bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Mitochondrial Toxicity in Human Cells Treated with Tenofovir: Comparison with Other Nucleoside Reverse Transcriptase Inhibitors
Authors: Birkus Gabriel , Hitchcock Michael J. M. , Cihlar Tomas
Published: July 2002

Drug-associated dysfunction of mitochondria is believed to play a role in the etiology of the various adverse symptoms that occur in human immunodeficiency virus (HIV)-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Tenofovir, a nucleotide analog recently approved for use in the treatment of HIV infection, was evaluated in vitro for its potential to cause mitochondrial toxicity and was compared to currently used NRTIs. Treatment with tenofovir (3 to 300 μM) for up to 3 weeks produced no significant changes in mitochondrial DNA (mtDNA) levels in human hepatoblastoma (HepG2) cells, skeletal muscle cells (SkMCs), or renal proximal tubule epithelial cells. The potencies of inhibition of mtDNA synthesis by the NRTIs tested were zalcitabine (ddC) > didanosine (ddI) > stavudine > zidovudine (ZDV) > lamivudine = abacavir = tenofovir, with comparable relative effects in the three cell types. Unlike ddC and ddI, tenofovir did not affect cellular expression of COX II and COX IV, two components of the mitochondrial cytochrome
c
oxidase complex. Lactate production was elevated by less than 20% in HepG2 cells or SkMCs following treatment with 300 μM tenofovir. In contrast, lactate synthesis increased by >200% in the presence of 300 μM ZDV. Thus, treatment of various human cell types with tenofovir at concentrations that greatly exceed those required for it both to have in vitro anti-HIV type 1 activity in peripheral blood mononuclear cells (50% effective concentration, 0.2 μM) and to achieve therapeutically relevant levels in plasma (maximum concentrations in plasma, 0.8 to 1.3 μM) is not associated with mitochondrial toxicity.
Antileishmanial Activities of Several Classes of Aromatic Dications
Authors: Brendle James J. , Outlaw Abram , Kumar Arvind , Boykin David W. , Patrick Donald A. , Tidwell Richard R. , Werbovetz Karl A.
Published: July 2002

Aromatic dicationic molecules possess impressive activity against a broad spectrum of microbial pathogens, including
Pneumocystis carinii
,
Cryptosporidium parvum
, and
Candida albicans
. In this work, 58 aromatic cations were examined for inhibitory activity against axenic amastigote-like
Leishmania donovani
parasites. In general, the most potent of the compounds were substituted diphenyl furan and thiophene dications. 2,5-Bis-(4-amidinophenyl)thiophene was the most active compound. This agent displayed a 50% inhibitory concentration (IC
50
) of 0.42 ± 0.08 μM against
L. donovani
and an in vitro antileishmanial potency 6.2-fold greater than that of the clinical antileishmanial dication pentamidine and was 155-fold more toxic to the parasites than to a mouse macrophage cell line. 2,4-Bis-(4-amidinopheny)furan was twice as active as pentamidine (IC
50
, 1.30 ± 0.21 μM), while 2,5-bis-(4-amidinopheny)furan and pentamidine were essentially equipotent in our in vitro antileishmanial assay. Carbazoles, dibenzofurans, dibenzothiophenes, and benzimidazoles containing amidine or substituted amidine groups were generally less active than the diphenyl furans and thiophenes. In all cases, aromatic dications possessing strong antileishmanial activity were severalfold more toxic to the parasites than to a cultured mouse macrophage cell line. These structure-activity relationships demonstrate the potent antileishmanial activity of several aromatic dications and provide valuable information for the future design and synthesis of more potent antiparasitic agents.
Extent of Cross-Resistance between Agents Used To Treat Human Immunodeficiency Virus Type 1 Infection in Clinically Derived Isolates
Authors: Harrigan P. Richard , Larder Brendan A.
Published: July 2002

The phenomenon of cross-resistance to antiretroviral agents used to treat human immunodeficiency virus type 1 infection is well known but so far has been only qualitatively described. Here, we quantitate the degree of cross-resistance among all commonly prescribed antiretroviral agents in almost 5,000 clinically derived recombinant isolates collected in the United States since January 2000.
Effects of Chemical and Physical Agents on Viability and Infectivity of
Encephalitozoon intestinalis
Determined by Cell Culture and Flow Cytometry
Authors: Santillana-Hayat Maud , Sarfati Claudine , Fournier Sandra , Chau Françoise , Porcher Raphaël , Molina Jean-Michel , Derouin Francis
Published: July 2002

We combined tissue culture and flow cytometry to assess the activities of various temperatures, chemicals, and disinfectants on the viability and infectivity of spores of
Encephalitozoon intestinalis
. Surfanios and benzalkonium chloride, disinfectants currently used in the hospital, were remarkably efficient in destroying spore viability and infectivity.
Rhizosphere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant Bacteria
Authors: Shim Hojae , Chauhan Sadhana , Ryoo Doohyun , Bowers Kally , Thomas Stuart M. , Canada Keith A. , Burken Joel G. , Wood Thomas K.
Published: July 2002

Indigenous bacteria from poplar tree (
Populus canadensis
var.
eugenei
‘Imperial Carolina’) and southern California shrub rhizospheres, as well as two tree-colonizing
Rhizobium
strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene
o
-monooxygenase (TOM) from
Burkholderia cepacia
G4 by integrating the
tom
locus into the chromosome. The poplar and
Rhizobium
recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 × 10
5
to 23 × 10
5
CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% ± 12% of all rhizosphere bacteria after 28 days (0.2 × 10
5
to 31 × 10
5
CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as
Pseudomonas
sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% ± 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.
Characterization of
Bacillus
Species Used for Oral Bacteriotherapy and Bacterioprophylaxis of Gastrointestinal Disorders
Authors: Hoa Ngo Thi , Baccigalupi Loredana , Huxham Ashley , Smertenko Andrei , Van Pham Hung , Ammendola Sergio , Ricca Ezio , Cutting Simon M.
Published: July 2002

Bacillus subtilis
spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since
B. subtilis
is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as
Lactobacillus
spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in
Marinosulfonomonas methylotropha
Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment
Authors: Baxter Nardia J. , Scanlan Julie , De Marco Paolo , Wood Ann P. , Murrell J. Colin
Published: July 2002

Marinosulfonomonas methylotropha
strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from
M. methylotropha
strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp
Hin
dIII fragment contained
msmA, msmB
, and
msmC
, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp
Hin
dIII fragment contained duplicate copies of
msmA
and
msmB
, as well as
msmD
, encoding the reductase component of methanesulfonate. Both sets of
msmA
and
msmB
genes were virtually identical, and the derived
msmA
and
msmB
sequences of
M. methylotropha
strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer
Methylosulfonomonas methylovora
strain M2 were found to be 82 and 69% identical. The
msmA
gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of
msmA
which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding
msmA
genes from newly isolated
Hyphomicrobium, Methylobacterium
, and
Pedomicrobium
species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these
msmA
gene fragments, compared to
msmA
sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.
Determination of Subunit Composition of
Clostridium cellulovorans
Cellulosomes That Degrade Plant Cell Walls
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H.
Published: July 2002

Clostridium cellulovorans
produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of
C. cellulovorans
and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.
Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1
Authors: Johnsen Anders R. , Bendixen Karen , Karlson Ulrich
Published: July 2002

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.
Bacteriophage Resistance of a Δ
thyA
Mutant of
Lactococcus lactis
Blocked in DNA Replication
Authors: Pedersen Martin B. , Jensen Peter R. , Janzen Thomas , Nilsson Dan
Published: July 2002

The
thyA
gene, which encodes thymidylate synthase (TS), of
Lactococcus lactis
CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of
thyA
by gene replacement. The resulting strain, MBP71 Δ
thyA
, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.
Humoral Immune Response in Chromoblastomycosis during and after Therapy
Authors: Esterre P. , Jahevitra M. , Andriantsimahavandy A.
Published: July 2002

A longitudinal study was carried out in Madagascar, the most important focus of chromoblastomycosis (P. Esterre, A. Andriantsimahavandy, E. Ramarcel, and J. L. Pecarrere, Am. J. Trop. Med. Hyg. 55:45–47, 1996), to investigate natural immunity to this disease. Sequential blood samples were obtained before, during, and at the end of a successful therapeutic trial with terbinafine, a new antifungal drug. Using enzyme-linked immunosorbent assay and immunoblot methods, detailed analyses of antibody concentration and antigen mapping were conducted for 136 serum samples and tentatively correlated to epidemiological and pathobiological data. Two different cytoplasmic antigens, corresponding to the two fungal species involved (
Fonsecaea pedrosoi
and
Cladophialophora carrionii
), were used to analyze the distribution of different classes of immunoglobulins. This was done with respect to the origin of the isolates, clinical and pathobiological. Although strong individual variations were noticed, some major antigens (one of 18.5 kDa specific for
F. pedrosoi
and two of 23.5 and 33 kDa, respectively, specific for
C. carrionii
) corresponded to high antibody prevalence and concentration. As some antigenic components were also detected by immunoglobulin M (IgM) and IgA antibodies, the role that these specific antibodies could play in the immune response is discussed.
Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting
Authors: La Scola Bernard , Rydkina Lena , Ndihokubwayo Jean-Bosco , Vene Sirkka , Raoult Didier
Published: July 2002

Differentiation of murine typhus due toRickettsia typhiand epidemic typhus due toRickettsia prowazekiiis critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) againstR. typhiandR. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekiiandR. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers againstR. typhiandR. prowazekiiallowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate betweenR. prowazekiiandR. typhiexposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.
Host Cell Responses to Genotypically SimilarHelicobacter pyloriIsolates from United States and Japan
Authors: Ando Takafumi , Peek Richard M. , Lee Yong-Chan , Krishna Uma , Kusugami Kazuo , Blaser Martin J.
Published: July 2002

Associations ofHelicobacter pylorigenotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-threecagA+vacAs1/m1H. pyloriisolates (United States,n= 24; Japan,n= 39) and eightcagA-negativevacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/orcagisland mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and JapanesecagA+vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frameoipAopen reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus,H. pylori cagA+vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by thecagisland in vitro. Assessment of apoptosis in vitro identified a group ofH. pyloriisolates that induce more severe gastric inflammation and atrophy.
Mycolic Acid Analysis by High-Performance Liquid Chromatography for Identification ofMycobacteriumSpecies
Authors: Butler W. Ray , Guthertz Linda S.
Published: July 2002

Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.
Regulation of Cathelicidin Gene Expression: Induction by Lipopolysaccharide, Interleukin-6, Retinoic Acid, and
Salmonella enterica
Serovar Typhimurium Infection
Authors: Wu Hua , Zhang Guolong , Minton J. Ernest , Ross Christopher R. , Blecha Frank
Published: July 2002

Cathelicidins are a family of antimicrobial peptides prominent in the host defense mechanisms of several mammalian species. In addition to their antimicrobial activities, these peptides have been implicated in wound healing, angiogenesis, and other innate immune mechanisms. To investigate the regulatory mechanisms of cathelicidin gene expression, we conducted in vitro experiments evaluating the bone marrow cell expression of two porcine cathelicidins, PR-39 and protegrin, and cloned and evaluated the promoter sequence of PR-39. In addition, we evaluated in vivo kinetics of cathelicidin gene expression in pigs during an infection with
Salmonella enterica
serovar Typhimurium. Lipopolysaccharide (LPS) increased PR-39 and protegrin mRNA expression, which was ameliorated by polymyxin B. Concentrations of PR-39 in supernatants from bone marrow cell cultures were increased 10-fold after LPS stimulation. Similarly, interleukin-6 (IL-6) and all-trans retinoic acid (RA) markedly induced cathelicidin gene expression. To verify the transcriptional activation of the PR-39 gene by these agents, we made a PR-39 promoter-luciferase construct containing the full-length PR-39 promoter driving luciferase gene expression and transiently transfected PK-15 epithelial cells. RA and IL-6 increased luciferase activity in PK-15 cells transfected with the PR-39 promoter-luciferase reporter. Similarly,
Salmonella
-challenged pigs showed increased expression of PR-39 and protegrin mRNA in bone marrow cells at 6 and 24 h postchallenge. Taken together, these findings show that bacterial products (LPS), IL-6, RA, and
Salmonella
infection enhance the expression of the cathelicidins, PR-39 and protegrin, in bone marrow progenitor cells, and we suggest that extrinsic modulation of this innate host defense mechanism may be possible.
Campylobacter fetus sap
Inversion Occurs in the Absence of RecA Function
Authors: Ray Kevin C. , Tu Zheng-Chao , Grogono-Thomas Rosemary , Newell Diane G. , Thompson Stuart A. , Blaser Martin J.
Published: July 2002

Phase variation of
Campylobacter fetus
surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique
sap
promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the
C. fetus sap
inversion system is RecA dependent. When we challenged a pregnant ewe with a
recA
mutant of wild-type
C. fetus
(strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the
aphA
insertion into
recA
was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the
sap
locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these
recA
mutants the
sap
inversion frequency is reduced by 2 to 3 log
10
units compared to that in the wild type. Thus, although the major
sap
inversion pathway in
C. fetus
is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.
IS
1414
, an
Escherichia coli
Insertion Sequence with a Heat-Stable Enterotoxin Gene Embedded in a Transposase-Like Gene
Authors: McVeigh Annette , Fasano Alessio , Scott Daniel A. , Jelacic Sandra , Moseley Steve L. , Robertson Donald C. , Savarino Stephen J.
Published: July 2002

Enteroaggregative
Escherichia coli
(EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic
E. coli
(ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of
astA
nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that
astA
is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the −1 reading frame in reference to the toxin gene. In vitro expression of the predicted
M
r
-∼46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS
285
from
Yersinia pestis
, IS
1356
from
Burkholderia cepacia
, and IS
Rm3
from
Rhizobium meliloti
. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS
1414
. Preliminary experiments to show IS
1414
translocation were unsuccessful. Overlapping genes of the type suggested by the IS
1414
core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.