bluuebunny/crossref_metadata_2025 huggingface dataset:
Clearance of Fungal Burden during Treatment of Disseminated Histoplasmosis with Liposomal Amphotericin B versus Itraconazole
Authors: Wheat L. Joseph , Cloud Gretchen , Johnson Philip C. , Connolly Patricia , Goldman Mitchell , Le Monte Ann , Fuller Deanna E. , Davis Thomas E. , Hafner Richard ,   The AIDS Clinical Trials Group The Mycoses Study Group Of NIAID
Published: July 2002

Animal studies have shown that fungal burden correlates with survival during treatment with new antifungal therapies for histoplasmosis. The purpose of this report is to compare the clearance of fungal burden in patients with histoplasmosis treated with liposomal amphotericin B versus itraconazole. In two separate closed clinical trials that evaluated the efficacy of liposomal amphotericin B and itraconazole treatment of disseminated histoplasmosis in patients with AIDS, blood was cultured for fungus and blood and urine were tested forHistoplasmaantigen. The clinical response rates were similar; 86% with liposomal amphotericin B (n= 51) versus 85% with itraconazole (n= 59). Of the patients with positive blood cultures at enrollment, after 2 weeks of therapy cultures were negative in over 85% of the liposomal amphotericin B group versus 53% of the itraconazole group (P= 0.0008). Furthermore, after 2 weeks, median antigen levels in serum fell by 1.6 U in the liposomal amphotericin B group versus 0.1 U in the itraconazole group (P= 0.02), and those in urine fell by 2.1 U in the liposomal amphotericin B group and 0.2 U in the itraconazole group (P= 0.0005). The more rapid clearance of fungemia supports the use of liposomal amphotericin B rather than itraconazole for initial treatment of moderately severe or severe histoplasmosis.
In Vitro Synergy of Caspofungin and Amphotericin B against
Aspergillus
and
Fusarium
spp
Authors: Arikan Sevtap , Lozano-Chiu Mario , Paetznick Victor , Rex John H.
Published: July 2002

We investigated the in vitro interaction of caspofungin and amphotericin B for clinical isolates of
Aspergillus
and
Fusarium
. Synergy tests were performed using the checkerboard method and following the NCCLS M38-P guidelines in Antibiotic Medium 3 broth supplemented to 2% glucose. Antagonism was not observed for any of the isolates tested. Caspofungin and amphotericin B were synergistic or synergistic to additive for at least half of the isolates.
Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro
Authors: Delmas Julien , Schorr Olivier , Jamard Catherine , Gibbs Craig , Trépo Christian , Hantz Olivier , Zoulim Fabien
Published: July 2002

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.
Plasma Protein Binding of Amphotericin B and Pharmacokinetics of Bound versus Unbound Amphotericin B after Administration of Intravenous Liposomal Amphotericin B (AmBisome) and Amphotericin B Deoxycholate
Authors: Bekersky Ihor , Fielding Robert M. , Dressler Dawna E. , Lee Jean W. , Buell Donald N. , Walsh Thomas J.
Published: July 2002

Unilamellar liposomal amphotericin B (AmBisome) (liposomal AMB) reduces the toxicity of this antifungal drug. The unique composition of liposomal AMB stabilizes the liposomes, producing higher sustained drug levels in plasma and reducing renal and hepatic excretion. When liposomes release their drug payload, unbound, protein-bound, and liposomal drug pools may exist simultaneously in the body. To determine the amounts of drug in these pools, we developed a procedure to measure unbound AMB in human plasma by ultrafiltration and then used it to characterize AMB binding in vitro and to assess the pharmacokinetics of nonliposomal pools of AMB in a phase IV study of liposomal AMB and AMB deoxycholate in healthy subjects. We confirmed that AMB is highly bound (>95%) in human plasma and showed that both human serum albumin and α
1
-acid glycoprotein contribute to this binding. AMB binding exhibited an unusual concentration dependence in plasma: the percentage of bound drug increased as the AMB concentration increased. This was attributed to the low solubility of AMB in plasma, which limits the unbound drug concentration to <1 μg/ml. Subjects given 2 mg of liposomal AMB/kg of body weight had lower exposures (as measured by the maximum concentration of drug in serum and the area under the concentration-time curve) to both unbound and nonliposomal drug than those receiving 0.6 mg of AMB deoxycholate/kg. Most of the AMB in plasma remained liposome associated (97% at 4 h, 55% at 168 h) after liposomal AMB administration, so that unbound drug concentrations remained at <25 ng/ml in all liposomal AMB-treated subjects. Although liposomal AMB markedly reduces the total urinary and fecal recoveries of AMB, urinary and fecal clearances based on unbound AMB were similar (94 to 121 ml h
−1
kg
−1
) for both formulations. Unbound drug urinary clearances were equal to the glomerular filtration rate, and tubular transit rates were <16% of the urinary excretion rate, suggesting that net filtration of unbound drug, with little secretion or reabsorption, is the mechanism of renal clearance for both conventional and liposomal AMB in humans. Unbound drug fecal clearances were also similar for the two formulations. Thus, liposomal AMB increases total AMB concentrations while decreasing unbound AMB concentrations in plasma as a result of sequestration of the drug in long-circulating liposomes.
Antitrypanosomal Activities of Tryptanthrins
Authors: Scovill John , Blank Elizabeth , Konnick Michael , Nenortas Elizabeth , Shapiro Theresa
Published: July 2002

New drugs and molecular targets are needed against
Trypanosoma brucei
, the protozoan that causes African sleeping sickness. Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione), a traditional antifungal agent, and 11 analogs were tested against
T. brucei
in vitro. The greatest activity was conferred by electron-withdrawing groups in the 8 position of the tryptanthrin ring system; the most potent compound had a 50% effective concentration of 0.40 μM.
Lack of Catheter Infection by the
efg1
/
efg1
cph1
/
cph1
Double-Null Mutant, a
Candida albicans
Strain That Is Defective in Filamentous Growth
Authors: Lewis Russell E. , Lo Hsiu-Jung , Raad Issam I. , Kontoyiannis Dimitrios P.
Published: July 2002

The molecular controls regulating the successful colonization of
Candida albicans
on foreign materials are not known. Here we show that a mutant
C. albicans
strain defective in filamentous growth and lacking the transcription factors Efg1p and Cph1p has a profoundly deficient potential for colonizing on polyurethane catheters.
Vesicle-Mediated Transfer of Virulence Genes from
Escherichia coli
O157:H7 to Other Enteric Bacteria
Authors: Yaron Sima , Kolling Glynis L. , Simon Lee , Matthews Karl R.
Published: July 2002

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen
Escherichia coli
O157:H7 facilitate the transfer of genes, which are then expressed by recipient
Salmonella enterica
serovar Enteritidis or
E. coli
JM109. Electron micrographs of purified DNA from
E. coli
O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (
hly
, L7095,
mobA
, and
gfp
), chromosomal DNA (
uidA
and
eaeA
), and phage DNA (
stx1
and
stx2
). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (
E. coli
JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10
3
transformants μg of DNA
−1
) and demonstrated that vesicles can deliver antibiotic resistance. Transformed
E. coli
JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.
Anaerobic Oxidation of
n
-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture
Authors: Kropp Kevin G. , Davidova Irene A. , Suflita Joseph M.
Published: July 2002

We identified trace metabolites produced during the anaerobic biodegradation of H
26
- and D
26
-
n
-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C—H or C—D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H
26
- and D
26
-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated
n
-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H
26
-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.
Effects of pH and Trace Minerals on Long-Term Starvation ofLeuconostoc mesenteroides
Authors: Kim Dong-Shik , Thomas Steven , Fogler H. Scott
Published: July 2002

Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production. Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion. For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria. This paper addresses the effects of pH and trace minerals on cell viability ofLeuconostoc mesenteroidesduring carbon source depletion. Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose. These substrates were chosen becauseL. mesenteroidesproduces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions. The effects of dextran on the cell viability were tested at different pH values with and without trace minerals. The rate of cell death followed an exponential-decay law for different values of the solution pH. The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above. The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested. The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions. The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7. For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals. It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells. The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.
Molecular Diversity within
Lactobacillus helveticus
as Revealed by Genotypic Characterization
Authors: Giraffa Giorgio , Gatti Monica , Rossetti Lia , Senini Lucia , Neviani Erasmo
Published: July 2002

Lactobacillus helveticus
is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian cheeses, such as Emmental, Grana, and Provolone cheeses. Substantial differences in several technologically important characteristics are found among
L. helveticus
strains isolated from natural dairy starter cultures. In the present study we investigated the genotypic diversity of 74 strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains. A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used as genotypic fingerprinting. A multivariate statistical analysis of the data enabled us to identify significant genotypic heterogeneity in
L. helveticus
. We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese). Our data contribute to the description of microbial heterogeneity in
L. helveticus
and provide a more solid basis for understanding the functional and ecological significance of the presence of different
L. helveticus
biotypes in natural dairy starter cultures.
Survival and Filamentation of
Salmonella enterica
Serovar Enteritidis PT4 and
Salmonella enterica
Serovar Typhimurium DT104 at Low Water Activity
Authors: Mattick K. L. , Jørgensen F. , Legan J. D. , Cole M. B. , Porter J. , Lappin-Scott H. M. , Humphrey T. J.
Published: July 2002

In this study we investigated the long-term survival of and morphological changes in
Salmonella
strains at low water activity (a
w
).
Salmonella enterica
serovar Enteritidis PT4 and
Salmonella enterica
serovar Typhimurium DT104 survived at low a
w
for long periods, but minimum humectant concentrations of 8% NaCl (a
w
, 0.95), 96% sucrose (a
w
, 0.94), and 32% glycerol (a
w
, 0.92) were bactericidal under most conditions.
Salmonella rpoS
mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a
w
, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a
w
values of 0.93 to 0.98, strains of
S. enterica
serovar Enteritidis and
S. enterica
serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of
rpoS
expression. When the preparations were returned to high-a
w
conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of
Salmonella
strains at low a
w
highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in
Salmonella
biomass at low a
w
(without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a
w
storage. If
Salmonella
strains form filaments in food products that have low a
w
values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.
Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane
Authors: Alakomi H.-L. , Skyttä E. , Saarela M. , Mattila-Sandholm T. , Latva-Kala K. , Helander I. M.
Published: July 2002

The effect of lactic acid on the outer membrane permeability of
Escherichia coli
O157:H7,
Pseudomonas aeruginosa
, and
Salmonella enterica
serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for
P. aeruginosa
, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl
2
. Lactic acid sensitized
E. coli
and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized
P. aeruginosa
to SDS and to Triton X-100.
P. aeruginosa
was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.
A New
Klebsiella planticola
Strain (Cd-1) Grows Anaerobically at High Cadmium Concentrations and Precipitates Cadmium Sulfide
Authors: Sharma Pramod K. , Balkwill David L. , Frenkel Anatoly , Vairavamurthy Murthy A.
Published: July 2002

Heavy metal resistance by bacteria is a topic of much importance to the bioremediation of contaminated soils and sediments. We report here the isolation of a highly cadmium-resistant
Klebsiella planticola
strain, Cd-1, from reducing salt marsh sediments. The strain grows in up to 15 mM CdCl
2
under a wide range of NaCl concentrations and at acidic or neutral pH. In growth medium amended with thiosulfate, it precipitated significant amounts of cadmium sulfide (CdS), as confirmed by x-absorption spectroscopy. In comparison with various other strains tested, Cd-1 is superior for precipitating CdS in cultures containing thiosulfate. Thus, our results suggest that Cd-1 is a good candidate for the accelerated bioremediation of systems contaminated by high levels of cadmium.
rpoB
-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity
Authors: Dahllöf Ingela , Baillie Harriet , Kjelleberg Staffan
Published: July 2002

Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (
rpoB
), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga
Delisea pulchra
and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of
rpoB
, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and
rpoB
PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from
D. pulchra
. Eight out of 14 isolates from
D. pulchra
, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when
rpoB
was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from
D. pulchra
proved to be more accurately described using
rpoB
, compared to the 16S rDNA-based PCR-DGGE.
Mathematical Analysis of Growth and Interaction Dynamics of Streptomycetes and a Bacteriophage in Soil
Authors: Burroughs N. J. , Marsh P. , Wellington E. M. H.
Published: July 2002

We observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its hostStreptomyces lividansTK24 in sterile soil microcosms. Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts. The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens. The interaction ofS. lividansand KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments. This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics. Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates. There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (>150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean [SEM], 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage. We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil.
Application of the 5′-Nuclease PCR Assay in Evaluation and Development of Methods for Quantitative Detection ofCampylobacter jejuni
Authors: Nogva Hege Karin , Bergh Anette , Holck Askild , Rudi Knut
Published: July 2002

Campylobacter jejuniis recognized as a leading human food-borne pathogen. Traditional diagnostic testing forC. jejuniis not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5′-nuclease PCR assay for quantitative detection ofC. jejuniand describe its evaluation. A probe including positions 381121 to 381206 of the publishedC. jejunistrain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates ofC. jejunitested (32 isolates, including the type strain) and negative for all otherCampylobacterspp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development ofC. jejunidetection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viableC. jejuniafter exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from deadC. jejuniand protected DNA in living bacteria.
Detection of EnterotoxicBacillus cereusandBacillus thuringiensisStrains by PCR Analysis
Authors: Hansen Bjarne Munk , Hendriksen Niels Bohse
Published: July 2002

Many strains ofBacillus cereuscause gastrointestinal diseases, and the closely related insect pathogenB. thuringiensishas also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22B. cereusand 41B. thuringiensisstrains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of theB. thuringiensisstrains, while sixB. cereusstrains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate thatbceTis widely distributed amongB. cereusandB. thuringiensisstrains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected thebceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceTgene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereusandB. thuringiensisbased on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection ofbceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.
Pantoea agglomeransStrain EH318 Produces Two Antibiotics That InhibitErwinia amylovoraIn Vitro
Authors: Wright Sandra A. I. , Zumoff Cathy H. , Schneider Lois , Beer Steven V.
Published: July 2002

Pantoea agglomerans(synonym:Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded withErwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred onEscherichia colithe ability to inhibit growth ofE. amylovora. The two cosmids conferred different antibiotic activities onE. coliDH5α and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-succinamic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.
Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA
Authors: Demanèche Sandrine , Jocteur-Monrozier Lucile , Quiquampoix Hervé , Simonet Pascal
Published: July 2002

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium
Acinetobacter
sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.
Genetic Engineering of
Escherichia coli
for Enhanced Uptake and Bioaccumulation of Mercury
Authors: Bae Weon , Mehra Rajesh K. , Mulchandani Ashok , Chen Wilfred
Published: July 2002

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)

n

Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg
2+
transport system with (Glu-Cys)
20
Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg
2+
. Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.
Identification of Bacteria Responsible for Ammonia Oxidation in Freshwater Aquaria
Authors: Burrell Paul C. , Phalen Carol M. , Hovanec Timothy A.
Published: July 2002

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the
Nitrosomonas marina
cluster, the
Nitrosospira
cluster, or the
Nitrosomonas europaea-Nitrosococcus mobilis
cluster of the β subdivision of the class
Proteobacteria
. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the
Nitrosomonas marina
-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the
Nitrosomonas marina
-like AOB strain was present in the original enrichment, even one with other AOB, only the
Nitrosomonas marina
-like AOB strain was present in aquaria after nitrification was established.
Nitrosomonas marina
-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.
Expression of
cmg1
, an Exo-β-1,3-Glucanase Gene from
Coniothyrium minitans
, Increases during Sclerotial Parasitism
Authors: Giczey Gábor , Kerényi Zoltán , Fülöp László , Hornok László
Published: July 2002

During sclerotial infection of
Sclerotinia sclerotiorum
the mycoparasite
Coniothyrium minitans
penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated
cmg1
, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73.
Saccharomyces cerevisiae
INVSc1 expressing the
cmg1
gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of
S. sclerotiorum
by 35 and 85% at concentrations of 300 and 600 μg ml
−1
, respectively. A single copy of the
cmg1
gene is present in the genome of
C. minitans
. Northern analyses indicated increases in the transcript levels of
cmg1
due to both carbon starvation and the presence of ground sclerotia of
S. sclerotiorum
; only slight repression was observed in the presence of 2% glucose. Expression of
cmg1
increased during parasitic interaction with
S. sclerotiorum
.
TheBacillus thuringiensis cytGenes for Hemolytic Endotoxins Constitute a Gene Family
Authors: Guerchicoff Alejandra , Delécluse Armelle , Rubinstein Clara P.
Published: July 2002

In the same way thatcrygenes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, thecytgenes for hemolytic toxins seem to compose another set of highly related genes inBacillus thuringiensis. Although the occurrence of Cyt hemolytic factors inB. thuringiensishas been typically associated with mosquitocidal strains, we have recently shown thatcytgenes are also present in strains with different pathotypes; this is the case for themorrisonisubspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such asB. thuringiensissubsp.israelensisand subsp.morrisoniPG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50B. thuringiensisstrains revealed thatcyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of manyB. thuringiensisstrains.
Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by
Lactobacillus acidophilus
Authors: Ogawa Jun , Matsumura Kenji , Kishino Shigenobu , Omura Yoriko , Shimizu Sakayu
Published: July 2002

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of
Lactobacillus acidophilus
AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as
cis
-9,
trans
-11- or
trans
-9,
cis
-11-octadecadienoic acid and
trans
-9,
trans
-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-
cis
-12-octadecaenoic acid and 10-hydroxy-
trans
-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-
cis
-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of
L. acidophilus
, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of
L. acidophilus
producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.
Tests of a Cellular Model for Constant Branch Distribution in the Filamentous Fungus
Neurospora crassa
Authors: Watters Michael K. , Griffiths Anthony J. F.
Published: July 2002

The growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. In
Neurospora crassa
, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. Much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by
N. crassa
has not been proposed. We described and tested a model in which the formation of a lateral branch in
N. crassa
was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate of deposition at the apex. If both rates are proportional to metabolic rate, then the model explains the known lack of dependence of branch interval on growth rate. We tested the model by manipulating the tip extension rate, first by shifting temperature in both the wild type and hyperbranching (colonial) mutants and also by observing the behavior of both tipless colonies and colonyless tips. We found that temperature shifts in either direction result in temporary changes in branching. We found that colonyless tips also pass through a temporary transition phase of branching. The tipless colonies produced a cluster of new tips near the point of damage. We also found that branching in colonial mutants is dependent on growth rate. The results of these tests are consistent with a model of branching in which branch initiation is controlled by the dynamics of tip growth while being independent of the actual rate of this growth.
Isolation and Characterization of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria Associated with the Rhizosphere of Salt Marsh Plants
Authors: Daane L. L. , Harjono I. , Zylstra G. J. , Häggblom M. M.
Published: July 2002

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group,
Paenibacillus
. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from
Pseudomonas putida
NCIB 9816-4,
Comamonas testosteroni
GZ42,
Sphingomonas yanoikuyae
B1, and
Mycobacterium
sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The
Paenibacillus
isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.
Ecological Physiology ofSynechococcussp. Strain SH-94-5, a Naturally Occurring Cyanobacterium Deficient in Nitrate Assimilation
Authors: Miller Scott R. , Castenholz Richard W.
Published: July 2002

Synechococcussp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.
Impact of Fumigants on Soil Microbial Communities
Authors: Ibekwe A. Mark , Papiernik Sharon K. , Gan Jianying , Yates Scott R. , Yang Ching-Hong , Crowley David E.
Published: July 2002

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity,
H,
was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (
H
decreased from 1.14 to 0.13). After 12 weeks of incubation,
H
increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to
Bacillus
spp. and
Heliothrix oregonensis
. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.