bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACTAphanothece 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTBoth 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

ABSTRACT

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

ABSTRACT

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.