bluuebunny/crossref_metadata_2025 huggingface dataset:
Activities of Bismuth Thiols against Staphylococci and Staphylococcal Biofilms
Authors: Domenico Philip , Baldassarri Lucilla , Schoch Paul E. , Kaehler Kristina , Sasatsu Masanori , Cunha Burke A.
Published: July 2002

Indwelling medical devices are associated with infectious complications. Incorporating antimicrobials into indwelling materials may reduce bacterial colonization. Bismuth thiols are antibiofilm agents with up to 1,000-fold-greater antibacterial activity than other bismuth salts. Staphylococci are particularly sensitive, as determined by agar diffusion and broth dilution susceptibility testing. Bismuth-ethanedithiol inhibited 10 methicillin-resistant
Staphylococcus epidermidis
strains at 0.9 to 1.8,
Staphylococcus aureus
ATCC 25923 at 2.4, and
S. epidermidis
ATCC 12228 at 0.1 μM Bi
3+
. Antiseptic-resistant
S. aureus
was sensitive to bismuth-2-3-dimercaptopropanol (BisBAL) at ≤7 μM Bi
3+
. Hydrogel-coated polyurethane rods soaked in BisBAL inhibited
S. epidermidis
for 39 days (inhibitory zone diameter in agar, ≥30 mm for >25 days). Slime from 16 slime-producing
S. epidermidis
strains was inhibited significantly by bismuth-3,4-dimercaptotoluene (BisTOL), but not by AgNO
3
, at subinhibitory concentrations. In conclusion, bismuth-thiols are bacteriostatic and bactericidal against staphylococci, including resistant organisms, but are also inhibitors of slime at subinhibitory concentrations. At subinhibitory concentrations, BisTOL may be useful in preventing the colonization and infection of indwelling intravascular lines, since staphylococci are important pathogens in this setting.
Serotype 19F Multiresistant Pneumococcal Clone Harboring Two Erythromycin Resistance Determinants [
erm
(B) and
mef
(A)] in South Africa
Authors: McGee Lesley , Klugman Keith P. , Wasas Avril , Capper Thora , Brink Adrian ,   The Antibiotics Surveillance Forum Of South Africa
Published: July 2002

One hundred eighteen erythromycin-resistant
Streptococcus pneumoniae
(ERSP) strains (MICs of ≥0.5 μg/ml) from five laboratories serving the private sector in South Africa were analyzed for the genes encoding resistance to macrolides. Sixty-seven ERSP strains (56.8%) contained the
erm
(B) gene, and 15 isolates (12.7%) contained the
mef
(A) gene. Thirty-six isolates (30.5%) harbored both the
erm
(B) and
mef
(A) genes and were highly resistant to erythromycin and clindamycin. DNA fingerprinting by BOX-PCR and pulsed-field gel electrophoresis identified 83% of these strains as belonging to a single multiresistant serotype 19F clone.
Safety and Bactericidal Activity of Rifalazil in Patients with Pulmonary Tuberculosis
Authors: Dietze Reynaldo , Teixeira Luciléia , Rocha Lia Márcia Canedo , Palaci Moisés , Johnson John L. , Wells Charles , Rose Lynn , Eisenach Kathleen , Ellner Jerrold J.
Published: July 2002

Rifalazil, also known as KRM-1648 or benzoxazinorifamycin, is a new semisynthetic rifamycin with a long half-life of approximately 60 h. Rifalazil has potent bactericidal activity against
Mycobacterium tuberculosis
in vitro and in animal models of tuberculosis (TB). Prior studies in healthy volunteers showed that once-weekly doses of 25 to 50 mg of rifalazil were well tolerated. In this randomized, open-label, active-controlled phase II clinical trial, 65 subjects with sputum smear-positive pulmonary TB received one of the following regimens for the first 2 weeks of therapy: 16 subjects received isoniazid (INH) (5 mg/kg of body weight) daily; 16 received INH (5 mg/kg) and rifampin (10 mg/kg) daily; 17 received INH (5 mg/kg) daily plus 10 mg of rifalazil once weekly; and 16 received INH (5 mg/kg) daily and 25 mg of rifalazil once weekly. All subjects were then put on 6 months of standard TB therapy. Pretreatment and day 15 sputum CFU of
M. tuberculosis
were measured to assess the bactericidal activity of each regimen. The number of drug-related adverse experiences was low and not significantly different among treatment arms. A transient decrease in absolute neutrophil count to less than 2,000 cells/mm
3
was detected in 10 to 20% of patients in the rifalazil- and rifampin-containing treatment arms without clinical consequences. Decreases in CFU counts were comparable among the four treatment arms; however, the CFU results were statistically inconclusive due to the variability in the control arms. Acquired drug resistance did not occur in any patient. Studies focused on determining a maximum tolerated dose will help elucidate the full anti-TB effect of rifalazil.
Antimicrobial Activity of Prosthetic Heart Valve Sewing Cuffs Coated With Minocycline and Rifampin
Authors: Darouiche Rabih O. , Fowler Vance G. , Adal Karim , Kielhofner Marcia , Mansouri David , Reller L. Barth
Published: July 2002

Prosthetic heart valve sewing cuffs coated with minocycline and rifampin exhibited in vitro zones of inhibition against all 52 tested clinical isolates responsible for prosthetic valve endocarditis. An in vitro elution study of these coated sewing cuffs demonstrated residual zones of inhibition against
Staphylococcus epidermidis
for at least 4 weeks.
Oxazolidinone Antibiotics Target the P Site onEscherichiacoliRibosomes
Authors: Aoki Hiroyuki , Ke Lizhu , Poppe Susan M. , Poel Toni J. , Weaver Elizabeth A. , Gadwood Robert C. , Thomas Richard C. , Shinabarger Dean L. , Ganoza M. Clelia
Published: July 2002

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance inHalobacteriumhalobium,Staphylococcusaureus, andEscherichiacolimap at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.
TEM Derivative-Producing
Enterobacter aerogenes
Strains: Dissemination of a Prevalent Clone
Authors: Dumarche P. , De Champs C. , Sirot D. , Chanal C. , Bonnet R. , Sirot J.
Published: July 2002

TEM-24 (CAZ-6) extended-spectrum β-lactamase (ESBL) was detected in 1988 in Clermont-Ferrand, France, in
Klebsiella pneumoniae
(
bla
TEM-24
) and
Enterobacter aerogenes
(
bla
TEM-24b
), and since 1994, a TEM-24-producing
E. aerogenes
clonal strain has been observed elsewhere in the country. To determine if the spread of this clonal strain was restricted to TEM-24-producing
E. aerogenes
strains, 84
E. aerogenes
strains (non-TEM/SHV-producing strains, TEM-1- or -2-producing strains, and different ESBL-producing strains), isolated from 1988 to 1999 in Clermont-Ferrand (
n
= 59) and in 11 other French hospitals in 1998 (
n
= 25), were studied. A clonal strain was found for TEM-24- but also for TEM-3- and TEM-1- or 2-producing isolates. This study shows that there is a clonal strain dependent on acquisition of the TEM-type enzyme (TEM-24 and other TEM types).
Comparative In Vitro Activities of Ertapenem (MK-0826) against 469 Less Frequently Identified Anaerobes Isolated from Human Infections
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen
Published: July 2002

We studied the in vitro activity of ertapenem against 469 less frequently identified anaerobes from 11 genera and 52 species isolated from human infections. Ertapenem was uniformly active against 460 of 469 (98%) strains at concentrations of ≤4 μg/ml. Only 4 of 14
Clostridium difficile
, 1 of 11
Clostridium innocuum
, and 4 of 6
Lactobacillus
sp. strains required ertapenem concentrations of ≥8 μg/ml for inhibition.
Characterization of Plasmids Carrying CMY-2 from Expanded-Spectrum Cephalosporin-Resistant
Salmonella
Strains Isolated in the United States between 1996 and 1998
Authors: Carattoli A. , Tosini F. , Giles W. P. , Rupp M. E. , Hinrichs S. H. , Angulo F. J. , Barrett T. J. , Fey P. D.
Published: July 2002

Sequencing of DNA from 15 expanded-spectrum cephalosporin (e.g., ceftriaxone)-resistant
Salmonella
isolates obtained in the United States revealed that resistance to ceftriaxone in all isolates was mediated by
cmy-2.
Hybridization patterns revealed three plasmid structures containing
cmy-2
in these 15 isolates. These data suggest that the spread of
cmy-2
among
Salmonella
strains is occurring through mobilization of the
cmy-2
gene into different plasmid backbones and consequent horizontal transfer by conjugation.
PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca
2+
Channel Blockers
Authors: Wenzel Uwe , Kuntz Sabine , Diestel Simone , Daniel Hannelore
Published: July 2002

Ca
2+
channel blockers like nifedipine have been shown to increase the oral bioavailability of β-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca
2+
channel blockers on β-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca
2+
ion (Ca
2+
in
) concentrations by Ca
2+
channel blockers or by Ca
2+
ionophores affect [
14
C]cefixime absorption. Reduction of Ca
2+
in
levels by Ca
2+
channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 μM led to 35 to 50% increases in the cellular uptake of 1 mM [
14
C]cefixime. Increases in Ca
2+
in
levels by Ca
2+
ionophores, on the other hand, led to 40% reductions in [
14
C]cefixime absorption. Nifedipine increased the
V
max
of cefixime transport by 67%, whereas the

K
m

of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H
+
-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca
2+
channel blocker increased the level of H
+
and cefixime cotransport. Increases in Ca
2+
in
levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H
+
and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca
2+
in
levels, e.g., by Ca
2+
channel blockers, affect pH regulatory systems, such as apical Na
+
and H
+
exchange, and thereby alter the H
+
gradient that serves as the driving force for uptake of β-lactams into intestinal epithelial cells.
Genetic Characterization of Soybean Rhizobia in Paraguay
Authors: Chen Lu Shi , Figueredo Antonio , Pedrosa Fábio O. , Hungria Mariangela
Published: July 2002

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the
Bradyrhizobium japonicum
and
Bradyrhizobium elkanii
species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with
Rhizobium
genomic species Q. However, two other fast growers showed high 16S rDNA identity with
Agrobacterium
spp., and both of these strains established efficient symbioses with soybean plants.
Characterization ofColletotrichum acutatumCausing Anthracnose of Anemone (Anemone coronariaL.)
Authors: Freeman Stanley , Shabi Ezra , Katan Talma
Published: July 2002

Anthracnose, or leaf-curl disease of anemone, caused byColletotrichumsp., has been reported to occur in Australia, western Europe, and Japan. Symptoms include tissue necrosis, corm rot, leaf crinkles, and characteristic spiral twisting of floral peduncles. Three epidemics of the disease have been recorded in Israel: in 1978, in 1990 to 1993, and in 1996 to 1998. We characterized 92Colletotrichumisolates associated with anthracnose of anemone (Anemone coronariaL.) for vegetative compatibility (72 isolates) and for molecular genotype (92 isolates) and virulence (4 isolates). Eighty-six of the isolates represented the three epidemics in Israel, one isolate was from Australia, and five isolates originated from western Europe. We divided these isolates into three vegetative-compatibility groups (VCGs). One VCG (ANE-A) included all 10 isolates from the first and second epidemics, and 13 of 62 examined isolates from the third epidemic in Israel, along with the isolate from Australia and 4 of 5 isolates from Europe. Another VCG (ANE-F) included most of the examined isolates (49 of the 62) from the third epidemic, as well asColletotrichum acutatumfrom strawberry, in Israel. Based on PCR amplification with species-specific primers, all of the anemone isolates were identified asC. acutatum. Anemone and strawberry isolates of the two VCGs were genotypically similar and indistinguishable when compared by arbitrarily primed PCR of genomic DNA. Only isolate NL-12 from The Netherlands, confirmed asC. acutatumbut not compatible with either VCG, had a distinct genotype; this isolate represents a third VCG ofC. acutatum. Isolates from anemone and strawberry could infect both plant species in artificial inoculations. VCG ANE-F was recovered from natural infections of both anemone and strawberry, but VCG ANE-A was recovered only from anemone. This study ofC. acutatumfrom anemone illustrates the potential of VCG analysis to reveal distinct subspecific groups within a pathogen population which appears to be genotypically homogeneous by molecular assays.
Carbon Dioxide and Nisin Act Synergistically on
Listeria monocytogenes
Authors: Nilsson Lilian , Chen Yuhuan , Chikindas Michael L. , Huss Hans Henrik , Gram Lone , Montville Thomas J.
Published: July 2002

This paper examines the synergistic action of carbon dioxide and nisin on
Listeria monocytogenes
Scott A wild-type and nisin-resistant (Nis
r
) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis
r
cells. Wild-type cells grown in 100% CO
2
were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nis
r
cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO
2
. There was a quantifiable synergistic action between nisin and CO
2
in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO
2
. This synergism between nisin and CO
2
was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO
2
and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO
2
atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO
2
and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO
2
is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.
Effect of Catalase on Hydrogen Peroxide Penetration into
Pseudomonas aeruginosa
Biofilms
Authors: Stewart Philip S. , Roe Frank , Rayner Joanna , Elkins James G. , Lewandowski Zbigniew , Ochsner Urs A. , Hassett Daniel J.
Published: July 2002

The penetration of hydrogen peroxide into biofilms formed by wild-type and catalase-deficient
Pseudomonas aeruginosa
strains was measured using microelectrodes. A flowing stream of hydrogen peroxide (50 mM, 1 h) was unable to penetrate or kill wild-type biofilms but did penetrate and partially kill biofilms formed by an isogenic strain in which the
katA
gene was knocked out. Catalase protects aggregated bacteria by preventing full penetration of hydrogen peroxide into the biofilm.
Targeted Disruption of the
kstD
Gene Encoding a 3-Ketosteroid Δ
1
-Dehydrogenase Isoenzyme of
Rhodococcus erythropolis
Strain SQ1
Authors: van der Geize R. , Hessels G. I. , van Gerwen R. , Vrijbloed J. W. , van der Meijden P. , Dijkhuizen L.
Published: July 2002

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ
1
-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of
Rhodococcus erythropolis
SQ1 revealed an open reading frame (
kstD
) showing similarity with known 3-ketosteroid Δ
1
-dehydrogenase genes. Heterologous expression of
kstD
yielded 3-ketosteroid Δ
1
-dehydrogenase (KSTD) activity under the control of the
lac
promoter in
Escherichia coli
. Targeted disruption of the
kstD
gene in
R. erythropolis
SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by the
kstD
gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ
1
-dehydrogenation in the
kstD
mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of
kstD
is not sufficient for inactivation of the Δ
1
-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that
R. erythropolis
SQ1 in fact harbors two activity bands, one of which is absent in the
kstD
mutant strain.
Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria
Authors: Fife Dee Jay , Bruhn Debby F. , Miller Karen S. , Stoner Daphne L.
Published: July 2002

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.
Genotypic Heterogeneity of
Streptococcus oralis
and Distinct Aciduric Subpopulations in Human Dental Plaque
Authors: Alam S. , Brailsford S. R. , Adams S. , Allison C. , Sheehy E. , Zoitopoulos L. , Kidd E. A. , Beighton D.
Published: July 2002

The genotypic heterogeneity of
Streptococcus oralis
isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual.
S. oralis
is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (χ
2
= 13.09;
P
= 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for
Streptococcus mitis
. Examination of
S. oralis
strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using
Salmonella enteritidis
repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of
S. oralis
implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.
Tandem Repeat of a Transcriptional Enhancer Upstream of the Sterol 14α-Demethylase Gene (
CYP51
) in
Penicillium digitatum
Authors: Hamamoto Hiroshi , Hasegawa Koji , Nakaune Ryoji , Lee Young Jin , Makizumi Yoshiyuki , Akutsu Katsumi , Hibi Tadaaki
Published: July 2002

We investigated the mechanism of resistance to demethylation inhibitors (DMI) in
Penicillium digitatum
by isolating the
CYP51
gene, which encodes the target enzyme (P450
14DM
) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of
CYP51
in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced
CYP51
, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased
CYP51
expression. We also found that if the number of copies of the repeat was reduced to two, resistance and
CYP51
expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances
CYP51
expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.
Numerical Analysis of Grassland Bacterial Community Structure under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns
Authors: McCaig Allison E. , Glover L. Anne , Prosser James I.
Published: July 2002

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ∼45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721–1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.
Cloning and Genetic Characterization of
dca
Genes Required for β-Oxidation of Straight-Chain Dicarboxylic Acids in
Acinetobacter
sp. Strain ADP1
Authors: Parke D. , Garcia M. A. , Ornston L. N.
Published: July 2002

A previous study of deletions in the protocatechuate (
pca
) region of the
Acinetobacter
sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the
pca
structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb
Sac
I fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known
pca
,
qui
, and
pob
genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of
Escherichia coli
DH5α, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in β-oxidation. Knockouts of three genes implicated in β-oxidation steps were introduced into the chromosome of
Acinetobacter
sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the
dca
(dicarboxylic acid) region. Mutant strains included one with a deletion in
dcaA
(encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in
dcaE
(encoding an enoyl-CoA hydratase homolog), and one with a deletion in
dcaH
(encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the
dca
region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the
Acinetobacter
chromosome.
Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from
Pyrococcus horikoshii
OT3
Authors: Ishikawa Kazuhiko , Ishida Hiroyasu , Matsui Ikuo , Kawarabayasi Yutaka , Kikuchi Hisasi
Published: July 2002

Genome sequencing of the thermophilic archaeon
Pyrococcus horikoshii
OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of
Sulfolobus solfataricus
and also to that encoding the aminoacylase from
Bacillus stearothermophilus
. The gene from
P. horikoshii
comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in
Escherichia coli
with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.
Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning
Authors: Qiu Xiaoyun , Wu Liyou , Huang Heshu , McDonel Patrick E. , Palumbo Anthony V. , Tiedje James M. , Zhou Jizhong
Published: July 2002

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division
Proteobacteria
as well as gram-positive bacterium, all of which could be distinguished by
Hha
I restriction digestion patterns. The overall PCR artifacts were significantly different among the three
Taq
DNA polymerases examined: 20% for Z-
Taq
, with the highest processitivity; 15% for LA-
Taq
, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, Ampli
Taq
. In contrast to the theoretical prediction, the frequency of chimeras for both Z-
Taq
(8.7%) and LA-
Taq
(6.2%) was higher than that for Ampli
Taq
(2.5%). The frequencies of chimeras and of heteroduplexes for Z-
Taq
were almost three times higher than those of Ampli
Taq
. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using Ampli
Taq
and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.
Enhanced Iron Uptake ofSaccharomyces cerevisiaeby Heterologous Expression of a Tadpole Ferritin Gene
Authors: Shin Young-Mi , Kwon Tae-Ho , Kim Kyung-Suk , Chae Keon-Sang , Kim Dae-Hyuk , Kim Jae-Ho , Yang Moon-Sik
Published: July 2002

We genetically engineeredSaccharomyces cerevisiaeto express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene(TFH)into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody againstEscherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.
Genetic Evidence that Loss of Virulence Associated with
gacS
or
gacA
Mutations in
Pseudomonas syringae
B728a Does Not Result from Effects on Alginate Production
Authors: Willis David K. , Holmstadt Jeremy J. , Kinscherf Thomas G.
Published: July 2002

Mutations in the global regulatory genes
gacS
and
gacA
render
Pseudomonas syringae
pv. syringae strain B728a completely nonpathogenic in foliar infiltration assays on bean plants. It had been previously demonstrated that
gac
genes regulate alginate production in
Pseudomonas
species, while other published work indicated that alginate is involved in the pathogenic interaction of
P. syringae
on bean plants. Together, these results suggested that the effects of
gacS
and
gacA
mutations on virulence in B728a might stem directly from a role in regulating alginate. In this report, we confirm a role for
gac
genes in both
algD
expression and alginate production in B728a. However, B728a mutants completely devoid of detectable alginate were as virulent as the wild-type strain in our assay. Thus, factors other than, or in addition to, a deficiency of alginate must be involved in the lack of pathogenicity observed with
gacS
and
gacA
mutants.
Characterization of Glycine Sarcosine
N
-Methyltransferase and Sarcosine Dimethylglycine
N
-Methyltransferase
Authors: Nyyssölä Antti , Reinikainen Tapani , Leisola Matti
Published: July 2002

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine
N
-methyltransferase (GSMT) and sarcosine dimethylglycine
N
-methyltransferase (SDMT) of
Ectothiorhodospira halochloris
catalyze the threefold methylation of glycine to betaine, with
S
-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in
Escherichia coli
and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent

K
m

and
V
max
values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product
S
-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.
Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture
Authors: Pernthaler Jakob , Posch Thomas , S̆imek Karel , Vrba Jaroslav , Pernthaler Annelie , Glöckner Frank Oliver , Nübel Ulrich , Psenner Roland , Amann Rudolf
Published: July 2002

We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte
Cryptomonas
sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (
Ochromonas
sp.) always resulted in an immediate bloom of a single phylotype population of members of the class
Actinobacteria
(Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate
Cyclidium glaucoma
. However, when the
Ochromonas
sp. was added after the establishment of
C. glaucoma
, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an
Aquabacterium
sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.
Inactivation of
Mycobacterium paratuberculosis
by Pulsed Electric Fields
Authors: Rowan Neil J. , MacGregor Scott J. , Anderson John G. , Cameron Douglas , Farish Owen
Published: July 2002

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of
Mycobacterium paratuberculosis
cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50°C (2,500 pulses at 30 kV/cm) reduced the level of viable
M. paratuberculosis
cells by approximately 5.3 and 5.9 log
10
CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of
M. paratuberculosis
at lower temperatures resulted in less lethality. Heating alone at 50°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of
M. paratuberculosis
of approximately 0.01 and 2.4 log
10
CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to
M. paratuberculosis
.
Detoxification of Corn Antimicrobial Compounds as the Basis for Isolating
Fusarium verticillioides
and Some Other
Fusarium
Species from Corn
Authors: Glenn A. E. , Hinton D. M. , Yates I. E. , Bacon C. W.
Published: July 2002

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively.
Fusarium verticillioides
(=
F. moniliforme
) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29
Fusarium
species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were
F. verticillioides
,
F. subglutinans
,
F. cerealis
(=
F. crookwellense
), and
F. graminearum
. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of
F. verticillioides
from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of
F. verticillioides
infection in field samples.
Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic
Escherichia coli
Groups
Authors: Grant Michael A. , Weagant Stephen D. , Feng Peter
Published: July 2002

The enzyme glutamate decarboxylase (GAD) is prevalent in
Escherichia coli
but few strains in the various pathogenic
E. coli
groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the
gadA
and
gadB
genes encoding GAD, we examined the distribution of the
gadAB
genes among enteric bacteria. Analysis of 173 pathogenic
E. coli
strains, including 125 enterohemorrhagic
E. coli
isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic
E. coli
, enterotoxigenic
E. coli
, enteroinvasive
E. coli
, and other Shiga toxin-producing
E. coli
(STEC) serotypes, showed that
gadAB
genes were present in all these strains. Among the 22 non-
E. coli
isolates tested, only the 6
Shigella
spp. carried
gadAB
. Analysis of naturally contaminated water and food samples using a
gadAB
-specific DNA probe that was labeled with digoxigenin showed that a
gadAB
-based assay is as reliable as standard methods that enumerate
E. coli
organisms on the basis of lactose fermentation. The presence of few
E. coli
cells initially seeded into produce rinsates could be detected by PCR to
gadA/B
genes after overnight enrichment. A multiplex PCR assay using the
gadAB
primers in combination with primers to Shiga toxin (Stx) genes
stx
1
and
stx
2
was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The
gadAB
primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic
E. coli
groups.
True Chemotaxis in Oxygen Gradients of the Sulfur-Oxidizing Bacterium
Thiovulum majus
Authors: Thar Roland , Fenchel Tom
Published: July 2002

Observations of free-swimming
Thiovulum majus
cells show that these bacteria exhibit a phobic response as well as true chemotaxis in oxygen gradients. Both phenomena of their chemotactic behavior are integrated into a single model of helical klinotaxis, which is demonstrated by computer simulations.
l
-Pyroglutamate Spontaneously Formed from
l
-Glutamate Inhibits Growth of the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Authors: Park Chan B. , Lee Sun Bok , Ryu Dewey D. Y.
Published: July 2002

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of
Sulfolobus solfataricus
in medium containing
l
-glutamate, we observed formation of
l
-pyroglutamic acid (PGA). PGA formed spontaneously from
l
-glutamate under culture conditions (78°C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of
S. solfataricus
growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of
l
-glutamate or
l
-aspartate to the medium. PGA was also produced from the
l
-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78°C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of
l
-glutamate, such as
l
-methionine sulfoxide, glutaric acid, succinic acid, and
l
-glutamic acid γ-methyl ester, also inhibited the growth of
S. solfataricus
, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues,
N
-acetyl-
l
-glutamate enhanced the growth of
S. solfataricus.
This compound was stable under cell culture conditions, and replacement of
l
-glutamate with
N
-acetyl-
l
-glutamate in the medium resulted in increased cell density.
Recovery of
Streptococcus iniae
from Diseased Fish Previously Vaccinated with a
Streptococcus
Vaccine
Authors: Bachrach Gilad , Zlotkin Amir , Hurvitz Avshalom , Evans Donald L. , Eldar Avi
Published: July 2002

Streptococcus iniae
was recovered from diseased rainbow trout (
Oncorhynchus mykiss
, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.
Survival of Fecal Coliforms in Dry-Composting Toilets
Authors: Redlinger Thomas , Graham Jay , Corella-Barud Verónica , Avitia Raquel
Published: July 2002

The dry-composting toilet, which uses neither water nor sewage infrastructure, is a practical solution in areas with inadequate sewage disposal and where water is limited. These systems are becoming increasingly popular and are promoted to sanitize human excreta and to recycle them into fertilizer for nonedible plants, yet there are few data on the safety of this technology. This study analyzed fecal coliform reduction in approximately 90 prefabricated, dry-composting toilets (Sistema Integral de Reciclamiento de Desechos Orgánicos [SIRDOs]) that were installed on the U.S.-Mexico border in Ciudad Juárez, Chihuahua, Mexico. The purpose of this study was to determine fecal coliform reduction over time and the most probable method of this reduction. Biosolid waste samples were collected and analyzed at approximately 3 and 6 months and were classified based on U.S. Environmental Protection Agency standards. Results showed that class A compost (high grade) was present in only 35.8% of SIRDOs after 6 months. The primary mechanism for fecal coliform reduction was found to be desiccation rather than biodegradation. There was a significant correlation (
P
= 0.008) between classification rating and percent moisture categories of the biosolid samples: drier samples had a greater proportion of class A samples. Solar exposure was critical for maximal class A biosolid end products (
P
= 0.001). This study only addressed fecal coliforms as an indicator organism, and further research is necessary to determine the safety of composting toilets with respect to other pathogenic microorganisms, some of which are more resistant to desiccation.
Molecular Characterization of Novel Red Green Nonsulfur Bacteria from Five Distinct Hot Spring Communities in Yellowstone National Park
Authors: Boomer Sarah M. , Lodge Daniel P. , Dutton Bryan E. , Pierson Beverly
Published: July 2002

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60°C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll
a
(Bchl
a
), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl
a
protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to
Roseiflexus castenholzii
, a red filamentous bacterium from Japan that also contains only Bchl
a
. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new “red” cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to
Roseiflexus
and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl
a
spectrum differences or obvious features of the habitat.