bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Tracing, byTaqNuclease Assays, of aSynechococcusEcotype in a Highly Diversified Natural Population
Authors: Becker Sven , Fahrbach Michael , Böger Peter , Ernst Anneliese
Published: August 2002

QuantitativeTaqnuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-richSynechococcusspp. in the pelagic zone of this lake. Cells passing a 3-μm-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-richSynechococcusspp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-richSynechococcusstrains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all knownSynechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolateSynechococcussp. strain BO 8807. During the growth period, genome numbers of knownSynechococcusspp. varied by 2 orders of magnitude (2.9 × 103to 3.1 × 105genomes per ml). The ecotypeSynechococcussp. strain BO 8807 was detected in every sample at concentrations between 1.6 × 101and 1.3 × 104genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.
Evidence for Natural Horizontal Transfer of thepcpBGene in the Evolution of Polychlorophenol-Degrading Sphingomonads
Authors: Tiirola Marja A. , Wang Hong , Paulin Lars , Kulomaa Markku S.
Published: August 2002

The chlorophenol degradation pathway inSphingobium chlorophenolicumis initiated by thepcpBgene product, pentachlorophenol-4-monooxygenase. The distribution of the gene was studied in a phylogenetically diverse group of polychlorophenol-degrading bacteria isolated from contaminated groundwater in Kärkölä, Finland. All the sphingomonads isolated were shown to sharepcpBgene homologs with 98.9 to 100% sequence identity. The gene product was expressed when the strains were induced by 2,3,4,6-tetrachlorophenol. A comparative analysis of the 16S rDNA andpcpBgene trees suggested that a recent horizontal transfer of thepcpBgene was involved in the evolution of the catabolic pathway in the Kärkölä sphingomonads. The full-length KärköläpcpBgene allele had approximately 70% identity with the threepcpBgenes previously sequenced from sphingomonads. It was very closely related to the environmental clones obtained from chlorophenol-enriched soil samples (M. Beaulieu, V. Becaert, L. Deschenes, and R. Villemur, Microbiol. Ecol.40:345-355, 2000). The gene was not present in polychlorophenol-degrading nonsphingomonads isolated from the Kärkölä source.
Rhamnolipid Stimulates Uptake of Hydrophobic Compounds by
Pseudomonas aeruginosa
Authors: Noordman Wouter H. , Janssen Dick B.
Published: August 2002

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (
Pseudomonas aeruginosa
UG2
, Acinetobacter calcoaceticus
RAG1,
Rhodococcus erythropolis
DSM 43066,
R. erythropolis
ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by
P. aeruginosa
was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that
P. aeruginosa
has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon α,ω-tetradecadiene by a cell suspension of
P. aeruginosa
. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of
P. aeruginosa
was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in
P. aeruginosa
strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.
Lipopeptide Production in
Pseudomonas
sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System
Authors: Koch Birgit , Nielsen Tommy H. , Sørensen Dan , Andersen Jens Bo , Christophersen Carsten , Molin Søren , Givskov Michael , Sørensen Jan , Nybroe Ole
Published: August 2002

Pseudomonas
sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn
5
::
luxAB
mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (
amsY
) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (
amsY
) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the
amsY
::
luxAB
reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional
gacS
locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.
Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain
Authors: Matsumoto Takeshi , Fukuda Hideki , Ueda Mitsuyoshi , Tanaka Atsuo , Kondo Akihiko
Published: August 2002

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (
FS
or
FL
gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter
UPR-ICL
(5′ upstream region of the isocitrate lyase of
Candida tropicalis
). Using this new system, recombinant lipase with a pro sequence from
Rhizopus oryzae
(rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.
Environmental Factors Influencing Human Viral Pathogens and Their Potential Indicator Organisms in the Blue Mussel,
Mytilus edulis
: the First Scandinavian Report
Authors: Hernroth Bodil E. , Conden-Hansson Ann-Christine , Rehnstam-Holm Ann-Sofi , Girones Rosina , Allard Annika K.
Published: August 2002

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting
Bacteroides fragilis
, and
Escherichia coli
. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites.
E. coli
counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of
E. coli
were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination,
E. coli
, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of “clean” mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.
Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by
Torula corallina
Authors: Lee Jung-Kul , Koo Bong-Seong , Kim Sang-Yong
Published: August 2002

Torula corallina
, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by
Torula corallina
with trace elements such as Cu
2+
has been thoroughly reported, but the mechanism by which Cu
2+
increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu
2+
enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in
T. corallina
, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu
2+
concentration. This suggests that supplemental Cu
2+
reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in
T. corallina
.
The Bacterivorous Soil Flagellate
Heteromita globosa
Reduces Bacterial Clogging under Denitrifying Conditions in Sand-Filled Aquifer Columns
Authors: Mattison Richard G. , Taki Hironori , Harayama Shigeaki
Published: August 2002

An exopolymer (slime)-producing soil bacterium
Pseudomonas
sp. (strain PS+) rapidly clogged sand-filled columns supplied with air-saturated artificial groundwater containing glucose (500 mg liter
−1
) as a sole carbon source and nitrate (300 mg liter
−1
) as an alternative electron acceptor. After 80 days of operation under denitrifying conditions, the effective porosity and saturated hydraulic conductivity (permeability) of sand in these columns had fallen by 2.5- and 26-fold, respectively. Bacterial biofilms appeared to induce clogging by occluding pore spaces with secreted exopolymer, although there may also have been a contribution from biogas generated during denitrification. The bacterivorous soil flagellate
Heteromita globosa
minimized reductions in effective porosity (1.6-fold) and permeability (13-fold), presumably due to grazing control of biofilms. Grazing may have limited growth of bacterial biomass and hence the rate of exopolymer and biogas secretion into pore spaces. Evidence for reduction in biogas production is suggested by increased nitrite efflux from columns containing flagellates, without a concomitant increase in nitrate consumption. There was no evidence that flagellates could improve flow conditions if added once clogging had occurred (60 days). Presumably, bacterial biofilms and their secretions were well established at that time. Nevertheless, this study provides evidence that bacterivorous flagellates may play a positive role in maintaining permeability in aquifers undergoing remediation treatments.
Enzymatic Properties and Intracellular Localization of the Novel
Trichoderma reesei
β-Glucosidase BGLII (Cel1A)
Authors: Saloheimo Markku , Kuja-Panula Juha , Ylösmäki Erkko , Ward Michael , Penttilä Merja
Published: August 2002

This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (
bgl2
) from the cellulolytic fungus
Trichoderma reesei
(
Hypocrea jecorina
). The expression pattern of
bgl2
is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the
cre1
gene. The BGLII protein was produced in
Escherichia coli
, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in
T. reesei
cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of
T. reesei
strains expressing
bgl2
from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase.
Unveiling the Organisms behind Novel Eukaryotic Ribosomal DNA Sequences from the Ocean
Authors: Massana Ramon , Guillou Laure , Díez Beatriz , Pedrós-Alió Carlos
Published: August 2002

Despite the fact that the smallest eukaryotes (cells less than 5 μm in diameter) play key roles in marine food webs, particularly in open oligotrophic areas, the study of their in situ diversity started just one year ago. Perhaps the most remarkable finding of the most recent studies has been the discovery of completely new phylogenetic lineages, such as novel clades belonging to the stramenopile and alveolate phyla. The two new groups account for a significant fraction of clones in genetic libraries from North Atlantic, equatorial Pacific, Antarctic, and Mediterranean Sea waters. However, the identities and ecological relevance of these organisms remain unknown. Here we investigate the phylogenetic relationships, morphology, in situ abundance, and ecological role of novel stramenopiles. They form at least eight independent clades within the stramenopile basal branches, indicating a large phylogenetic diversity within the group. Two lineages were visualized and enumerated in field samples and enrichments by fluorescent in situ hybridization using specific rRNA-targeted oligonucleotide probes. The targeted organisms were 2- to 3-μm-diameter, round-shaped, nonpigmented flagellates. Further, they were found to be bacterivorous. One lineage accounted for up to 46% (average during an annual cycle, 19%) of heterotrophic flagellates in a coastal environment, providing evidence that novel stramenopiles are important and unrecognized components of the total stock of bacterial grazers.
Rickettsia monacensis
sp. nov., a Spotted Fever Group Rickettsia, from Ticks (
Ixodes ricinus
) Collected in a European City Park
Authors: Simser Jason A. , Palmer Ann T. , Fingerle Volker , Wilske Bettina , Kurtti Timothy J. , Munderloh Ulrike G.
Published: August 2002

We describe the isolation and characterization of
Rickettsia monacensis
sp. nov. (type strain, IrR/Munich
T
) from an
Ixodes ricinus
tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with
Ixodes scapularis
cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with
I. ricinus
. Phylogenetic analysis of partial
rompA
sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae.
R. monacensis
also replicated in cell lines derived from the ticks
I. ricinus
(IRE11) and
Dermacentor andersoni
(DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with
R. monacensis
had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA.
R. monacensis
induced actin tails in both tick and mammalian cells similar to those reported for
R. rickettsii. R. monacensis
joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.
Identification and Isolation of Two Ascomycete Fungi from Spores of the Arbuscular Mycorrhizal Fungus
Scutellospora castanea
Authors: Hijri Mohamed , Redecker Dirk , Petetot Jean A. MacDonald-Comber , Voigt Kerstin , Wöstemeyer Johannes , Sanders Ian R.
Published: August 2002

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF)
Scutellospora castanea
. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus
Nectria
, and the second, obtained from both healthy and dead spores, was assigned to
Leptosphaeria
, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of
S. castanea
was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and
S. castanea
. The role of this fungus in
S. castanea
spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.
Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of
Tuber borchii
Authors: Lacourt Isabelle , Duplessis Sébastien , Abbà Simona , Bonfante Paola , Martin Francis
Published: August 2002

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle
Tuber borchii
, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.
Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of
Spodoptera litura
with Insecticidal Crystal Protein Cry1C
Authors: Agrawal Neema , Malhotra Pawan , Bhatnagar Raj K.
Published: August 2002

Insecticidal toxins produced by
Bacillus thuringiensis
interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of
B. thuringiensis
toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest,
Spodoptera litura
. The
S. litura
APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing
S. litura
APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.
Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse
Chloroflexaceae
-Related Bacteria in Hot Spring Microbial Mats
Authors: Nübel Ulrich , Bateson Mary M. , Vandieken Verona , Wieland Andrea , Kühl Michael , Ward David M.
Published: August 2002

We investigated the diversity, distribution, and phenotypes of uncultivated
Chloroflexaceae
-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus
Chloroflexus
or with the type C cluster, a group of previously discovered
Chloroflexaceae
relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll
a. Chloroflexus
sp. filaments were rare at 60°C but were more abundant at 70°C, where they were confined to the upper millimeter of the mat. Both type C organisms and
Chloroflexus
spp. were observed to assimilate radiolabeled acetate under in situ conditions.
Construction and Characterization of a
proU
-
gfp
Transcriptional Fusion That Measures Water Availability in a Microbial Habitat
Authors: Axtell Catherine A. , Beattie Gwyn A.
Published: August 2002

We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the
proU
promoter from
Escherichia coli
and the reporter gene
gfp
was introduced into strains of
E. coli
,
Pantoea agglomerans
, and
Pseudomonas syringae
. The
proU
-
gfp
fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na
2
SO
4
, KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced
proU
-
gfp
expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of
P. agglomerans
biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a
proU
-
gfp
-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of
proU
-
gfp
in multiple bacterial species illustrates the potential for tailoring
proU
-
gfp
-based biosensors to specific habitats.
Isolation of Tellurite- and Selenite-Resistant Bacteria from Hydrothermal Vents of the Juan de Fuca Ridge in the Pacific Ocean
Authors: Rathgeber Christopher , Yurkova Natalia , Stackebrandt Erko , Beatty J. Thomas , Yurkov Vladimir
Published: August 2002

Deep-ocean hydrothermal-vent environments are rich in heavy metals and metalloids and present excellent sites for the isolation of metal-resistant microorganisms. Both metalloid-oxide-resistant and metalloid-oxide-reducing bacteria were found. Tellurite- and selenite-reducing strains were isolated in high numbers from ocean water near hydrothermal vents, bacterial films, and sulfide-rich rocks. Growth of these isolates in media containing K
2
TeO
3
or Na
2
SeO
3
resulted in the accumulation of metallic tellurium or selenium. The MIC of K
2
TeO
3
ranged from 1,500 to greater than 2,500 μg/ml, and the MIC of Na
2
SeO
3
ranged from 6,000 to greater than 7,000 μg/ml for 10 strains. Phylogenetic analysis of 4 of these 10 strains revealed that they form a branch closely related to members of the genus
Pseudoalteromonas
, within the γ-3 subclass of the
Proteobacteria
. All 10 strains were found to be salt tolerant, pH tolerant, and thermotolerant. The metalloid resistance and morphological, physiological, and phylogenetic characteristics of newly isolated strains are described.
Aquatic Insects as a Vector for
Mycobacterium ulcerans
Authors: Marsollier Laurent , Robert Raymond , Aubry Jacques , Saint André Jean-Paul , Kouakou Henri , Legras Pierre , Manceau Anne-Lise , Mahaza Chetaou , Carbonnelle Bernard
Published: August 2002

Mycobacterium ulcerans
is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking
M. ulcerans
to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of
M. ulcerans
, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that
M. ulcerans
may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition,
M. ulcerans
appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated
M. ulcerans
from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of
M. ulcerans
.
Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography
Authors: Nielsen Jeppe Lund , Juretschko Stefan , Wagner Michael , Nielsen Per Halkjær
Published: August 2002

Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 × 10
8
cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4′,6′-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma
Proteobacteria
, and 10% were assigned to the delta subclass of
Proteobacteria
.
Endosymbiotic Bacteria in the Esophageal Organ of Glossiphoniid Leeches
Authors: Kikuchi Yoshitomo , Fukatsu Takema
Published: August 2002

We characterized the intracellular symbiotic bacteria of the hematophagous glossiphoniid leeches
Placobdelloides siamensis
and a
Parabdella
sp. These leeches have a specialized structure called an “esophageal organ,” the cells of which harbor bacterial symbionts. From the esophageal organ of each species, a 1.5-kb eubacterial 16S rRNA gene segment was amplified by PCR, cloned, and sequenced. Diagnostic PCR detected the symbiont in the esophageal organ and intestine. Phylogenetic analysis of the 16S rRNA gene(s) demonstrated that the symbionts from the leeches formed a monophyletic group in a well-defined clade containing endosymbiotic bacteria of plant sap-feeding insects in the γ-subdivision of the
Proteobacteria
. The nucleotide compositions of the 16S rRNA gene from the leech symbionts were highly AT biased (53.7%).
Effect of
Weissella confusa
Strain PL9001 on the Adherence and Growth of
Helicobacter pylori
Authors: Nam Hyeran , Ha Misun , Bae On , Lee Yeonhee
Published: August 2002

Viable and nonviable
Weissella confusa
strain PL9001 inhibited the binding of
Helicobacter pylori
to human gastric-cell line MKN-45 cells by more than 90%. Spent culture supernatant of PL9001 rapidly decreased the viability of
H. pylori
, rupturing cell walls. The results suggest that PL9001 is a probiotic that can reduce the infectivity and persistence of
H. pylori
.
Infection of Mice with Aerosolized
Mycobacterium tuberculosis
: Use of a Nose-Only Apparatus for Delivery of Low Doses of Inocula and Design of an Ultrasafe Facility
Authors: Schwebach J. Reid , Chen Bing , Glatman-Freedman Aharona , Casadevall Arturo , McKinney John D. , Harb John L. , McGuire Patrick J. , Barkley W. Emmett , Bloom Barry R. , Jacobs William R.
Published: August 2002

Aerosolized delivery of virulent or hypervirulent
Mycobacterium tuberculosis
requires careful consideration of methodology and safety. To maximize safety, we installed a nose-only aerosol apparatus that can reproducibly deliver a low dose (<100 CFU per mouse) of
M. tuberculosis
in a carefully designed biohazard facility.
Potential for Misidentification of a Spore-Forming
Paenibacillus polymyxa
Isolate as an Endophyte by Using Culture-Based Methods
Authors: Bent Elizabeth , Chanway Christopher P.
Published: August 2002

While
Paenibacillus polymyxa
strain Pw-2 has been identified as an endophyte of lodgepole pine (M. Shishido, B. M. Loeb, and C. P. Chanway, Can. J. Microbiol. 41:707-713, 1995),
P. polymyxa
strain L6 has not, a distinction that could be explained by the differential abilities of these isolates to form spores, rather than the differential abilities to colonize the interior tissues of lodgepole pine. Chemical disinfection was used to destroy bacteria on the root exterior, but bacterial endospores are known for their ability to withstand chemical disinfection, and strain Pw-2 was found to produce 300 to 11,000 times more germinating endospores than strain L6 under the experimental conditions used by Shishido et al. (Can. J. Microbiol. 41:707-713, 1995). Attempts to identify strain Pw-2 within lodgepole pine root tissues by using confocal microscopy techniques failed. We discuss the possibility that spore-forming bacteria can be mistakenly identified as endophytes when culture-based methods alone are used.
Characterization of the Lipopolysaccharides and Capsules of
Shewanella
spp
Authors: Korenevsky Anton A. , Vinogradov Evgeny , Gorby Yuri , Beveridge Terry J.
Published: August 2002

Electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining and
1
H,
13
C, and
31
P-nuclear magnetic resonance (NMR) were used to detect and characterize the lipopolysaccharides (LPSs) of several
Shewanella
species. Many expressed only rough LPS; however, approximately one-half produced smooth LPS (and/or capsular polysaccharides). Some LPSs were affected by growth temperature with increased chain length observed below 25°C. Maximum LPS heterogeneity was found at 15 to 20°C. Thin sections of freeze-substituted cells revealed that
Shewanella oneidensis
,
S. algae
,
S. frigidimarina
, and
Shewanella
sp. strain MR-4 possessed either O-side chains or capsular fringes ranging from 20 to 130 nm in thickness depending on the species. NMR detected unusual sugars in
S. putrefaciens
CN32 and
S. algae
BrY
DL
. It is possible that the ability of
Shewanella
to adhere to solid mineral phases (such as iron oxides) could be affected by the composition and length of surface polysaccharide polymers. These same polymers in
S. algae
may also contribute to this opportunistic pathogen's ability to promote infection.
Gene Disruption by Homologous Recombination in the
Xylella fastidiosa
Citrus Variegated Chlorosis Strain
Authors: Gaurivaud Patrice , Souza Leonardo C. A. , Virgílio Andrea C. D. , Mariano Anelise G. , Palma Renê R. , Monteiro Patrícia B.
Published: August 2002

Mutagenesis by homologous recombination was evaluated in
Xylella fastidiosa
by using the
bga
gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of
bga
and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a
cvaB
mutant.
Quorum Sensing and Production of Autoinducer-2 in
Campylobacter
spp.,
Escherichia coli
O157:H7, and
Salmonella enterica
Serovar Typhimurium in Foods
Authors: Cloak Orla M. , Solow Barbara T. , Briggs Connie E. , Chen Chin-Yi , Fratamico Pina M.
Published: August 2002

Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that
Campylobacter jejuni
and
Campylobacter coli
possessed
luxS
, which is responsible for autoinducer-2 (AI-2) production. Using a
Vibrio harveyi
luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by
C. coli
,
C. jejuni
,
Salmonella enterica
serovar Typhimurium, and
Escherichia coli
O157:H7 under different conditions.
Enzymatic Degradation of Chlorodiamino-
s
-Triazine
Authors: Seffernick Jennifer L. , Shapir Nir , Schoeb Michael , Johnson Gilbert , Sadowsky Michael J. , Wackett Lawrence P.
Published: August 2002

2-Chloro-4,6-diamino-
s
-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-
s
-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by
N
-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.
Escherichia coli
O157 and Non-O157 Isolates Are More Susceptible to
l
-Lactate than to
d
-Lactate
Authors: McWilliam Leitch E. C. , Stewart C. S.
Published: August 2002

The antimicrobial effect of
l
-lactate was much greater than that of
d
-lactate over a range of concentrations for
Escherichia coli
O157 and non-O157 strains. Despite this, the intracellular pHs and membrane potentials of
l
-lactate- and
d
-lactate-treated cells were similar, suggesting that these factors are not involved in the antimicrobial action of
l
-lactate.
Comparison of Models Describing Light Dependence of N2Fixation in Heterocystous Cyanobacteria
Authors: Staal Marc , te Lintel Hekkert Sacco , Herman Peter , Stal Lucas J.
Published: August 2002

The abilities of four models to describe nitrogenase light-response curves were compared, using the heterocystous cyanobacteriumNodularia spumigenaand a cyanobacterial bloom from the Baltic Sea as examples. All tested models gave a good fit of the data, and the rectangular hyperbola model is recommended for fitting nitrogenase-light response curves. This model describes an enzymatic process, while the others are empirical. It was possible to convert the process parameters between the four models and compare N2fixation with photosynthesis. The physiological meanings of the process parameters are discussed and compared to those of photosynthesis.
Enhanced Degradation of an Endocrine-Disrupting Chemical, Butyl Benzyl Phthalate, byFusarium oxysporumf. sp.pisiCutinase
Authors: Kim Yang-Hoon , Lee Jeewon , Ahn Ji-Young , Gu Man Bock , Moon Seung-Hyeon
Published: August 2002

Compared to yeast esterase, fungal cutinase degraded butyl benzyl phthalate (BBP) far more efficiently; i.e., almost 60% of the BBP disappeared within 7.5 h. Also, the final chemical composition significantly depended on the enzyme used. Toxicity monitoring using bioluminescent bacteria showed that butyl methyl phthalate, a major product of degradation by esterase, was an oxidative toxic hazard.
Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Human Gut
Authors: Pereira Dora I. A. , Gibson Glenn R.
Published: August 2002

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found:
Lactobacillus fermentum
strains F53 and KC5b,
Bifidobacterium infantis
ATCC 15697,
Streptococcus bovis
ATCC 43143,
Enterococcus durans
DSM 20633,
Enterococcus gallinarum
, and
Enterococcus faecalis
. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate
L. fermentum
KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.
Diversity of
Sinorhizobium meliloti
from the Central Asian Alfalfa Gene Center
Authors: Roumiantseva Marina L. , Andronov Evgeny E. , Sharypova Larissa A. , Dammann-Kalinowski Tatjana , Keller Mathias , Young J. Peter W. , Simarov Boris V.
Published: August 2002

Sinorhizobium meliloti
was isolated from nodules and soil from western Tajikistan, a center of diversity of the host plants (
Medicago
,
Melilotus
, and
Trigonella
species). There was evidence of recombination, but significant disequilibrium, between and within the chromosome and megaplasmids. The most frequent alleles matched those in the published genome sequence.
Comparison between the Skin Snip Test and Simple Dot Blot Assay as Potential Rapid Assessment Tools for Onchocerciasis in the Postcontrol Era in Ghana
Authors: Guzmán G. E. , Awadzi K. , Opoku N. , Narayanan R. B. , Akuffo H. O.
Published: August 2002

Successful control of onchocerciasis through mass distribution of ivermectin needs to be coupled with reliable, sensitive, specific, yet affordable diagnostic methods to monitor and ensure the efficacy of such measures. The effort put into the development of diagnostic methods for onchocerciasis that can substitute for or work in combination with the present “gold standard,” the skin snip test, has resulted in the discovery of a number of immunogenic proteins with potential use as diagnostic tools in the postcontrol era. Most of these proteins have now been produced through recombinant DNA techniques. However, when costs are not a trivial issue, none of them have yet found their way into the areas where the disease still exists. In the present study, we have evaluated the performance of a simple dot blot assay which uses a mixture of native proteins designated PakF as a serious contender in the quest for a less invasive and more sensitive method to detectOnchocerca volvulusinfection in areas with diverse endemicities. Our results indicate that the assay we propose is more sensitive than the skin snip test and shows high specificity, both characteristics required for a suitable tool for the monitoring of onchocerciasis in the postcontrol era.
Calcium Phosphate Nanoparticles Induce Mucosal Immunity and Protection against Herpes Simplex Virus Type 2
Authors: He Qing , Mitchell Alaina , Morcol Tulin , Bell Steve J. D.
Published: August 2002

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.
Specificities and Opsonophagocytic Activities of Antibodies to Pneumococcal Capsular Polysaccharides in Sera of Unimmunized Young Children
Authors: Soininen Anu , Karpala Maijastiina , Wahlman Sirkka-Liisa , Lehtonen Hannele , Käyhty Helena
Published: August 2002

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.