bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective and Sensitive Method for PCR Amplification of
Escherichia coli
16S rRNA Genes in Soil
Authors: Sabat G. , Rose P. , Hickey W. J. , Harkin J. M. 
Published: July 2002

ABSTRACT

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of
Escherichia coli
16S rRNA genes. The method was capable of discriminating
E. coli
from other enteric bacteria, including its closest relative,
Shigella
. Selective amplification of
E. coli
occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl
2
concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting
E. coli
DNA in heterogeneous DNA samples, such as those extracted from soil.
Interactions between Pyruvate and Lactate Metabolism in
Propionibacterium freudenreichii
subsp.
shermanii
: In Vivo
13
C Nuclear Magnetic Resonance Studies
Authors: Deborde Catherine , Boyaval Patrick 
Published: July 2002

ABSTRACT

In vivo
13
C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of
Propionibacterium freudenreichii
subsp.
shermanii
. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 μmol · min
−1
· g
−1
(cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 μmol · min
−1
· g
−1
when it was cometabolized with pyruvate.
P. shermanii
used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO
2
, succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.
Biocontrol of the Sugarcane Borer
Eldana saccharina
by Expression of the
Bacillus thuringiensis cry1Ac7
and
Serratia marcescens chiA
Genes in Sugarcane-Associated Bacteria
Authors: Downing Katrina J. , Leslie Graeme , Thomson Jennifer A. 
Published: July 2002

ABSTRACT

The
cry1Ac7
gene of
Bacillus thuringiensis
strain 234, showing activity against the sugarcane borer
Eldana saccharina
, was cloned under the control of the
tac
promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the
tac
promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a
Pseudomonas fluorescens
strain isolated from the phylloplane of sugarcane and the endophytic bacterium
Herbaspirillum seropedicae
found in sugarcane. The p
tac-cry1Ac7
construct was introduced into the chromosome of
P. fluorescens
using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated
cry1Ac7
gene were much higher under the control of the
tac
promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in
P. fluorescens
and
H. seropedicae
of p
tac-cry1Ac7
carried on pKT240 caused plasmid instability with no detectable protein expression. In
H. seropedicae
, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm
r
promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of
E. saccharina
larvae than the latter.
P. fluorescens
14::p
tac-tox
resulted in higher mortality of larvae than did
P. fluorescens
14::
tox
. An increased toxic effect was observed when
P. fluorescens
14::p
tac-tox
was combined with
P. fluorescens
carrying the
Serratia marcescens
chitinase gene
chiA
, under the control of the
tac
promoter, integrated into the chromosome.
Studies on the Production of Fungal Peroxidases in
Aspergillus niger
Authors: Conesa Ana , van den Hondel Cees A. M. J. J. , Punt Peter J. 
Published: July 2002

ABSTRACT

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the
Phanerochaete chrysosporium
lignin peroxidase H8 (
lipA
) and manganese peroxidase (MnP) H4 (
mnp1
) genes in
Aspergillus niger
has been studied. For this purpose, a protease-deficient
A. niger
strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the
lipA
and
mnp1
genes fused to the
A. niger
glucoamylase gene did not result in improved production yields.
Use of Self-Assembled Monolayers of Different Wettabilities To Study Surface Selection and Primary Adhesion Processes of Green Algal (
Enteromorpha
) Zoospores
Authors: Callow Maureen E. , Callow J. A. , Ista Linnea K. , Coleman Sarah E. , Nolasco Aleece C. , López Gabriel P. 
Published: July 2002

ABSTRACT

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (
Enteromorpha
) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH
3
) or hydroxyl (OH) groups or mixtures of CH
3
- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and “searching” above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to “sense” that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.
Thermostable Chitosanase from
Bacillus
sp. Strain CK4: Cloning and Expression of the Gene and Characterization of the Enzyme
Authors: Yoon Ho-Geun , Kim Hee-Yun , Lim Young-Hee , Kim Hye-Kyung , Shin Dong-Hoon , Hong Bum-Shik , Cho Hong-Yon 
Published: July 2002

ABSTRACT

A thermostable chitosanase gene from the environmental isolate
Bacillus
sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from
Bacillus
sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from
Bacillus subtilis
,
Bacillus ehemensis
, and
Bacillus circulans
, respectively. C-terminal homology analysis shows that
Bacillus
sp. strain CK4 belongs to cluster III with
B. subtilis
. The gene was similar in size to that of the mesophile
B. subtilis
but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80°C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.
Isolation of an Aldehyde Dehydrogenase Involved in the Oxidation of Fluoroacetaldehyde to Fluoroacetate in
Streptomyces cattleya
Authors: Murphy Cormac D. , Moss Steven J. , O'Hagan David 
Published: July 2002

ABSTRACT

Streptomyces cattleya
is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD
+
-dependent fluoroacetaldehyde dehydrogenase from
S. cattleya
that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde.
Identification of Disulfides from the Biodegradation of Dibenzothiophene
Authors: Bressler David C. , Fedorak Phillip M. 
Published: July 2002

ABSTRACT

Several investigations have identified benzothiophene-2,3-dione in the organic solvent extracts of acidified cultures degrading dibenzothiophene via the Kodama pathway. In solution at neutral pH, the 2,3-dione exists as 2-mercaptophenylglyoxylate, which cyclizes upon acidification and is extracted as the 2,3-dione. The fate of these compounds in microbial cultures has never been determined. This study investigated the abiotic reactions of 2-mercaptophenylglyoxylate incubated aerobically in mineral salts medium at neutral pH. Oxidation led to the formation of 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, formed from two molecules of 2-mercaptophenylglyoxylate. Two sequential abiotic, net losses of both a carbon and an oxygen atom produced two additional disulfides, 2-oxo-2-(2-thiophenyl)ethanoic acid 2-benzoic acid disulfide and 2,2′-dithiosalicylic acid. The methods developed to extract and detect these three disulfides were then used for the analysis of a culture of
Pseudomonas
sp. strain BT1d grown on dibenzothiophene as its sole carbon and energy source. All three of the disulfides were detected, indicating that 2-mercaptophenylglyoxylate is an important, short-lived intermediate in the breakdown of dibenzothiophene via the Kodama pathway. The disulfides eluded previous investigations because of (i) their high polarity, being dicarboxylic acids; (ii) the need to lower the pH of the aqueous medium to <1 to extract them into an organic solvent such as dichloromethane; (iii) their poor solubility in organic solvents, (iv) their removal from organic extracts of cultures during filtration through the commonly used drying agent anhydrous sodium sulfate; and (v) their high molecular masses (362, 334, and 306 Da) compared to that of dibenzothiophene (184 Da).
Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1
Authors: Bohuslavek Jan , Payne Jason W. , Liu Yong , Bolton Harvey , Xun Luying 
Published: July 2002

ABSTRACT

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes,
emoA
and
emoB
, were expressed in
Escherichia coli
, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The
emoA
and
emoB
genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.
Specific Growth Rate Plays a Critical Role in Hydrogen Peroxide Resistance of the Marine Oligotrophic Ultramicrobacterium
Sphingomonas alaskensis
Strain RB2256
Authors: Ostrowski Martin , Cavicchioli Ricardo , Blaauw Maarten , Gottschal Jan C. 
Published: July 2002

ABSTRACT

The marine oligotrophic ultramicrobacterium
Sphingomonas alaskensis
RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as
Vibrio angustum
S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of
S. alaskensis
RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of
S. alaskensis
RB2256 at a specific growth rate of 0.02 to 0.13 h
−1
exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h
−1
or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast,
V. angustum
S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h
−1
. Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of
S. alaskensis
RB2256, especially under oxidative stress conditions.
Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments
Authors: Ringelberg David B. , Talley Jeffrey W. , Perkins Edward J. , Tucker Samuel G. , Luthy Richard G. , Bouwer Edward J. , Fredrickson Herbert L. 
Published: July 2002

ABSTRACT

Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of
14
C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonical
r
2
of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e.,
Rhodococcus
spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.
Biotransformation of Biphenyl by
Paecilomyces lilacinus
and Characterization of Ring Cleavage Products
Authors: Gesell Manuela , Hammer Elke , Specht Michael , Francke Wittko , Schauer Frieder 
Published: July 2002

ABSTRACT

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus
Paecilomyces lilacinus
. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via
ortho
-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4′-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that
P. lilacinus
has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.
Cattle Water Troughs as Reservoirs of
Escherichia coli
O157
Authors: LeJeune Jeffrey T. , Besser Thomas E. , Hancock Dale D. 
Published: July 2002

ABSTRACT

Environmental survival of
Escherichia coli
O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious
E. coli
O157 was studied using microcosms simulating cattle water troughs. Culturable
E. coli
O157 survived for at least 245 days in the microcosm sediments. Furthermore,
E. coli
O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of
E. coli
O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting
E. coli
O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.
Physiological Characterization of
Pseudomonas putida
DOT-T1E Tolerance to
p
-Hydroxybenzoate
Authors: Ramos-González Marı́a-Isabel , Godoy Patricia , Alaminos Miguel , Ben-Bassat Arie , Ramos Juan-Luis 
Published: July 2002

ABSTRACT

Pseudomonas putida
DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of
p
-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the
cis/trans
ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.
Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of
Helicobacter pylori
Authors: Nilsson Hans-Olof , Blom Jens , Al-Soud Waleed Abu , Ljungh Åsa , Andersen Leif P. , Wadström Torkel 
Published: July 2002

ABSTRACT

Helicobacter pylori
can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of
H. pylori
in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable
H. pylori
were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and
ureA
mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (
P
= 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable
H. pylori
, indicating viability of such morphological forms.