bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Dye Decolorization Method for Screening Potential Wood Preservatives
Authors: Borokhov Olga , Rothenburger Stephen
Published: July 2002

We developed a new screening method for potential wood preservatives based on decolorization of the dye Remazol Brilliant Blue R by extracellular oxidative agents produced by wood decay fungi. Oxidative biodegradation of lignin yielded decolorized zones around and under fungal cultures on a dyed agar medium. Inhibitory effects were detected by direct observation and measurement of the decolorized zones.
The
Streptococcus thermophilus
Autolytic Phenotype Results from a Leaky Prophage
Authors: Husson-Kao Clara , Mengaud Jérôme , Cesselin Bénédicte , van Sinderen Douwe , Benbadis Laurent , Chapot-Chartier Marie-Pierre
Published: July 2002

Streptococcus thermophilus
autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage φ01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569–577, 1999). It appears from our results that the autolytic properties are conferred to the
S. thermophilus
strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins.
Influence of Osmotic and Nutritional Stress on Physiology of
Penicillium fellutanum
in Removal of Phosphocholine and Modification of Phospho-1-
O
-[
N
-Peptidyl-(2-Aminoethanol)] Phosphodiesters of Peptidophosphogalactomannan Species
Authors: Park Yong-Il , Bonetti Sandra J. , Gander John E.
Published: July 2002

Addition of 3 M NaCl to 72-h cultures of
Penicillium fellutanum
in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP
x
GM
iii
) and a decrease in that of pP
x
GM
ii
. The magnitude of
31
P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1-
O
-[
N
-peptidyl-(2-aminoethanol)] phosphodiesters pP
x
GM
ii
and pP
x
GM
iii
decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6-
O
-phospho-1′-
O
-[
N
-peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.
Propachlor Removal by
Pseudomonas
Strain GCH1 in an Immobilized-Cell System
Authors: Martín M. , Mengs G. , Plaza E. , Garbi C. , Sánchez M. , Gibello A. , Gutierrez F. , Ferrer E.
Published: July 2002

A bacterial strain capable of growing on propachlor (2-chloro-
N
-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genus
Pseudomonas
. Washed-cell suspensions of strain GCH1 accumulated
N
-isopropylacetanilide, acetanilide, acetamide, and catechol.
Pseudomonas
strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 ± 0.15 μmol h
−1
. Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 ± 0.02 mg of protein μmol
−1
. GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.
Reductive Cleavage of Demeton-
S
-Methyl by
Corynebacterium glutamicum
in Cometabolism on More Readily Metabolizable Substrates
Authors: Girbal Laurence , Hilaire Didier , Leduc Sébastien , Delery Laure , Rols Jean-Luc , Lindley Nicholas D.
Published: July 2002

Corynebacterium glutamicum
is able to biotransform demeton-
S
-methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. Among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton-
S
-methyl biotransformation. The reaction mechanism of demeton-
S
-methyl biotransformation involves reductive cleavage of an S-C bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.
Transient Growth Requirement in
Bacillus subtilis
following the Cessation of Exponential Growth
Authors: Sung Huang-Mo , Yasbin Ronald E.
Published: July 2002

During an investigation of the parameters controlling mutations in
Bacillus subtilis
we observed that this bacterium exhibits a transient growth requirement for two nonessential amino acids (glutamic acid and isoleucine) during a type of postexponential growth on a minimal medium.
Genetic and Biochemical Approach for Characterization of Resistance to
Bacillus thuringiensis
Toxin Cry1Ac in a Field Population of the Diamondback Moth,
Plutella xylostella
Authors: Sayyed Ali H. , Haward Robert , Herrero Salvador , Ferré Juan , Wright Denis J.
Published: July 2002

Four subpopulations of a
Plutella xylostella
(L.) strain from Malaysia (F
4
to F
8
) were selected with
Bacillus thuringiensis
subsp.
kurstaki
HD-1,
Bacillus thuringiensis
subsp.
aizawai
, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F
9
found that selection with Cry1Ac, Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
in UNSEL-MEL declined significantly by F
9
. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
(5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-,
B. thuringiensis
subsp.
kurstaki
-, and
B. thuringiensis
subsp.
aizawai
-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F
9
to F
13
) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with
125
I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F
15
). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F
2
progeny from a backcross of F
1
progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
In Situ Reverse Transcription-PCR for Monitoring Gene Expression in Individual
Methanosarcina mazei
S-6 Cells
Authors: Lange Marianne , Tolker-Nielsen Tim , Molin Søren , Ahring Birgitte K.
Published: July 2002

An in situ reverse transcription-PCR protocol for detecting specific mRNA in
Methanosarcina mazei
S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene
dnaK
. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate–Fast Red detection system and binding of anti-digoxigenin–alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the
dnaK
transcript.
A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides
Authors: Soong Chee-Leong , Ogawa Jun , Shimizu Sakayu
Published: July 2002

A novel amidase involved in bacterial cyclic imide metabolism was purified from
Blastobacter
sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (

K
m

= 6.2 mM;
k
cat
= 5.76 s
−1
) and glutaramic acid (

K
m

= 2.8 mM;
k
cat
= 2.23 s
−1
). However, the substrates of known amidases such as short-chain (C
2
to C
4
) aliphatic amides, long-chain (above C
16
) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides,
N
-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an
M
r
of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.
Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in
Rhodococcus opacus
1cp
Authors: Finkelstein Zoya I. , Baskunov Boris P. , Boersma Marelle G. , Vervoort Jacques , Golovlev Eugene L. , van Berkel Willem J. H. , Golovleva Ludmila A. , Rietjens Ivonne M. C. M.
Published: July 2002

The transformation of monofluorophenols by whole cells of
Rhodococcus opacus
1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and
19
F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The
19
F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in
R. opacus
1cp.
Fortified Sera and Their Use in Environmental Virology
Authors: Hoyt Jonathan L. , Margolin Aaron B.
Published: July 2002

Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay.
Development of a Novel, Rapid Integrated
Cryptosporidium parvum
Detection Assay
Authors: Kozwich Diane , Johansen Kristine A. , Landau Keli , Roehl Christopher A. , Woronoff Sam , Roehl Patrick A.
Published: July 2002

The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of
Cryptosporidium parvum
. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-stranded RNA (dsRNA) specific for
C. parvum
. The Xtra Bind Capture System interfaced with pellets concentrated from water samples collected with previously developed filtration devices. The pellets were resuspended in reagent water (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The dsRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were removed by washing the Xtra Bind material several times with buffer. The Xtra Bind material with its bound dsRNA was added directly to an amplification reaction mixture, and the target was amplified without elution from the Xtra Bind material. A PCR was performed in the presence of the Xtra Bind Capture System, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods typically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, we were able to reproducibly and correctly identify 10 oocysts added to 0.5 ml of reagent water. When the protocol was evaluated with a small set of environmental samples, the level of detection was as low as 1 oocyst/liter. The total time from resuspension of the pellet to detection was about 3 h, which is considerably less than the 5 h required for immunomagnetic separation followed by an indirect immunofluorescence assay and microscopy.
Exposure of Phytopathogenic
Xanthomonas
spp. to Lethal Concentrations of Multiple Oxidants Affects Bacterial Survival in a Complex Manner
Authors: Sriprang Rutchadaporn , Vattanaviboon Paiboon , Mongkolsuk Skorn
Published: July 2002

During plant-microbe interactions and in the environment,
Xanthomonas campestris
pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (
tert
-butyl hydroperoxide) by 1,000-fold; conversely, treatment of cells with menadione plus H
2
O
2
resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of
X. campestris
with a combination of H
2
O
2
and
tert
-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H
2
O
2
and
tert
-butyl hydroperoxide plus H
2
O
2
. These data suggest that H
2
O
2
is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus
tert
-butyl hydroperoxide. However,
X. campestris
cells in the stationary phase and a spontaneous H
2
O
2
-resistant mutant (
X. campestris
pv. phaseoli HR) were more resistant to killing by menadione plus
tert
-butyl hydroperoxide. These findings give new insight into oxidant killing of
Xanthomonas
spp. that could be generally applied to other bacteria.
Pigment Production by
Streptococcus agalactiae
in Quasi-Defined Media
Authors: Rosa-Fraile Manuel , Sampedro Antonio , Rodrı́guez-Granger Javier , Garcı́a-Peña Maria Luisa , Ruiz-Bravo Alfonso , Haı̈dour Ali
Published: July 2002

A quasi-defined medium that supports the growth of
Streptococcus agalactiae
as pigmented colonies has been developed. The medium contains starch, a peptic digest of albumin, amino acids, nucleosides, vitamins, and salts. The presence of free cysteine, which could be replaced with other sulphur-containing compounds and to a lesser degree by reducing agents, was required for pigment formation.
Cross-Epithelial Hydrogen Transfer from the Midgut Compartment Drives Methanogenesis in the Hindgut of Cockroaches
Authors: Lemke Thorsten , van Alen Theo , Hackstein Johannes H. P. , Brune Andreas
Published: July 2002

In the intestinal tracts of animals, methanogenesis from CO
2
and other C
1
compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach
Blaberus
sp. showed that molecular hydrogen strongly accumulated in the midgut (H
2
partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH
4
[105 ± 49 nmol (g of cockroach)
−1
h
−1
] but only traces of H
2
. In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H
2
, whereas hindguts emitted only CH
4
[106 ± 58 and 71 ± 50 nmol (g of cockroach)
−1
h
−1
, respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H
2
accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H
2
(20 kPa) identified the hindgut as an efficient sink for externally supplied H
2
. A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H
2
concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.
Homologous Expression of the Lipase and ABC Transporter Gene Cluster,
tliDEFA
, Enhances Lipase Secretion in
Pseudomonas
spp
Authors: Ahn Jung Hoon , Pan Jae Gu , Rhee Joon Shick
Published: July 2002

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in
Pseudomonas fluorescens
. For the enhanced secretion of the lipase, we tried to coexpress
tliA
and
tliDEF
in various
Pseudomonas
species. Whereas the coexpression of
tliA
and
tliDEF
was required for the lipase secretion in
P. fragi
, the expression of
tliA
was sufficient for the lipase secretion in
P. fluorescens
,
P. syringae
, and
P. putida
, indicating the existence of compatible ABC transporter in these species. However,
P. fluorescens
harboring
tliDEFA
secreted much more lipase than
P. fluorescens
harboring only
tliA
, but the
tliDEF
was functional only at temperatures below 30°C. The recombinant
P. fluorescens
overexpressing
tliDEFA
showed the highest secretion level, 217 U/ml · OD (optical density) (28 μg/ml · OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that
P. fluorescens
can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.
Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment
Authors: Wu Liyou , Thompson Dorothea K. , Li Guangshan , Hurt Richard A. , Tiedje James M. , Zhou Jizhong
Published: July 2002

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (
nirS
and
nirK
) genes, ammonia mono-oxygenase (
amoA
) genes, and methane mono-oxygenase (
pmoA
) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65°C). The detection limit for
nirS
genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (
r
2
= 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of
nir
and
amo
A and
pmo
A gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.
Burkholderia cepacia
Genomovar III Is a Common Plant-Associated Bacterium
Authors: Balandreau Jacques , Viallard Veronique , Cournoyer Benoit , Coenye Tom , Laevens Severine , Vandamme Peter
Published: July 2002

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of
Burkholderia cepacia
-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to
B. cepacia
genomovar III, a genomic species associated with “cepacia syndrome” in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the
B. cepacia
complex consists of two independent phylogenetic lineages.
Applications of Gene Replacement Technology to
Streptomyces clavuligerus
Strain Development for Clavulanic Acid Production
Authors: Paradkar A. S. , Mosher R. H. , Anders C. , Griffin A. , Griffin J. , Hughes C. , Greaves P. , Barton B. , Jensen S. E.
Published: July 2002

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism
Streptomyces clavuligerus
by targeted disruption of the gene (
lat
) encoding lysine ɛ-aminotransferase. Specific production of clavulanic acid increased in the
lat
mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by
S. clavuligerus
. Therefore, mutations in
lat
and in
cvm1
, a gene involved in clavam production, were introduced into a high-titer industrial strain of
S. clavuligerus
to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.
Quantitative Shedding of Two Genotypes of
Cryptosporidium parvum
in California Ground Squirrels (
Spermophilus beecheyi
)
Authors: Atwill Edward R. , Camargo Sergio Maldonado , Phillips Ralph , Alonso Laura Herrera , Tate Kenneth W. , Jensen Wayne A. , Bennet Joe , Little Scott , Salmon Terrell P.
Published: July 2002

Sixteen percent of California ground squirrels (
Spermophilus beecheyi
) were found to be shedding an average of 53,875
Cryptosporidium parvum
oocysts/g of feces. Male squirrels had a higher prevalence and higher intensity of shedding than did female squirrels. The majority of
C. parvum
isolates matched a bovine-murine genotype, with a few isolates resembling a porcine genotype. Higher intensities of shedding by males may enhance dissemination and genotypic mixing of this protozoa given males' proclivity to disperse to nonnatal colonies.
Nitrogen-Fixing Nodules with
Ensifer adhaerens
Harboring
Rhizobium tropici
Symbiotic Plasmids
Authors: Rogel M. Antonio , Hernández-Lucas Ismael , Kuykendall L. David , Balkwill David L. , Martinez-Romero Esperanza
Published: July 2002

Ensifer adhaerens
is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene,
E. adhaerens
is related to
Sinorhizobium
spp.
E. adhaerens
ATCC 33499 did not nodulate
Phaseolus vulgaris
(bean) or
Leucaena leucocephala
, but with symbiotic plasmids from
Rhizobium tropici
CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as
E. adhaerens
isolates by growth on selective media.
Quantitative Analysis of Bacterial Gene Expression by Using the
gusA
Reporter Gene System
Authors: Sun Jun , Smets Ilse , Bernaerts Kristel , Van Impe Jan , Vanderleyden Jos , Marchal Kathleen
Published: July 2002

An
Azospirillum brasilense
Sp7 strain containing a plasmid-borne translational
cytN-gusA
fusion was grown in a continuous culture to quantitatively evaluate the influence of extracellular signals (such as O
2
) on expression of the
cytNOQP
operon. The dissolved oxygen concentration was shifted at regular time intervals before the steady state was reached. The measured β-glucuronidase activity was used to monitor
cytN
gene expression. However, as the β-glucuronidase activity in the experimental setup not only depended on altered transcription of the hybrid gene when the signal was varied but was also influenced by cellular accumulation, degradation, and dilution of the hybrid fusion protein, a mathematical method was developed to describe the intrinsic properties of the dynamic bioprocess. After identification and validation of the mathematical model, the apparent specific rate of expression of the fusion, which was independent of the experimental setup, could be deduced from the model and used to quantify gene expression regulated by extracellular environmental signals. In principle, this approach can be generalized to assess the effects of external signals on bacterial gene expression.
Brown Pigments Produced by
Yarrowia lipolytica
Result from Extracellular Accumulation of Homogentisic Acid
Authors: Carreira Alexandra , Ferreira Luı́sa M. , Loureiro Virgı́lio
Published: July 2002

Yarrowia lipolytica
produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid,
p
-hydroxyphenylethanol, and
p
-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and
p
-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1).
p
-Hydroxyphenylethanol and
p
-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde,
p
-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in
Y. lipolytica
involves the formation of
p
-hydroxyphenylacetaldehyde, which is oxidized to
p
-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn
2+
accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn
2+
. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.
Isolation of an Insertion Sequence from
Ralstonia solanacearum
Race 1 and Its Potential Use for Strain Characterization and Detection
Authors: Lee Yung-An , Fan Shu-Chung , Chiu Ling-Ya , Hsia Kuo-Chiang
Published: July 2002

A new insertion sequence (IS), IS
1405
, was isolated and characterized from a
Ralstonia solanacearum
race 1 strain by the method of insertional inactivation of the
sacB
gene. Sequence analysis indicated that the IS is closely related to the members of IS
5
family, but the extent of nucleotide sequence identity in 5′ and 3′ noncoding regions between IS
1405
and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all
R. solanacearum
race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS
1405
and additional five endogenous IS
1405
s that reside in the chromosome of
R. solanacearum
race 1 strains indicated that IS
1405
prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS
1405
as a probe revealed extensive genetic variation among strains of
R. solanacearum
race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of
R. solanacearum
through a host plant community. Furthermore, specific insertion sites of IS
1405
in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of
R. solanacearum
, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.
Susceptibility of a Field-Derived,
Bacillus thuringiensis
-Resistant Strain of Diamondback Moth to In Vitro-Activated Cry1Ac Toxin
Authors: Sayyed Ali H. , Gatsi Roxani , Kouskoura Thaleia , Wright Denis J. , Crickmore Neil
Published: July 2002

Resistant and susceptible populations of the diamondback moth (
Plutella xylostella
) were tested with crystalline, solubilized, and partially and fully activated forms of the
Bacillus thuringiensis
Cry1Ac δ-endotoxin. Fully activated toxin greatly reduced the resistance ratio (ratio of the 50% lethal concentration for the resistant population to that for the susceptible population) of the resistant population, suggesting that a defect in toxin activation is a major resistance mechanism.
Microbial Diversity of a Heavily Polluted Microbial Mat and Its Community Changes following Degradation of Petroleum Compounds
Authors: Abed Raeid M. M. , Safi Nimer M. D. , Köster Jürgen , de Beer Dirk , El-Nahhal Yasser , Rullkötter Jürgen , Garcia-Pichel Ferran
Published: July 2002

We studied the microbial diversity of benthic cyanobacterial mats inhabiting a heavily polluted site in a coastal stream (Wadi Gaza) and monitored the microbial community response induced by exposure to and degradation of four model petroleum compounds in the laboratory.
Phormidium-
and
Oscillatoria
-like cyanobacterial morphotypes were dominant in the field. Bacteria belonging to different groups, mainly the
Cytophaga
-
Flavobacterium
-
Bacteriodes
group, the γ and β subclasses of the class
Proteobacteria
, and the green nonsulfur bacteria, were also detected. In slurry experiments, these communities efficiently degraded phenanthrene and dibenzothiophene completely in 7 days both in the light and in the dark.
n
-Octadecane and pristane were degraded to 25 and 34% of their original levels, respectively, within 7 days, but there was no further degradation until 40 days. Both cyanobacterial and bacterial communities exhibited noticeable changes concomitant with degradation of the compounds. The populations enriched by exposure to petroleum compounds included a cyanobacterium affiliated phylogenetically with
Halomicronema
. Bacteria enriched both in the light and in the dark, but not bacteria enriched in any of the controls, belonged to the newly described
Holophaga
-
Geothrix
-
Acidobacterium
phylum. In addition, another bacterial population, found to be a member of green nonsulfur bacteria, was detected only in the bacteria treated in the light. All or some of the populations may play a significant role in metabolizing the petroleum compounds. We concluded that the microbial mats from Wadi Gaza are rich in microorganisms with high biodegradative potential.
Identification and Characterization of
Leuconostoc fallax
Strains Isolated from an Industrial Sauerkraut Fermentation
Authors: Barrangou Rodolphe , Yoon Sung-Sik , Breidt, Frederick , Fleming Henry P. , Klaenhammer Todd R.
Published: July 2002

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of
Leuconostoc mesenteroides
. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were
Leuconostoc fallax
strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the
L. fallax
isolates were highly similar to those of
L. mesenteroides
. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the
L. fallax
strains exhibited the malolactic reaction, which is characteristic of most
L. mesenteroides
strains. These results indicated that in addition to
L. mesenteroides
, a variety of
L. fallax
strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of
L. fallax
require further investigation.
oriC
Region and Replication Termination Site,
dif
, of the
Xanthomonas campestris
pv. campestris 17 Chromosome
Authors: Yen Ming-Ren , Lin Nien-Tsung , Hung Chih-Hsin , Choy Ka-Tim , Weng Shu-Fen , Tseng Yi-Hsiung
Published: July 2002

A 13-kb DNA fragment containing
oriC
and the flanking genes
thdF
,
orf900
,
yidC
,
rnpA
,
rpmH
,
oriC
,
dnaA
,
dnaN
,
recF
, and
gyrB
was cloned from the gram-negative plant pathogen
Xanthomonas campestris
pv. campestris 17. These genes are conserved in order with other eubacterial
oriC
genes and code for proteins that share high degrees of identity with their homologues, except for
orf900
, which has a homologue only in
Xylella fastidiosa
. The
dnaA
/
dnaN
intergenic region (273 bp) identified to be the minimal
oriC
region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical
oriC
s contain four or more DnaA boxes located upstream of the
dnaA
gene. The
X. campestris
pv. campestris 17
attB
site required for site-specific integration of cloned fragments from filamentous phage φLf replicative form DNA was identified to be a
dif
site on the basis of similarities in nucleotide sequence and function with the
Escherichia coli dif
site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The
oriC
and
dif
sites were located at 12:00 and 6:00, respectively, on the circular
X. campestris
pv. campestris 17 chromosome map, similar to the locations found for
E. coli
sites. Computer searches revealed the presence of both the
dif
site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the
dif
sites are located far away from the 6:00 point instead of being placed opposite the cognate
oriC
. The differences in the relative position suggest that mechanisms different from that of
E. coli
may participate in the control of chromosome replication.
Differential Expression of Neurokinin-1 Receptor by Human Mucosal and Peripheral Lymphoid Cells
Authors: Goode Triona , O'Connell Joe , Ho Wen-Zhe , O'Sullivan Gerald C. , Collins J. Kevin , Douglas Steven D. , Shanahan Fergus
Published: July 2002

Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.
Intestinal Secretory Immunoglobulin A Response to Enteroaggregative
Escherichia coli
in Travelers with Diarrhea
Authors: Sutjita Made , Bouckenooghe Alain R. , Adachi Javier A. , Jiang Zhi Dong , Mathewson John J. , Ericsson Charles D. , DuPont Herbert L.
Published: July 2002

We examined stool samples from travelers for secretory immunoglobulin A (sIgA) to enteroaggregative
Escherichia coli
(EAEC) during episodes of acute diarrhea. Ten paired samples from 10 patients with diarrhea caused by EAEC were examined for the presence of specific sIgA by dot blot and Western blot immunoassays. Five samples were positive by dot blotting, and two samples were positive by Western blotting.
Serological Detection of Infection with Diverse Human and Simian Immunodeficiency Viruses Using Consensus
env
Peptides
Authors: Masciotra Silvina , Rudolph Donna L. , van der Groen Guido , Yang Chunfu , Lal Renu B.
Published: July 2002

Cross-species transmission has been shown to play an important role in the emergence of human retroviruses. We developed a generic enzyme immunoassay using synthetic peptides from gp41 and C2V3 consensus sequences (human immunodeficiency virus [HIV] type 1 [HIV-1] groups M, O, and N and the homologous region of simian immunodeficiency virus [SIV] strains from chimpanzees [SIVcpz], SIVcpzGAB1 and SIVcpzANT) to detect divergent HIV and SIV. A cocktail of peptides from gp41 and C2V3 (M-O) detected all HIV-1 group M and O sera and showed cross-reactivity with SIVcpz sera. Further, a mixture of C2V3 peptides (GAB1-ANT) failed to detect HIV-1 infections but reacted with all SIVcpz sera, allowing discrimination of SIVcpz from HIV-1 infections. Since most SIVcpz sera cross-reacted with HIV-1 peptides, we next evaluated SIVcpz serum reactivity with rapid tests for HIV-1/2. SIVcpzANT and SIVcpzUS sera reacted with the Sero-strip and Multispot assays. Both tests are sensitive in detecting group M (97 100%, respectively), although Multispot has lower sensitivity for group O detection (67%) than does Sero-strip (100%). The limited volume and time required to perform these assays make them a generic tool for field screening. The
env
peptide-based assay and rapid tests should allow for the identification of emerging variants of HIV and SIV.
An Immunoblot Assay for Detection of Immunoglobulin M Antibody to Human Herpesvirus 6
Authors: LaCroix Steve , Stewart John A. , Thouless Margaret E. , Black Jodi B.
Published: July 2002

We identified the human herpesvirus 6 (HHV-6)-dominant immunoglobulin M (IgM)-reactive virion protein as being the same 101-kDa protein (101K) previously identified as the major IgG immunoreactive protein and a specific serologic marker of HHV-6 infection. An immunoblot assay (IB) to detect HHV-6-specific IgM antibodies against the 101K protein in human serum samples was developed. The assay was validated by using acute- and convalescent-phase serum collected from children under 2 years of age in which we previously detected IgG seroconversion to the HHV-6 101K protein. Of 32 serum pairs which previously demonstrated IgG seroconversion to the 101K protein, 29 had IgM reactivity to the same protein in the acute-phase sample and the remaining 3 had reactivity in the convalescent-phase sample. We also detected HHV-6 IgM activity in sera collected from individuals ≥4 years of age who were also IgM seropositive to measles or rubella. Results of cross-adsorption studies using measles virus-, rubella virus-, and HHV-6-infected cells as the adsorbing antigen indicated no cross-reactivity between measles or rubella IgM and HHV-6 IgM in human serum samples. The IgM IB detected HHV-6-specific IgM antibody to the 101K protein in 78% (63 of 81) of tested acute-phase serum collected from young children with an undifferentiated rash illness by using a single serum dilution.
Inhibition of Human Peripheral Blood Neutrophil Respiratory Burst by Alcohol-Based Venipuncture Site Disinfection
Authors: Reinisch Norbert , Wiedermann Christian J. , Ricevuti Giovanni
Published: July 2002

Ethanol inhibits the respiratory burst of neutrophils. Therefore, the effects of alcohol-based skin disinfection on oxygen metabolism in neutrophils were tested using 70% ethanol or an ethanol–isopropanol–
n
-propanol mixture. Neutrophil respiratory burst activity as assessed fluorometrically by oxidation of 2′,7′-dichlorofluorescein diacetate increased at 10 min after disinfection with 70% ethanol compared to the activity at 30 s. The increase was significant for triggering oxidative burst with formylpeptide but not with phorbol myristate acetate.
Multicenter Evaluation of a Rapid and Convenient Method for Determination of Cytomegalovirus Immunoglobulin G Avidity
Authors: Baccard-Longere Monique , Freymuth Francois , Cointe Denis , Seigneurin Jean Marie , Grangeot-Keros Liliane
Published: July 2002

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.
Cytokine Expression in the Mouse Brain in Response to Immune Activation by
Corynebacterium parvum
Authors: Sheng W. S. , Hu S. , Ding J. M. , Chao C. C. , Peterson P. K.
Published: July 2002

Cytokine expression in the brain has been suggested to mediate various sickness behaviors. Here we report that intraperitoneal injection of a
Corynebacterium parvum
antigen in C57BL/6 mice was followed by prolonged upregulation of cytokines in the cerebral cortex and subcortical structures in a time course that coincided with reduced spontaneous running activity.
Pathogenic Potential of Canine Parvovirus Types 2a and 2c in Domestic Cats
Authors: Nakamura Kazuya , Sakamoto Maki , Ikeda Yasuhiro , Sato Eiji , Kawakami Kazuo , Miyazawa Takayuki , Tohya Yukinobu , Takahashi Eiji , Mikami Takeshi , Mochizuki Masami
Published: July 2002

The in vivo pathogenicity of canine parvovirus (CPV) type 2c (strain V203) and of CPV type 2a (strain V154) against cats was investigated. Our results indicate that both types of CPV have the potential to induce disease in cats.
Reactivity of MEST-1 (Antigalactofuranose) with
Trypanosoma cruzi
Glycosylinositol Phosphorylceramides (GIPCs): Immunolocalization of GIPCs in Acidic Vesicles of Epimastigotes
Authors: Suzuki Erika , Mortara Renato A. , Takahashi Helio K. , Straus Anita H.
Published: July 2002

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various
Trypanosoma cruzi
forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. In epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.