bluuebunny/crossref_metadata_2025 huggingface dataset:
Rational Dose Selection for a Nonnucleoside Reverse Transcriptase Inhibitor through Use of Population Pharmacokinetic Modeling and Monte Carlo Simulation
Authors: Drusano G. L. , Moore K. H. P. , Kleim J. P. , Prince W. , Bye A.
Published: July 2002

In order to choose a rational dose for GW 420867X, we first set a goal of therapy. We hypothesized that, for optimal antiretroviral activity, the trough free drug concentration should remain above the 90% effective concentration (EC
90
) of human immunodeficiency virus type 1. We performed population pharmacokinetic analysis on three different doses of GW 420867X (50, 100, and 200 mg). Monte Carlo simulation was performed, assuming a log-normal distribution for 1,000 simulated subjects for each dose, and was repeated three times. The trough concentrations were divided by 76 to account for protein binding and for the difference between EC
50
and EC
90
. We then determined the fraction of the simulated population whose free drug trough concentrations would exceed an EC
90
over a broad range of values. The target attainment for all three doses exceeded 95% out to a starting EC
50
of 10 nM. For 16 viral isolates, the EC
50
range encountered for GW 420867X did not exceed 8 nM, implying that the three doses could not be differentiated by effect in a clinical trial in naive patients. This prediction was shown to be correct in a randomized, double-blind trial with 1 week of monotherapy with GW 420867X.
A Tight-Binding Mode of Inhibition Is Essential for Anti-Human Immunodeficiency Virus Type 1 Virucidal Activity of Nonnucleoside Reverse Transcriptase Inhibitors
Authors: Motakis Dimitrios , Parniak Michael A.
Published: July 2002

It was previously found that certain nonnucleoside reverse transcriptase inhibitors (NNRTI) possess virucidal activity against human immunodeficiency virus type 1 (HIV-1), and it was suggested that the tight-binding mode of inhibition of reverse transcriptase might be important for this virucidal activity (Borkow et al., J. Virol. 71:3023-3030, 1997). To test this, we compared six different NNRTI, including three tight-binding NNRTI, namely UC781, efavirenz (EFV) (Sustiva), and 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC), and three rapid-equilibrium NNRTI, delavirdine (DLV) (Rescriptor), nevirapine (NVP) (Viramune), and UC84, in a variety of virucidal tests. Incubation of isolated HIV-1 virions with UC781, EFV, or CSIC rapidly inactivated the virus, whereas DLV, NVP, and UC84 were ineffective in this respect. Exposure of H9+ cells chronically infected by HIV-1 to the tight-binding NNRTI abolished the infectivity of nascent virus subsequently produced by these cells following removal of extracellular drug, thereby preventing cell-to-cell virus transmission in the absence of exogenous drug. In contrast, cell-to-cell transmission of HIV was blocked by DLV, NVP, and UC84 only when the drug remained in the extracellular medium. Pretreatment of uninfected lymphocytoid cells with UC781, EFV, or CSIC, but not DLV, NVP, or UC84, protected these cells from subsequent HIV-1 infection in the absence of extracellular drug. The protective effect was dependent on both the dose of NNRTI and the viral load. The overall virucidal efficacy of the tight-binding NNRTI tested was CSIC > UC781 ≃ EFV. We conclude that the tight-binding mode of inhibition is an essential characteristic for virucidal NNRTI and that antiviral potency is an insufficient predictor for virucidal utility of NNRTI.
Efficacy of Colistin versus β-Lactams, Aminoglycosides, and Rifampin as Monotherapy in a Mouse Model of Pneumonia Caused by Multiresistant
Acinetobacter baumannii
Authors: Montero A. , Ariza J. , Corbella X. , Doménech A. , Cabellos C. , Ayats J. , Tubau F. , Ardanuy C. , Gudiol F.
Published: July 2002

The treatment of life-threatening infections due to carbapenem-resistant
Acinetobacter baumannii
has become a serious challenge for physicians worldwide. Often, only colistin shows in general good in vitro activity against these carbapenem-resistant strains, but its antibacterial efficacy in comparison with the antibiotics most used in clinical practice is not well known. We studied the efficacy of colistin versus those of imipenem, sulbactam, tobramycin, and rifampin in an experimental pneumonia model with immunocompetent mice. We used three strains of
A. baumannii
corresponding to the main clones (A, D, and E) involved in the outbreaks of our hospital, with different grades of resistance to imipenem (imipenem MICs of 1, 8, and 512 μg/ml, respectively) and to the other antibiotics. The MIC of colistin was 0.5 μg/ml for the three strains. Reduction of log
10
CFU/g in lung bacterial counts, clearance of bacteremia, and survival versus results with controls were used as parameters of efficacy. Imipenem and sulbactam (Δlung counts: −5.38 and −4.64 log
10
CFU/ml) showed the highest level of bactericidal efficacy in infections by susceptible and even intermediate strains. Tobramycin and rifampin (−4.16 and −5.15 log
10
CFU/ml) provided good results against intermediate or moderately resistant strains, in agreement with killing curves and pharmacodynamics. On the contrary, colistin showed the weakest antibacterial effect among the antibiotics tested, both in killing curves and in the in vivo model (−2.39 log
10
CFU/ml;
P
< 0.05). We conclude that colistin did not appear as a good option for treatment of patients with pneumonia due to carbapenem-resistant
A. baumannii
strains. Other alternatives, including combinations with rifampin, may offer better therapeutic profiles and thus should be studied.
A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil
Authors: Hay Anthony G. , Rice James F. , Applegate Bruce M. , Bright Nathan G. , Sayler Gary S.
Published: July 2002

A bioreporter was made containing a

tfdRP
DII
-luxCDABE

fusion in a modified mini-Tn
5
construct. When it was introduced into the chromosome of
Ralstonia eutropha
JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (
R
2
= 0.9825) in the range of 2.0 μM to 1.1 × 10
2
μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 10
2
μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.
Genetic Control of Resistance to the Sterol 14α-Demethylase Inhibitor Fungicide Prochloraz in the Cereal Eyespot Pathogen
Tapesia yallundae
Authors: Dyer Paul S. , Hansen Jacqueline , Delaney Andrew , Lucas John A.
Published: July 2002

Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen
Tapesia yallundae
. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% {IG
50
} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG
50
= 0.5 mg/liter], PR1 [IG
50
= 1.0 mg/liter], and 11-3-18 [IG
50
= 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny. This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses, crosses between F
1
progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic components responsible for the increased IG
50
s of the more resistant isolates. A further cross was made between isolate PR11 and an F
1
progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.
Identification and Characterization of an ATP Binding Cassette
l
-Carnitine Transporter in
Listeria monocytogenes
Authors: Fraser Katy R. , Harvie Duncan , Coote Peter J. , O'Byrne Conor P.
Published: July 2002

We identified an operon in
Listeria monocytogenes
EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from
Bacillus subtilis
, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated
opuC
, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An
opuC
-defective mutant was generated by the insertional inactivation of the
opuCA
gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of
l
-carnitine. We conclude that the
opuC
operon in
L. monocytogenes
encodes an ABC compatible solute transporter which is capable of transporting
l
-carnitine and which plays an important role in osmoregulation in this pathogen.
Apparent Contradiction: Psychrotolerant Bacteria from Hydrocarbon-Contaminated Arctic Tundra Soils That Degrade Diterpenoids Synthesized by Trees
Authors: Yu Zhongtang , Stewart Gordon R. , Mohn William W.
Published: July 2002

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82°N, 62°W). According to most-probable-number assays, resin acid degraders were abundant (10
3
to 10
4
propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10
6
to 10
7
propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus
Pseudomonas
. Strain DhA-95 is a member of the genus
Sphingomonas
. All four strains are psychrotolerant, with growth temperature ranges of 4°C to 30°C (DhA-91 and DhA-95) or 4°C to 22°C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22°C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two
Mycobacterium
sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.
β-Cyanoalanine Production by Marine Bacteria on Cyanide-Free Medium and Its Specific Inhibitory Activity toward Cyanobacteria
Authors: Yoshikawa Kazuhiro , Adachi Kyoko , Nishijima Miyuki , Takadera Takahide , Tamaki Seiji , Harada Ken-ichi , Mochida Kenichi , Sano Hiroshi
Published: July 2002

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against
Oscillatoria amphibia
NIES-361. One strain, C-979, identified as a
Vibrio
sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be β-cyano-
l
-alanine (
l
-CNAla) by instrumental analyses and the application of the advanced Marfey method.
l
-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to
l
-CNAla at a concentration of 0.4 to 25 μg/ml. The effect of
l
-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.
High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe
Thermoanaerobacter ethanolicus
39E
Authors: Jones C. R. , Ray M. , Dawson K. A. , Strobel H. J.
Published: July 2002

Thermoanaerobacter ethanolicus
is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of
T. ethanolicus
maltose-grown cells with [
14
C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and α-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that
T. ethanolicus
possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and α-trehalose.
Poly(3-Hydroxyvalerate) Depolymerase of
Pseudomonas lemoignei
Authors: Schöber Ute , Thiel Christian , Jendrossek Dieter
Published: July 2002

Pseudomonas lemoignei
is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (
phaZ1
to
phaZ5
) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA
SCL
) as the sole sources of carbon and energy. Four genes (
phaZ1
,
phaZ2
,
phaZ3
, and
phaZ5
) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene,
phaZ4
, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596–607, 1995). However, in this study, we show that
phaZ4
codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of
phaZ4
, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene,
phaZ6
, was identified by screening a genomic library of
P. lemoignei
in
Escherichia coli
for clearing zone formation on PHV agar. The DNA sequence of
phaZ6
contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant
E. coli
and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA
SCL
depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [
M
r
], 43,610 Da) resembles precursors of other extracellular PHA
SCL
depolymerases (28 to 50% identical amino acids). The mature protein (
M
r
, 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S
136
, D
211
, and H
269
similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA
SCL
depolymerases. Differences in the codon usage of
phaZ6
for some codons from the average codon usage of
P. lemoignei
indicated that
phaZ6
might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA
SCL
depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA
SCL
depolymerases.
Design and Application of a Biosensor for Monitoring Toxicity of Compounds to Eukaryotes
Authors: Hollis R. P. , Killham K. , Glover L. A.
Published: July 2002

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast
Saccharomyces cerevisiae
was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of
S. cerevisiae
cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.
Genotypic and Phenotypic Diversity of
phlD
-Containing
Pseudomonas
Strains Isolated from the Rhizosphere of Wheat
Authors: McSpadden Gardener B. B. , Schroeder K. L. , Kalloger S. E. , Raaijmakers J. M. , Thomashow L. S. , Weller D. M.
Published: July 2002

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent
Pseudomonas
spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent
Pseudomonas
strains containing
phlD
, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.
Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene
Authors: Nishino Shirley F. , Paoli George C. , Spain Jim C.
Published: July 2002

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by
Burkholderia
sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway.
Burkholderia cepacia
strain JS850 and
Hydrogenophaga palleronii
strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.
Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination
Authors: Martín M. Cruz , Alonso Juan C. , Suárez Juan E. , Alvarez Miguel A.
Published: July 2002

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (
attP-attB
and
six
, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented
six
sites, a multiple cloning site where passenger DNA could be inserted (e.g., the
c
I gene of bacteriophage A2), the
int
gene, and the
attP
site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into
Lactobacillus casei
ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the
attB
site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade
L. casei
ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.
Contamination of Foods by Food Handlers: Experiments on Hepatitis A Virus Transfer to Food and Its Interruption
Authors: Bidawid S. , Farber J. M. , Sattar S. A.
Published: July 2002

Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm
2
resulted in transfer of 9.2% ± 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% ± 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.
Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis
Authors: Marsh Terence L. , Saxman Paul , Cole James , Tiedje James
Published: July 2002

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (
http://www.cme.msu.edu/RDP/html/analyses.html
) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.
Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer
Authors: Annweiler Eva , Michaelis Walter , Meckenstock Rainer U.
Published: July 2002

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [
13
C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the
13
C content of the carboxyl group were formed, indicating the addition of a C
1
unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.
Successional Changes in an Evolving Anaerobic Chlorophenol-Degrading Community Used To Infer Relationships between Population Structure and System-Level Processes
Authors: Becker Jennifer G. , Berardesco Gina , Rittmann Bruce E. , Stahl David A.
Published: July 2002

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H
2
levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from “
Desulfovibrionaceae
” populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of
Syntrophus
-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H
2
and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the
Syntrophus
-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.
Fungal Metabolism of Toluene: Monitoring of Fluorinated Analogs by
19
F Nuclear Magnetic Resonance Spectroscopy
Authors: Prenafeta-Boldú Francesc X. , Luykx Dion M. A. M. , Vervoort Jacques , de Bont Jan A. M.
Published: July 2002

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (
Cunninghamella echinulata
and
Aspergillus niger
) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by
19
F nuclear magnetic resonance. Oxidation of fluorotoluene by
C. echinulata
was initiated either at the aromatic ring, resulting in fluorinated
o-
cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the
19
F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.
Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of
Bacillus cereus
to Nisin and Pulsed-Electric-Field Treatment
Authors: Pol Irene E. , van Arendonk Willy G. C. , Mastwijk Hennie C. , Krommer Judit , Smid Eddy J. , Moezelaar Roy
Published: July 2002

Treatment of
Bacillus cereus
spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.
Purification and Characterization of Cellobiose Dehydrogenase from the Plant PathogenSclerotium(Athelia)rolfsii
Authors: Baminger Ursula , Subramaniam Sai S. , Renganathan V. , Haltrich Dietmar
Published: July 2002

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochromec, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungusSclerotium rolfsii(teleomorph:Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme fromS. rolfsiicontains one hemeband one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochromec(equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochromec. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.
Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses
Authors: De Angelis M. , Corsetti A. , Tosti N. , Rossi J. , Corbo M. R. , Gobbetti M.
Published: July 2002

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive
Lactobacillus
isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32%
Lactobacillus plantarum
isolates, 15%
L. brevis
isolates, 12%
L. paracasei
subsp.
paracasei
isolates, 9%
L. curvatus
isolates, 6%
L. fermentum
isolates, 6%
L. casei
subsp.
casei
isolates, 5%
L. pentosus
isolates, 3%
L. casei
subsp.
pseudoplantarum
isolates, and 1%
L. rhamnosus
isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as
L. plantarum
and
L. pentosus
or as
L. casei
subsp.
casei
and
L. paracasei
subsp.
paracasei
, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.
Degradation of Substituted Phenylurea Herbicides by
Arthrobacter globiformis
Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene,
puhA
Authors: Turnbull Gillian A. , Ousley Margaret , Walker Allan , Shaw Eve , Morgan J. Alun W.
Published: July 2002

Arthrobacter globiformis
D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to
A. globiformis
D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb
Eco
RI fragment of plasmid pHRIM620 was expressed in
Escherichia coli
and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in
E. coli
coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (
puhA
gene).
Microbial Populations Associated with Treatment of an Industrial Dye Effluent in an Anaerobic Baffled Reactor
Authors: Plumb Jason J. , Bell Joanne , Stuckey David C.
Published: July 2002

Fluorescent in situ hybridization (FISH) using 16S and 23S rRNA-targeted probes together with construction of an archaeal 16S ribosomal DNA (rDNA) clone library was used to characterize the microbial populations of an anaerobic baffled reactor successfully treating industrial dye waste. Wastewater produced during the manufacture of food dyes containing several different azo and other dye compounds was decolorized and degraded under sulfidogenic and methanogenic conditions. Use of molecular methods to describe microbial populations showed that a diverse group of
Bacteria
and
Archaea
was involved in this treatment process. FISH enumeration showed that members of the gamma subclass of the class
Proteobacteria
and bacteria in the
Cytophaga-Flexibacter-Bacteroides
phylum, together with sulfate-reducing bacteria, were prominent members of a mixed bacterial population. A combination of FISH probing and analysis of 98 archaeal 16S rDNA clone inserts revealed that together with the bacterial population, a methanogenic population dominated by
Methanosaeta
species and containing species of
Methanobacterium
and
Methanospirillum
and a relatively unstudied methanogen,
Methanomethylovorans hollandica
, contributed to successful anaerobic treatment of the industrial waste. We suggest that sulfate reducers, or more accurately sulfidogenic bacteria, together with
M. hollandica
contribute considerably to the treatment process through metabolism of dye-associated sulfonate groups and subsequent conversion of sulfur compounds to carbon dioxide and methane.
Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays
Authors: Cho Jae-Chang , Tiedje James M.
Published: July 2002

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent
Pseudomonas
species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent
Pseudomonas
strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.
Isolation and Regulation of
Sinorhizobium meliloti
1021 Loci Induced by Oxygen Limitation
Authors: Trzebiatowski Jodi R. , Ragatz Daniel M. , de Bruijn Frans J.
Published: July 2002

Eleven
Sinorhizobium meliloti
1021 loci whose expression was induced under low oxygen concentrations were identified in a collection of 5,000 strains carrying Tn
5
-
1063
(
luxAB
) transcriptional reporter gene fusions. The 11 Tn
5
-
1063
-tagged loci were cloned and characterized. The dependence of the expression of the tagged loci on the FixL/FixJ oxygen-sensing two-component regulatory system was examined. Three of the loci were found to be dependent upon
fixL
and
fixJ
for their expression, while one locus showed a partial dependence. The remaining seven loci showed
fixL
- and
fixJ
-independent induction of expression in response to oxygen limitation. This suggests that in
S. meliloti
, additional regulatory system(s) exist that respond either directly or indirectly to oxygen limitation conditions.
Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid
Emiliania huxleyi
Authors: Laguna Richard , Romo Jesus , Read Betsy A. , Wahlund Thomas M.
Published: July 2002

Emiliania huxleyi
is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of
E. huxleyi
and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of
E. huxleyi
. Plating
E. huxleyi
C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of
E. huxleyi
under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.
Dissolved Organic Nitrogen Hydrolysis Rates in Axenic Cultures of
Aureococcus anophagefferens
(Pelagophyceae): Comparison with Heterotrophic Bacteria
Authors: Berg Gry Mine , Repeta Daniel J. , Laroche Julie
Published: July 2002

The marine autotroph
Aureococcus anophagefferens
(Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.