bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrogen Fixation Genes in an Endosymbiotic
Burkholderia
Strain
Authors: Minerdi Daniela , Fani Renato , Gallo Romina , Boarino Alessandra , Bonfante Paola
Published: July 2002

In this paper we report the identification and characterization of a DNA region containing putative
nif
genes and belonging to a
Burkholderia
endosymbiont of the arbuscular mycorrhizal fungus
Gigaspora margarita
. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with
Azospirillum brasilense nifHDK
genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the
Burkholderia nifHDK
genes and Southern blot analysis demonstrate that they actually belong to the genome of the
G. margarita
endosymbiont. They offer, therefore, the first sequence for the
nif
operon described for
Burkholderia
. Reverse transcriptase PCR experiments with primers designed on the
Burkholderia nifH
and
nifD
genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available
nifK
sequences placed the endosymbiotic
Burkholderia
close to
A. brasilense
.
Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate
Authors: McLean Jeff , Beveridge Terry J.
Published: July 2002

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter
−1
and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a

K
m

of 23 mg liter
−1
(437 μM) and a
V
max
of 0.98 mg of Cr h
−1
mg of protein
−1
(317 nmol min
−1
mg of protein
−1
). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.
Temperature-Driven Adaptation of the Bacterial Community in Peat Measured by Using Thymidine and Leucine Incorporation
Authors: Ranneklev Sissel Brit , Bååth Erland
Published: July 2002

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, or 55°C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55°C (thermophilic activity) and 25°C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25°C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log
10
ratio of TdR incorporation at 55 and 25°C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55°C 3 or 11 days before incubation at 25°C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.
Simultaneous Measurement of Denitrification and Nitrogen Fixation Using Isotope Pairing with Membrane Inlet Mass Spectrometry Analysis
Authors: An Soonmo , Gardner Wayne S. , Kana Todd
Published: July 2002

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N2gas species (28N2,29N2, and30N2) were monitored during incubation experiments after the addition of15NO3−. Formulas were developed to estimate the production (denitrification) and consumption (N2fixation) of N2gas from the fluxes of the different isotopic forms of N2. Proportions of the three isotopic forms produced from15NO3−and14NO3−agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 μg-atoms of N m−2h−1. They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N2production rates due to denitrification could be separated from high N2consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 μg-atoms of N m−2h−1) were lower than nitrogen fixation rates (average, 60 μg-atoms of N m−2h−1). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N2isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.
Relationship of Hydrogen Bioavailability to Chromate Reduction in Aquifer Sediments
Authors: Marsh Tamara L. , McInerney Michael J.
Published: July 2002

Biological Cr(VI) reduction was studied in anaerobic sediments from an aquifer in Norman, Okla. Microcosms containing sediment and mineral medium were amended with various electron donors to determine those most important for biological Cr(VI) reduction. Cr(VI) (about 340 μM) was reduced with endogenous substrates (no donor), or acetate was added. The addition of formate, hydrogen, and glucose stimulated Cr(VI) reduction compared with reduction in unamended controls. From these sediments, an anaerobic Cr(VI)-utilizing enrichment was obtained that was dependent upon hydrogen for both growth and Cr(VI) reduction. No methane was produced by the enrichment, which reduced about 750 μM Cr(VI) in less than six days. The dissolved hydrogen concentration was used as an indicator of the terminal electron accepting process occurring in the sediments. Microcosms with sediments, groundwater, and chromate metabolized hydrogen to a concentration below the detection limits of the mercury vapor gas chromatograph. In microcosms without chromate, the hydrogen concentration was about 8 nM, a concentration comparable to that under methanogenic conditions. When these microcosms were amended with 500 μM Cr(VI), the dissolved hydrogen concentration quickly fell below the detection limits. These results showed that the hydrogen concentration under chromate-reducing conditions became very low, as low as that reported under nitrate- and manganese-reducing conditions, a result consistent with the free energy changes for these reactions. The utilization of formate, lactate, hydrogen, and glucose as electron donors for Cr(VI) reduction indicates that increasing the availability of hydrogen results in a greater capacity for Cr(VI) reduction. This conclusion is supported by the existence of an enrichment dependent upon hydrogen for growth and Cr(VI) reduction.
Phylogenetic and Morphological Diversity of Cyanobacteria in Soil Desert Crusts from the Colorado Plateau
Authors: Garcia-Pichel Ferran , López-Cortés Alejandro , Nübel Ulrich
Published: July 2002

We compared the community structures of cyanobacteria in four biological desert crusts from Utah's Colorado Plateau developing on different substrata. We analyzed natural samples, cultures, and cyanobacterial filaments or colonies retrieved by micromanipulation from field samples using microscopy, denaturing gradient gel electrophoresis, and sequencing of 16S rRNA genes. While microscopic analyses apparently underestimated the biodiversity of thin filamentous cyanobacteria, molecular analyses failed to retrieve signals for otherwise conspicuous heterocystous cyanobacteria with thick sheaths. The diversity found in desert crusts was underrepresented in currently available nucleotide sequence databases, and several novel phylogenetic clusters could be identified. Morphotypes fitting the description of
Microcoleus vaginatus
Gomont, dominant in most samples, corresponded to a tight phylogenetic cluster of probable cosmopolitan distribution, which was well differentiated from other cyanobacteria traditionally classified within the same genus. A new, diverse phylogenetic cluster, named “Xeronema,” grouped a series of thin filamentous
Phormidium
-like cyanobacteria. These were also ubiquitous in our samples and probably correspond to various botanical
Phormidium
and
Schizothrix
spp., but they are phylogenetically distant from thin filamentous cyanobacteria from other environments. Significant differences in community structure were found among soil types, indicating that soil characteristics may select for specific cyanobacteria. Gypsum crusts were most deviant from the rest, while sandy, silt, and shale crusts were relatively more similar among themselves.
Oxygen Requirements of the Food Spoilage Yeast
Zygosaccharomyces bailii
in Synthetic and Complex Media
Authors: Rodrigues Fernando , Côrte-Real Manuela , Leão Cecı́lia , van Dijken Johannes P. , Pronk Jack T.
Published: July 2002

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast
Zygosaccharomyces bailii
at a constant pH, pH 3.0. In aerobic, glucose-grown cultures,
Z. bailii
exhibited aerobic alcoholic fermentation similar to that of
Saccharomyces cerevisiae
and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol,
S. cerevisiae
exhibited rapid exponential growth. Growth of
Z. bailii
under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of
Z. bailii
in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast
Candida utilis
. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium,
Z. bailii
exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by
Z. bailii
. In contrast to the growth of
Z. bailii
, anaerobic growth of
S. cerevisiae
on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.
Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate
Bacillus anthracis
Strains
Authors: Brumlik Michael J. , Szymajda Urszula , Zakowska Dorota , Liang Xudong , Redkar Rajendra J. , Patra Guy , Del Vecchio Vito G.
Published: July 2002

The genome of
Bacillus anthracis
is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various
B. anthracis
strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105
B. anthracis
strains of diverse geographical origins. All
B. anthracis
strains produced fingerprints comprising seven to eight bands, referred to as “skeleton” bands, while one to three “diagnostic” bands differentiated between
B. anthracis
strains. LR REP-PCR fingerprints of
B. anthracis
strains showed very little in common with those of other closely related species such as
B. cereus, B. thuringiensis
, and
B. mycoides
, suggesting relative heterogeneity among the non-
B. anthracis
strains. Fingerprints from transitional non-
B. anthracis
strains, which possessed the
B. anthracis
chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct
B. anthracis
groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating
B. anthracis
strains.
Effect of Genetically Modified
Pseudomonas putida
WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat
Authors: Glandorf Debora C. M. , Verheggen Patrick , Jansen Timo , Jorritsma Jan-Willem , Smit Eric , Leeflang Paula , Wernars Karel , Thomashow Linda S. , Laureijs Eric , Thomas-Oates Jane E. , Bakker Peter A. H. M. , van Loon Leendert C.
Published: July 2002

We released genetically modified
Pseudomonas putida
WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the
phz
biosynthetic gene locus of strain
P. fluorescens
2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10
7
CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10
7
CFU per g of rhizosphere sample to below the limit of detection (10
2
CFU per g) 1 month after harvest of the wheat plants. The
phz
genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly
Fusarium
spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.
Impact of 2,4-Diacetylphloroglucinol-Producing Biocontrol Strain
Pseudomonas fluorescens
F113 on Intraspecific Diversity of Resident Culturable Fluorescent Pseudomonads Associated with the Roots of Field-Grown Sugar Beet Seedlings
Authors: Moënne-Loccoz Yvan , Tichy Hans-Volker , O'Donnell Anne , Simon Reinhard , O'Gara Fergal
Published: July 2002

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent
Pseudomonas fluorescens
F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent
Pseudomonas
community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and
l
-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent
Pseudomonas
community. These results suggest that the root-associated
Pseudomonas
community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.
Putative Virulence Factor Expression by Clinical and Food Isolates of
Bacillus
spp. after Growth in Reconstituted Infant Milk Formulae
Authors: Rowan Neil J. , Deans Karen , Anderson John G. , Gemmell Curtis G. , Hunter Iain S. , Chaithong Thararat
Published: July 2002

Forty-seven strains representing 14 different
Bacillus
species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21
Bacillus cereus
strains examined, 5 (24%) were invasive. A larger percentage of clinically derived
Bacillus
species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected
Bacillus
species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different
Bacillus
species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and
B. cereus
enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47
Bacillus
isolates examined, 3 isolates of
B. cereus
and 1 isolate of
B. subtilis
produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species
B. cereus
,
B. licheniformis
,
B. circulans
, and
B. megaterium
were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the
B. cereus
enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several
Bacillus
species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.
Rapid and Efficient Extraction Method for Reverse Transcription-PCR Detection of Hepatitis A and Norwalk-Like Viruses in Shellfish
Authors: Kingsley David H. , Richards Gary P.
Published: July 2002

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine–PEG–Tri-reagent–poly(dT) method can be performed in less than 8 h on hard-shell clams (
Mercenaria mercenaria
) and Eastern oysters (
Crassostrea virginica
) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR
50
units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as
Ruditapes philippinarum
) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.
Biosorption of Copper by a Bacterial Biofilm on a Flexible Polyvinyl Chloride Conduit
Authors: Qureshi Fouad M. , Badar Uzma , Ahmed Nuzhat
Published: July 2002

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.
Isolation and Initial Characterization of a Bacterial Consortium Able To Mineralize Fluorobenzene
Authors: Carvalho M.F. , Alves C.C.T. , Ferreira M.I.M. , De Marco P. , Castro P.M.L.
Published: July 2002

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the
Sphingobacterium/Flavobacterium
group, while the third (F4) falls in the β-
Proteobacteria
group, clustering with
Alcaligenes
species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter
−1
and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.
Natural Genetic Transformation of Clinical Isolates of
Escherichia coli
in Urine and Water
Authors: Woegerbauer Markus , Jenni Bernard , Thalhammer Florian , Graninger Wolfgang , Burgmann Heinz
Published: July 2002

Transfer of plasmid-borne antibiotic resistance genes in
Escherichia coli
wild-type strains is possible by transformation under naturally occurring conditions in oligotrophic, aquatic environments containing physiologic concentrations of calcium. In contrast, transformation is suppressed in nitrogen-rich body fluids like urine, a common habitat of uropathogenic strains. Current knowledge indicates that transformation of these
E. coli
wild-type strains is of no relevance for the acquisition of resistance in this clinically important environment.
Sequence Analysis of Marine Virus Communities Reveals that Groups of Related Algal Viruses Are Widely Distributed in Nature
Authors: Short Steven M. , Suttle Curtis A.
Published: July 2002

Algal-virus-specific PCR primers were used to amplify DNA polymerase (
pol
) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic peninsula. Genetic fingerprints of algal virus communities were generated using denaturing gradient gel electrophoresis (DGGE). Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino acid motif indicating that the sequences were
pol
gene fragments. Furthermore, the 25
pol
sequences were related to
pol
gene fragments from known algal viruses. In addition, similar virus sequences (>98% sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that DGGE with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. DGGE is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations.
Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader
Comamonas testosteroni
TK102 by Flow Cytometry
Authors: Hiraoka Yoshinori , Kimbara Kazuhide
Published: July 2002

The viability of the polychlorinated biphenyl-degrading bacterium
Comamonas testosteroni
TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (
y
= 0.9872
x
+ 0.18;
R
2
= 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (
y
= −0.9776
x
+ 98.36;
R
2
= 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (≤1 h) and distinguishes correctly among live, dead, and permeabilized cells.
Differential Regulation of
mnp2
, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus
Trametes versicolor
PRL 572
Authors: Johansson Tomas , Nyman Per Olof , Cullen Daniel
Published: July 2002

A peroxidase-encoding gene,
mnp2
, and its corresponding cDNA were characterized from the white-rot basidiomycete
Trametes versicolor
PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify
mnp2
transcripts in nutrient-limited stationary cultures. Although
mnp2
lacks upstream metal response elements (MREs), addition of MnSO
4
to cultures increased
mnp2
transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of
T. versicolor
,
mnp1
, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in
T. versicolor
are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the
mnp1
and
mnp2
polypeptides to have internal homology.
Fate of
Escherichia coli
O157:H7 in Manure-Amended Soil
Authors: Jiang Xiuping , Morgan Jennie , Doyle Michael P.
Published: July 2002

Escherichia coli
O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms.
E. coli
O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.
Cloning and Characterization of the
Zymobacter palmae
Pyruvate Decarboxylase Gene (
pdc
) and Comparison to Bacterial Homologues
Authors: Raj Krishnan Chandra , Talarico Lee A. , Ingram Lonnie O. , Maupin-Furlow Julie A.
Published: July 2002

Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial
pdc
genes have been previously described and expressed as active recombinant proteins. The
pdc
gene from
Zymomonas mobilis
has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial
pdc
gene from
Zymobacter palmae
. The pattern of codon usage for this gene appears quite similar to that for
Escherichia coli
genes. In
E. coli
recombinants, the
Z. palmae
PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the
Z. palmae
enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the
Z. palmae
enzyme exhibited the highest specific activity (130 U mg of protein
−1
) and the lowest

K
m

for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.
Anticardiolipin Antibodies in Patients with Chronic Hepatitis C Virus Infection: Characterization in Relation to Antiphospholipid Syndrome
Authors: Ordi-Ros Josep , Villarreal Julieta , Monegal Francesc , Sauleda Silvia , Esteban Ignacio , Vilardell Miquel
Published: July 2002

The antiphospholipid syndrome (APS) is usually defined by the association of clinical manifestations that comprise venous and/or arterial thrombosis, recurrent fetal losses, and thrombocytopenia, along with the presence of anticardiolipin (aCL) antibodies and/or lupus anticoagulant. Various infectious diseases can induce aCL; however, these antibodies are not usually associated with thrombotic events, as happens with autoimmune diseases, in which these antibodies need the presence of β
2
-glycoprotein I. Levels of immunoglobulin G (IgG) and IgM aCL antibodies were determined by enzyme-linked immunosorbent assay for 243 patients with chronic hepatitis C virus (HCV) infection and 100 healthy controls. Clinical events of APS, the level of β
2
-glycoprotein dependence of aCL, the presence of cryoglobulins and other autoantibodies, and cross-reactivity between purified aCL and HCV were evaluated. Positive results for aCL antibodies were found more frequently (3.3%) for the patients with HCV infection than for healthy controls (0%). All positive aCL antibodies were β
2
-glycoprotein I independent. No significant association was found between aCL antibodies and clinical manifestations of APS, neither was one found between the presence of other autoantibodies or cryoglobulins and that of aCL. Finally, no cross-reactivity between aCL antibodies and HCV antigens was observed. As previously reported, aCL antibodies seem to be an epiphenomenon, and they do not have clinical or laboratory significance in HCV patients.
A Double-Blind, Adjuvant-Controlled Trial of Human Immunodeficiency Virus Type 1 (HIV-1) Immunogen (Remune) Monotherapy in Asymptomatic, HIV-1-Infected Thai Subjects with CD4-Cell Counts of >300
Authors: Churdboonchart V. , Sakondhavat C. , Kulpradist S. , Na Ayudthya B. Isarangkura , Chandeying V. , Rugpao S. , Boonshuyar C. , Sukeepaisarncharoen W. , Sirawaraporn W. , Carlo D. J. , Moss R.
Published: July 2002

We examined the effect of a human immunodeficiency virus (HIV)-specific immune-based therapy in Thailand, where access to antiviral drug therapy is limited. A 40-week trial was conducted with 297 asymptomatic, HIV-infected Thai subjects with CD4-cell counts greater than 300 μl/mm
3
. Subjects were randomized to receive either HIV type 1 (HIV-1) immunogen (Remune; inactivated HIV-1 from which gp120 is depleted in incomplete Freund's adjuvant or adjuvant control at 0, 12, 24, and 36 weeks at five different clinical sites in Thailand. Neither group received antiviral drug therapy. The a priori primary endpoint for the trial was changes in CD4-cell counts with secondary parameters of percent changes in CD8-cell counts (percent CD4, CD8, and CD4/CD8) and body weight. Subsets of subjects were also examined for changes in plasma HIV-1 RNA levels, Western blot immunoreactivity, and HIV-1 delayed-type hypersensitivity (DTH) skin test reactivity. There was a significant difference in changes in CD4-cell counts that favored the HIV-1 immunogen-treated group compared to those for the adjuvant-treated control group (
P
< 0.05). On average, for HIV-1 immunogen-treated subjects CD4-cell counts increased by 84 cells by week 40, whereas the increase for the control group was 38 cells by week 40. This increase in CD4-cell count was associated with increased HIV-specific immunogenicity, as shown by Western blotting and enhanced HIV-1 DTH skin reactivity. No significant differences in adverse events were observed between the groups. The results of this trial suggest that HIV-1 immunogen is safe and significantly increases CD4-cell counts and HIV-specific immunity compared to those achieved with the adjuvant control in asymptomatic HIV-1-infected subjects not taking antiviral drugs.
Fecal Excretion of a Novel Human Circovirus, TT Virus, in Healthy Children
Authors: Lin C.-L. , Kyono W. , Tongson J. , Chua P. K. , Easa D. , Yanagihara R. , Nerurkar V. R.
Published: July 2002

The role of TT virus (TTV) as a human pathogen is unclear, as is the mode of TTV transmission. To determine the prevalence of TTV infection and the possible fecal-oral route of transmission, we analyzed fecal specimens from 67 healthy, nontransfused children for TTV DNA sequences by heminested PCR, using the NG and T primer sets. The overall prevalence of TTV fecal excretion was 22.4% (15 of 67), with the T primer set (19.4%) being more sensitive than the NG primer set (10.4%). TTV prevalence based on gender or ethnicity showed no significant differences. None of seven children in the 0- to 6-month age group had detectable TTV in feces. Of three sets of siblings, two unrelated sets of twins, ages 33 and 37 months, were negative for fecal TTV DNA, while the third set of siblings, ages 99 and 35 months, was positive. The absence of TTV in the feces of children younger than 6 months and the high prevalence (40%) in children 7 to 12 months of age is consistent with age-specific acquisition of TTV infection by the nonparenteral route. TTV genotypes 1, 3, 4, and 5 were represented in our study population. TTV-positive siblings had TTV genotypes 1 and 4, suggesting unrelated environmental sources of TTV infection. This observation suggests a possible time frame for TTV acquisition in children which coincides with increased interaction with their environment and increased susceptibility to infectious agents.
Determination of
Helicobacter pylori
Virulence by Simple Gene Analysis of the
cag
Pathogenicity Island
Authors: Ikenoue Tsuneo , Maeda Shin , Ogura Keiji , Akanuma Masao , Mitsuno Yuzo , Imai Yasuo , Yoshida Haruhiko , Shiratori Yasushi , Omata Masao
Published: July 2002

Nucleic acid amplification was performed for five loci in the
cag
pathogenicity island (PAI) of
Helicobacter pylori
(comprising
cagA
, the
cagA
promoter region,
cagE
,
cagT
, and the left end of
cag
II [LEC]), and gastric inflammation in patients was evaluated. Of 204
H. pylori
isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only
cagA
and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The
cagE
gene was a more accurate marker of an intact
cag
PAI than the
cagA
gene, and
cagE
seemed to be more useful in discriminating between
H. pylori
strains causing different rates of disease progression.
Importance of Complement Source in Measuring Meningococcal Bactericidal Titers
Authors: Santos George F. , Deck R. Randall , Donnelly John , Blackwelder William , Granoff Dan M.
Published: July 2002

Complement-mediated bactericidal antibodies in serum confer protection against meningococcal disease. The minimum protective titer is estimated to be between 1:4 and 1:8 when measured by the Goldschneider assay performed with human complement, the assay used in the 1960s to establish the correlation between bactericidal antibodies and protection. A more recently described bactericidal assay standardized by an international consortium uses rabbit complement, which is known to augment bactericidal titers. To define a protective titer measured by the standardized assay, we compared bactericidal titers against serogroup C strains measured by this assay to titers measured by the assay described by Goldschneider et al. A titer of ≥1:128 measured by the standardized assay was needed to predict with ≥80% certainty a positive titer of ≥1:4 as measured by the Goldschneider assay. However, the majority of samples with titers of 1:4 measured by the Goldschneider assay had titers of <1:128 when measured by the standardized assay. Therefore, by the results of the standardized assay such persons would be falsely categorized as being susceptible to disease. In conclusion, high bactericidal titers measured with the standardized assay performed with rabbit complement are predictive of protection, but no threshold titer is both sensitive and specific for predicting a positive titer measured by the Goldschneider assay using human complement. Up to 10% of the U.S. adult population lacks intrinsic bactericidal activity against serogroup C strains in serum and can serve as complement donors. Therefore, use of the Goldschneider assay or an equivalent assay performed with human complement is preferred over assays that use rabbit complement.
Invasive Pneumococcal Infections in Denmark from 1995 to 1999: Epidemiology, Serotypes, and Resistance
Authors: Konradsen Helle Bossen , Kaltoft Margit Staum
Published: July 2002

Danish nationwide surveillance data on invasive pneumococcal disease from the 5-year period from 1995 to 1999, including 5,452 isolates, are presented and described. Annual overall incidence rates, serotype distribution, and antimicrobial susceptibility patterns of the isolates were monitored. Major changes in the total annual incidence rate from 27/100,000 in 1996 to 17/100,000 in 1999 and a significant change in the proportion of invasive isolates belonging to types 1 and 12F were observed. The serotype coverage rate by the 23-valent polysaccharide vaccine among the elderly was 92.9%, and the serotype coverage rate by the 7-, 9-, and 11-valent pneumococcal conjugate vaccines among children less than 2 years old were 71.7, 75.2, and 81.4%, respectively. Invasive isolates with reduced susceptibility to penicillin or erythromycin increased from 1995 to 1999, with a high proportion of the penicillin-nonsusceptible invasive isolates originating from people 60 years old or older (57.0%). These observations underline the importance of adequate surveillance systems of invasive pneumococcal disease to introduce and maintain national vaccine strategies and adequate antibiotic policy.
Characterization of the
Yersinia pestis
Yfu ABC Inorganic Iron Transport System
Authors: Gong Shimei , Bearden Scott W. , Geoffroy Valerie A. , Fetherston Jacqueline D. , Perry Robert D.
Published: July 2002

In
Yersinia pestis
, the causative agent of plague, two inorganic iron transport systems have been partially characterized. The yersiniabactin (Ybt) system is a siderophore-dependent transport system required for full virulence. Yfe is an ABC transport system that accumulates both iron and manganese. We have identified and cloned a
Y. pestis yfuABC
operon. The YfuABC system is a member of the cluster of bacterial ABC iron transporters that include Sfu of
Serratia
, Hit of
Haemophilus
, and Yfu of
Yersinia enterocolitica
. The
Y. pestis
KIM6+ system is most homologous to that in
Y. enterocolitica
, showing identities of 84% for YfuA (periplasmic binding protein), 87% for YfuB (inner membrane permease), and 75% for YfuC (ATP hydrolase). We constructed a
yfuABC
promoter-
lacZ
fusion to examine regulation of transcription. This promoter contains a potential Fur binding sequence and is iron and Fur regulated. Significant expression from the
yfuABC
promoter occurred during iron-deficient growth conditions. In vitro transcription and translation of a recombinant plasmid encoding
yfuABC
indicates that YfuABC proteins are expressed.
Escherichia coli
1017 (an enterobactin-deficient mutant) carrying this plasmid was able to grow in an iron-restrictive complex medium. We constructed a deletion encompassing the
yfuABC
promoter and most of
yfuA
. This mutation was introduced into strains with mutations in Ybt, Yfe, or both systems to examine the role of Yfu in iron acquisition in
Y. pestis
. Growth of the
yfu
mutants in a deferrated, defined medium (PMH2) at 26 and 37°C failed to identify a growth or iron transport defect due to the
yfu
mutation. Fifty percent lethal dose studies in mice did not demonstrate a role for the Yfu system in mammalian virulence.
Human Lactoferrin and Peptides Derived from a Surface-Exposed Helical Region Reduce ExperimentalEscherichia coliUrinary Tract Infection in Mice
Authors: Håversen Liliana A. , Engberg Inga , Baltzer Lars , Dolphin Gunnar , Hanson Lars Å. , Mattsby-Baltzer Inger
Published: July 2002

Lactoferrin (LF) is a multifunctional immunoregulatory protein that has been associated with host defense at mucosal surfaces through its antibacterial properties. The antibacterial and anti-inflammatory properties of LF were further explored with an animal model of experimental urinary tract infection. Bovine LF (bLF), human LF (hLF), and synthetic peptide sequences based on the antibacterial region of hLF (amino acid residues 16 to 40 [HLD1] and 18 to 40 [HLD2]) were given orally to female mice 30 min after the instillation of 108Escherichia colibacteria into the urinary bladder. The control groups received phosphate-buffered saline or water. C3H/Tif mice were treated with hLF or bLF, and C3H/HeN mice were treated with bLF only. The numbers of bacteria in the kidneys and bladder of C3H/Tif and C3H/HeN mice were significantly reduced 24 h later by the LF treatments compared to the findings for the control group. The hLF-treated group showed the strongest reduction compared with the vehicle-treated-group (Pvalues were 0.009 and 0.0001 for the kidneys and bladder, respectively). The urinary leukocyte response was diminished in the hLF-treated group. The hLF treatment also significantly reduced the urinary interleukin-6 (IL-6) levels at 2 h and the systemic IL-6 levels at 24 h after infection (Pvalues were 0.04 and < 0.002, respectively). In the bLF-treated animals, no such strong anti-inflammatory effects were obtained. In another series of experiments, C3H/Tif mice perorally treated with HLD1 or HLD2 also showed reduced numbers of bacteria in the kidneys compared with the vehicle-treated mice, although the results were significantly different only for HLD2 (P< 0.01). Analysis of urine from hLF-fed C3H/Tif mice showed that hLF was excreted into the urinary tract at 2 h after feeding. Testing of the in vitro bactericidal activity of LF (1 mg/ml) or the peptides (0.1 mg/ml) in mouse urine against theE. colibacteria revealed moderate killing only by HLD2. In conclusion, these results demonstrate for the first time that oral administration of hLF or peptides thereof is effective in reducing infection and inflammation at a remote site, the urinary tract, possibly through transfer of hLF or its peptides to the site of infection via renal secretion. The antibacterial mechanism is suggested to involve bactericidal capacities of LF, fragments thereof, or its peptides.
Mechanisms of Intracellular Killing ofRickettsia conoriiin Infected Human Endothelial Cells, Hepatocytes, and Macrophages
Authors: Feng Hui-Min , Walker David H.
Published: July 2002

The mechanism of killing of obligately intracellularRickettsia conoriiwithin human target cells, mainly endothelium and, to a lesser extent, macrophages and hepatocytes, has not been determined. It has been a controversial issue as to whether or not human cells produce nitric oxide. AKN-1 cells (human hepatocytes) stimulated by gamma interferon, tumor necrosis factor alpha, interleukin 1β, and RANTES (regulated by activation, normal T-cell-expressed and -secreted chemokine) killed intracellular rickettsiae by a nitric oxide-dependent mechanism. Human umbilical vein endothelial cells (HUVECs), when stimulated with the same concentrations of cytokines and RANTES, differed in their capacity to kill rickettsiae by a nitric oxide-dependent mechanism and in the quantity of nitric oxide synthesized. Hydrogen peroxide-dependent intracellular killing ofR. conoriiwas demonstrated in HUVECs, THP-1 cells (human macrophages), and human peripheral blood monocytes activated with the cytokines. Rickettsial killing in the human macrophage cell line was also mediated by a limitation of the availability of tryptophan in association with the expression of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase. The rates of survival of all of the cell types investigated under the conditions of activation and infection in these experiments indicated that death of the host cells was not the explanation for the control of rickettsial infection. This finding represents the first demonstration that activated human hepatocytes and, in some cases, endothelium can kill intracellular pathogens via nitric oxide and that RANTES plays a role in immunity to rickettsiae. Human cells are capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by one or a combination of three mechanisms involving nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation.