purification and initial biochemical and structural characterization of a family M1 aminopeptidase (designated ColAP) produced by the marine psychrophile
Colwellia psychrerythraea
strain 34H. The 71-kDa enzyme displayed a low optimum temperature (19°C) and narrow pH range (pH 6 to 8.5) for activity and greater thermolability than other extracellular proteases. Sequencing of the gene encoding ColAP revealed a predicted amino acid sequence with the highest levels of identity (45 to 55%) to M1 aminopeptidases from mesophilic members of the γ subclass of the
Proteobacteria
and the next highest levels of identity (35 to 36%) to leukotriene A
4
hydrolases from mammalian sources. Compared to mesophilic homologs, ColAP had structural differences thought to increase the flexibility for activity in the cold; for example, it had fewer proline residues, fewer ion pairs, and a lower hydrophobic residue content. In addition to intrinsic properties that determine enzyme activity and stability, we also investigated effects of extracellular polymeric substances (EPS) from spent culture medium of strain 34H on ColAP activity at an environmentally relevant temperature (0°C) and at 45°C (the maximum temperature for activity). In both cases, ColAP stability increased significantly in the presence of EPS, indicating the importance of considering environmentally relevant extrinsic factors when enzyme structure and function are investigated.
Establishment of a Pure Culture of the Hitherto Uncultured Unicellular CyanobacteriumAphanothece sacrum, and Phylogenetic Position of the Organism
Authors: Fujishiro Tsuneo , Ogawa Takahira , Matsuoka Masayoshi , Nagahama Kazuhiro , Takeshima Yasunobu , Hagiwara Hideaki
Published: June 2004

Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained inA. sacrumcells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells ∼6 μm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of naturalA. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from naturalA. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed thatA. sacrumoccupies a phylogenetically unique position among the cyanobacteria.
Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in
Escherichia coli
Authors: Hwang Dong Soo , Yoo Hyo Jin , Jun Jong Hyub , Moon Won Kyu , Cha Hyung Joon
Published: June 2004

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for
Mytilus galloprovincialis
foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in
Escherichia coli
and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.
Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by
Pichia pastoris
Authors: Woo Jung Hee , Liu Yuan Yi , Stavrou Scott , Neville David M.
Published: June 2004

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G
4
S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive
Pichia pastoris
JW102 (

Mut
+

) under control of the
AOX1
promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for
P. pastoris
and the limited capacity of
P. pastoris
to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive
P. pastoris
.
Aquaporin-Mediated Improvement of Freeze Tolerance of
Saccharomyces cerevisiae
Is Restricted to Rapid Freezing Conditions
Authors: Tanghe An , Van Dijck Patrick , Colavizza Didier , Thevelein Johan M.
Published: June 2004

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (
Saccharomyces cerevisiae
) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes
AQY1
-
1
and
AQY2
-
1
improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.
Richness and Diversity of Bacterioplankton Species along an Estuarine Gradient in Moreton Bay, Australia
Authors: Hewson Ian , Fuhrman Jed A.
Published: June 2004

Bacterioplankton community diversity was investigated in the subtropical Brisbane River-Moreton Bay estuary, Australia (27°25′S, 153°5′E). Bacterial communities were studied using automated rRNA intergenic spacer analysis (ARISA), which amplifies 16S-23S ribosomal DNA internally transcribed spacer regions from mixed-community DNA and detects the separated products on a fragment analyzer. Samples were collected from eight sites throughout the estuary and east to the East Australian Current (Coral Sea). Bacterioplankton communities had the highest operational taxonomic unit (OTU) richness, as measured by ARISA at eastern bay stations (
S
[total richness] = 84 to 85 OTU) and the lowest richness in the Coral Sea (
S
= 39 to 59 OTU). Richness correlated positively with bacterial abundance; however, there were no strong correlations between diversity and salinity, NO
3
−
and PO
4
3−
concentrations, or chlorophyll
a
concentration. Bacterioplankton communities at the riverine stations were different from communities in the bay or Coral Sea. The main differences in OTU richness between stations were in taxa that each represented 0.1% (the detection limit) to 0.5% of the total amplified DNA, i.e., the “tail” of the distribution. We found that some bacterioplankton taxa are specific to distinct environments while others have a ubiquitous distribution from river to sea. Bacterioplankton richness and diversity patterns in the estuary are potentially a consequence of greater niche availability, mixing of local and adjacent environment communities, or intermediate disturbance. Furthermore, these results contrast with previous reports of spatially homogeneous bacterioplankton communities in other coastal waters.
Extracellular Iron Reduction Is Mediated in Part by Neutral Red and Hydrogenase inEscherichia coli
Authors: McKinlay James B. , Zeikus J. Gregory
Published: June 2004

Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, byEscherichia coli,ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts ofE. coliwere used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. AnE. coli hypEmutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.
Effects of Physical Properties of Feed on Microbial Ecology and Survival of
Salmonella enterica
Serovar Typhimurium in the Pig Gastrointestinal Tract
Authors: Mikkelsen Lene Lind , Naughton Patrick J. , Hedemann Mette S. , Jensen Bent Borg
Published: June 2004

A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of
Salmonella enterica
serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (
P
< 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of
S. enterica
serovar Typhimurium DT12 in content from the stomach (
P
< 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (
P
< 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of
S. enterica
serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (
P
< 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (
P
< 0.05) and colon (
P
< 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of
Salmonella
during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.
Analysis of the Peptidoglycan Hydrolase Complement of
Lactococcus lactis
: Identification of a Third
N-
Acetylglucosaminidase, AcmC
Authors: Huard Carine , Miranda Guy , Redko Yulia , Wessner Françoise , Foster Simon J. , Chapot-Chartier Marie-Pierre
Published: June 2004

The peptidoglycan hydrolase (PGH) complement of
Lactococcus lactis
was identified by amino acid sequence similarity searching of the
L. lactis
IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in
L. lactis
subsp.
cremoris
MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has
N
-acetylglucosaminidase activity rather than the
N
-acetylmuramidase activity predicted by sequence similarity.
The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of
Aspergillus niger
through Intracellular Acidification
Authors: Plumridge Andrew , Hesse Stephan J. A. , Watson Adrian J. , Lowe Kenneth C. , Stratford Malcolm , Archer David B.
Published: June 2004

The growth of the filamentous fungus
Aspergillus niger
, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (
trans-trans
-2,4-hexadienoic acid). Conidia inoculated at 10
5
/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size.
A. niger
is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using
31
P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH
cyt
) by more than 1 pH unit and a depression of vacuolar pH (pH
vac
) in
A. niger
occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH
cyt
. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of
A. niger
mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.
Prevalence of the
stx
2
Gene in Coliform Populations from Aquatic Environments
Authors: García-Aljaro Cristina , Muniesa Maite , Jofre Juan , Blanch Anicet R.
Published: June 2004

Shiga toxin-producing
Escherichia coli
strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the
stx
2
gene in coliform bacteria in sewage samples of different origins. The presence of the
stx
2
gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10
2
genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the
stx
2
gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to
E. coli
serotypes other than
E. coli
O157, suggesting a low prevalence of strains of this serotype carrying the
stx
2
gene in the wastewater studied.
Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins
Authors: Williams Richard E. , Rathbone Deborah A. , Scrutton Nigel S. , Bruce Neil C.
Published: June 2004

Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.
Medium-Chain Fatty Acids Decrease Colonization and Invasion through
hilA
Suppression Shortly after Infection of Chickens with
Salmonella enterica
Serovar Enteritidis
Authors: Van Immerseel F. , De Buck J. , Boyen F. , Bohez L. , Pasmans F. , Volf J. , Sevcik M. , Rychlik I. , Haesebrouck F. , Ducatelle R.
Published: June 2004

The most common source of
Salmonella
infections in humans is food of poultry origin.
Salmonella enterica
serovar Enteritidis has a particular affinity for the contamination of the egg supply. In this study, the medium-chain fatty acids (MCFA), caproic, caprylic, and capric acid, were evaluated for the control of
Salmonella
serovar Enteritidis in chickens. All MCFA were growth inhibiting at low concentrations in vitro, with caproic acid being the most potent. Contact of
Salmonella
serovar Enteritidis with low concentrations of MCFA decreased invasion in the intestinal epithelial cell line T84. By using transcriptional fusions between the promoter of the regulatory gene of the
Salmonella
pathogenicity island I,
hilA
, and
luxCDABE
genes, it was shown that all MCFA decreased the expression of
hilA
, a key regulator related to the invasive capacity of
Salmonella
. The addition of caproic acid (3 g/kg of feed) to the feed of chicks led to a significant decrease in the level of colonization of ceca and internal organs by
Salmonella
serovar Enteritidis at 3 days after infection of 5-day-old chicks. These results suggest that MCFA have a synergistic ability to suppress the expression of the genes required for invasion and to reduce the numbers of bacteria in vivo. Thus, MCFA are potentially useful products for reducing the level of colonization of chicks and could ultimately aid in the reduction of the number of contaminated eggs in the food supply.
Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
Authors: Josefsen M. H. , Jacobsen N. R. , Hoorfar J.
Published: June 2004

As part of a large international project for standardization of PCR (Food-PCR;
www.pcr.dk
), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10
2
to 10
7
CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 × 10
3
and 2 × 10
3
CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 × 10
1
to 1 × 10
7
(
R
2
= 1.00) for the RotorGene and 10
3
to 10
7
(
R
2
= 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (
R
2
) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.
Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using
Salmonella
as a Model Organism
Authors: Fey Axel , Eichler Stefan , Flavier Sébastien , Christen Richard , Höfle Manfred G. , Guzmán Carlos A.
Published: June 2004

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of
Salmonella enterica
serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards.
Salmonella
cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.
Biotransformation in Double-Phase Systems: Physiological Responses of
Pseudomonas putida
DOT-T1E to a Double Phase Made of Aliphatic Alcohols and Biosynthesis of Substituted Catechols
Authors: Rojas Antonia , Duque Estrella , Schmid Andreas , Hurtado Ana , Ramos Juan-Luis , Segura Ana
Published: June 2004

Pseudomonas putida
strain DOT-T1E is highly tolerant to organic solvents, with a logP
ow
(the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of ≥2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested
P. putida
DOT-T1E tolerance to different aliphatic alcohols with a logP
ow
value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP
ow
around 1.5 are produced.
P. putida
DOT-T1E responds to aliphatic alcohols as the second phase through
cis-
to-
trans
isomerization of unsaturated
cis
fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow
P. putida
DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP
ow
of aliphatic alcohols correlates with their toxic effects, as octanol (logP
ow
= 2.9) has more negative effects in
P. putida
cells than 1-nonanol (logP
ow
= 3.4) or 1-decanol (logP
ow
= 4). A
P. putida
DOT-T1E derivative bearing plasmid pWW0-
xylE
::Km transforms
m-
xylene (logP
ow
= 3.2) into 3-methylcatechol (logP
ow
= 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.
Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms
Authors: Uyeno Yutaka , Sekiguchi Yuji , Sunaga Akiko , Yoshida Hiroki , Kamagata Yoichi
Published: June 2004

A
rapid and simple approach to the small-subunit (SSU) rRNA-based
quantitative detection of a specific group of microorganisms in complex
ecosystems has been developed. The method employs sequence-specific
cleavage of rRNA molecules with oligonucleotides and RNase H. Defined
mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to
as a “scissor probe”) that was specifically designed to
hybridize with a particular site of targeted rRNA and were subsequently
digested with RNase H to proceed to sequence-dependent rRNA scission at
the hybridization site. Under appropriate reaction conditions, the
targeted rRNAs were correctly cut into two fragments, whereas
nontargeted rRNAs remained intact under the same conditions. The
specificity of the cleavage could be properly adjusted by controlling
the hybridization stringency between the rRNA and the oligonucleotides,
i.e., by controlling either the temperature of the reaction or the
formamide concentration in the hybridization-digestion buffer used for
the reaction. This enabled the reliable discrimination of completely
matched rRNA sequences from single-base mismatched sequences. For the
detection of targeted rRNAs, the resulting RNA fragment patterns were
analyzed by gel electrophoresis with nucleotide-staining fluorescent
dyes in order to separate cleaved and intact rRNA molecules. The
relative abundance of the targeted SSU rRNA fragments in the total SSU
rRNA could easily be calculated without the use of an external standard
by determining the signal intensity of individual SSU rRNA bands in the
electropherogram. This approach provides a fast and easy means of
identification, detection, and quantification of a particular group of
microbes in clinical and environmental specimens based on
rRNA.
Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from
Lactobacillus fermentum
BR11 and Its Use as a Peptide Fusion Partner in
Lactobacillus
and
Lactococcus
Authors: Turner Mark S. , Hafner Louise M. , Walsh Terry , Giffard Philip M.
Published: June 2004

Examination of supernatant fractions from broth cultures of
Lactobacillus fermentum
BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of
Lactobacillus reuteri
,
Enterococcus faecium
,
Streptococcus pneumoniae
,
Streptococcus agalactiae
, and
Lactobacillus plantarum
. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of
Lactobacillus gasseri
and
Lactobacillus johnsonii
. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in
L. fermentum
,
Lactobacillus rhamnosus
GG and
Lactococcus lactis
MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the
L. fermentum
,
L. rhamnosus
, and
L. lactis
hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in
L. fermentum
or
L. rhamnosus
a fragment of human E-cadherin, which contains the receptor region for
Listeria monocytogenes
. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.
Endogenous Xylose Pathway in
Saccharomyces cerevisiae
Authors: Toivari Mervi H. , Salusjärvi Laura , Ruohonen Laura , Penttilä Merja
Published: June 2004

The baker's yeast
Saccharomyces cerevisiae
is generally classified as a non-xylose-utilizing organism. We found that
S. cerevisiae
can grow on
d
-xylose when only the endogenous genes
GRE3
(
YHR104w
), coding for a nonspecific aldose reductase, and
XYL2
(
YLR070c
,
ScXYL2
), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed
S. cerevisiae
strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of
SOR1
, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An
S. cerevisiae
strain carrying the XR and XDH enzymes from the xylose-utilizing yeast
Pichia stipitis
grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the
GRE3
and
ScXYL2
genes in the
S. cerevisiae
CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter
−1
h
−1
and a xylitol yield of 55% when xylose was the main carbon source.
Uptake and Replication ofSalmonella entericainAcanthamoeba rhysodes
Authors: Tezcan-Merdol Dilek , Ljungström Marianne , Winiecka-Krusnell Jadwiga , Linder Ewert , Engstrand Lars , Rhen Mikael
Published: June 2004

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars ofSalmonella entericaand five different isolates of axenicAcanthamoebaspp. In gentamicin protection assays,Salmonella entericaserovar Dublin was internalized more efficiently thanSalmonella entericaserovar Enteritidis orSalmonella entericaserovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized byAcanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation ofhilA, a key regulator in the expression of the invasion-associatedSalmonellapathogenicity island 1. Microscopy of infectedA. rhysodesrevealed thatS. entericaresided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated withhilAand theSalmonellavirulence plasmid. The data show thatAcanthamoebaspp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by definedSalmonellavirulence loci.