bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes
Authors: Zahn James A. , Higgs Richard E. , Hilton Matthew D.
Published: July 2002

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired
t
test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography–ES-MS–evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.
Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)
Authors: Ravenschlag Katrin , Sahm Kerstin , Amann Rudolf
Published: July 2002

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the
Cytophaga-Flavobacterium
cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order
Planctomycetales
accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.
Comparative Study of Bacterial Groups within the Human Cecal and Fecal Microbiota
Authors: Marteau Philippe , Pochart Philippe , Doré Joël , Béra-Maillet Christel , Bernalier Annick , Corthier Gérard
Published: July 2002

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and
Bacteroides
were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria,
Bacteroides
, and members of the
Clostridium coccoides
group and
Clostridium leptum
subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.
Effects of Low Temperature and Freeze-Thaw Cycles on Hydrocarbon Biodegradation in Arctic Tundra Soil
Authors: Eriksson Mikael , Ka Jong-Ok , Mohn William W.
Published: July 2002

Degradation of petroleum hydrocarbons was monitored in microcosms with diesel fuel-contaminated Arctic tundra soil incubated for 48 days at low temperatures (−5, 0, and 7°C). An additional treatment was incubation for alternating 24-h periods at 7 and −5°C. Hydrocarbons were biodegraded at or above 0°C, and freeze-thaw cycles may have actually stimulated hydrocarbon biodegradation. Total petroleum hydrocarbon (TPH) removal over 48 days in the 7, 0, and 7 and −5°C treatments, respectively, was 450, 300, and 600 μg/g of soil. No TPH removal was observed at −5°C. Total carbon dioxide production suggested that TPH removal was due to biological mineralization. Bacterial metabolic activity, indicated by RNA/DNA ratios, was higher in the middle of the experiment (day 21) than at the start, in agreement with measured hydrocarbon removal and carbon dioxide production activities. The total numbers of culturable heterotrophs and of hydrocarbon degraders did not change significantly over the 48 days of incubation in any of the treatments. At the end of the experiment, bacterial community structure, evaluated by ribosomal intergenic spacer length analysis, was very similar in all of the treatments but the alternating 7 and −5°C treatment.
Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms
Authors: Stoodley Paul , Wilson Suzanne , Hall-Stoodley Luanne , Boyle John D. , Lappin-Scott Hilary M. , Costerton J. W.
Published: July 2002

Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.
Incidence, Distribution, and Spread of Tetracycline Resistance Determinants and Integron-Associated Antibiotic Resistance Genes among Motile Aeromonads from a Fish Farming Environment
Authors: Schmidt Anja S. , Bruun Morten S. , Dalsgaard Inger , Larsen Jens L.
Published: July 2002

A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908–4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had “empty” integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (
n
= 6), or E (
n
= 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to
Escherichia coli
recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly
tetA
positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and
tetA
among the OTC-resistant aeromonads,
tetE
and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.
Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms
Authors: Derakshani Manigee , Lukow Thomas , Liesack Werner
Published: July 2002

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus
Pirellula
within the
Planctomycetales
, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order
Planctomycetales
, while one lineage was affiliated with the division
Verrucomicrobia
and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.
α- and β-
Proteobacteria
Control the Consumption and Release of Amino Acids on Lake Snow Aggregates
Authors: Schweitzer Bernhard , Huber Ingrid , Amann Rudolf , Ludwig Wolfgang , Simon Meinhard
Published: July 2002

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4′,6′-diamidino-2-phenylindole) were detected as
Bacteria
by the probe EUB338. At a depth of 25 m, 10.5% ± 7.9% and 14.2% ± 10.2% of the DAPI cell counts were detected by probes specific for α- and β-
Proteobacteria
. These proportions increased to 12.0% ± 3.3% and 54.0% ± 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% ± 1.4% and 41.1% ± 8.4%, indicating a clear dominance of β-
Proteobacteria
at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a
Cytophaga/Flavobacteria
-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. γ-
Proteobacteria
always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to
Sphingomonas
species and
Brevundimonas diminuta
, we identified between 16 and 60% of the α-
Proteobacteria
. In addition, with three probes highly specific for close relatives of the β-
Proteobacteria Duganella zoogloeoides
(formerly
Zoogloea ramigera
),
Acidovorax facilis
, and
Hydrogenophaga palleroni
, bacteria common in activated sludge, 42 to 70% of the β-
Proteobacteria
were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of β-
Proteobacteria
. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of α- and β-
Proteobacteria
, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.
Bacterial Diversity and Community Structure in an Aerated Lagoon Revealed by Ribosomal Intergenic Spacer Analyses and 16S Ribosomal DNA Sequencing
Authors: Yu Zhongtang , Mohn William W.
Published: July 2002

We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35°C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus
Methylobacillus
, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus
Azospirillum
.
Dual-Bioaugmentation Strategy To Enhance Remediation of Cocontaminated Soil
Authors: Roane T. M. , Josephson K. L. , Pepper I. L.
Published: July 2002

Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 μg ml
−1
, these isolates represented the common soil genera
Arthrobacter
,
Bacillus,
and
Pseudomonas
. Isolates
Pseudomonas
sp. strain H1 and
Bacillus
sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates
Arthrobacter
strain D9 and
Pseudomonas
strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-μg ml
−1
2,4-D by the cadmium-sensitive 2,4-D degrader
Ralstonia eutropha
JMP134. Degradation occurred in the presence of up to 24 μg of cadmium ml
−1
in pure culture and up to 60 μg of cadmium g
−1
in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate
Pseudomonas
strain H1 enhanced degradation of 2,4-D in reactors inoculated with
R. eutropha
JMP134 in the presence of 60 μg of cadmium g
−1
. Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.
Examination of Recovery In Vitro and In Vivo of Nonculturable
Escherichia coli
O157:H7
Authors: Kolling Glynis L. , Matthews Karl R.
Published: July 2002

Escherichia coli
O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable
E. coli
O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved
E. coli
O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable
E. coli
O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable
E. coli
O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced)
E. coli
O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.
UDP-
N
-Acetylglucosamine 4-Epimerase Activity Indicates the Presence of
N
-Acetylgalactosamine in Exopolysaccharides of
Streptococcus thermophilus
Strains
Authors: Degeest Bart , Vaningelgem Frederik , Laws Andrew P. , De Vuyst Luc
Published: July 2002

The monomer composition of the exopolysaccharides (EPS) produced by
Streptococcus thermophilus
LY03 and
S. thermophilus
Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and
N
-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-
N
-acetylglucosamine 4-epimerase, converting UDP-
N
-acetylglucosamine into UDP-
N
-acetylgalactosamine, is responsible for the presence of
N
-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-
N
-acetylglucosamine 4-epimerase was higher in both
S. thermophilus
strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during
S. thermophilus
Sfi20 fermentations. A correlation between activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for
S. thermophilus
LY03, although
S. thermophilus
Sfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.
Bacteriophage Latent-Period Evolution as a Response to Resource Availability
Authors: Abedon Stephen T. , Herschler Troy D. , Stopar David
Published: July 2002

Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models—in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy—can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (∼108cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (107cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 108cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 105cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.
Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland
Authors: Marteinsson Viggó Thór , Hauksdóttir Sigurbjörg , Hobel Cédric F. V. , Kristmannsdóttir Hrefna , Hreggvidsson Gudmundur Oli , Kristjánsson Jakob K.
Published: July 2002

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C).
Archaea
and
Bacteria
were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the
Archaea
and
Bacteria
domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with
Desulfurococcales
relatives. Two strains belonging to
Desulfurococcus mobilis
and to the
Thermus
/
Deinococcus
group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.
Electrotransformation of
Lactobacillus delbrueckii
subsp.
bulgaricus
and
L. delbrueckii
subsp.
lactis
with Various Plasmids
Authors: Serror Pascale , Sasaki Takashi , Ehrlich S. Dusko , Maguin Emmanuelle
Published: July 2002

We describe, for the first time, a detailed electroporation procedure for
Lactobacillus delbrueckii
. Three
L. delbrueckii
strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10
4
transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in
L. delbrueckii
and integrated an integrative vector based on phage integrative elements into the
L. delbrueckii
subsp.
bulgaricus
chromosome. These vectors provide a good basis for developing molecular tools for
L. delbrueckii
and open the field of genetic studies in
L. delbrueckii
.
Hindgut Fermentation in Three Species of Marine Herbivorous Fish
Authors: Mountfort Douglas O. , Campbell Jane , Clements Kendall D.
Published: July 2002

Symbioses with gut microorganisms provides a means by which terrestrial herbivores are able to obtain energy. These microorganisms ferment cell wall materials of plants to short-chain fatty acids (SCFA), which are then absorbed and used by the host animal. Many marine herbivorous fishes contain SCFA (predominantly acetate) in their hindgut, indicative of gut microbial activity, but rates of SCFA production have not been measured. Such information is an important prerequisite to understanding the contribution that gut microorganisms make in satisfying the energy needs of the fish. We have estimated the rates of acetate production in the gut of three species of temperate marine herbivorous fish from northeastern New Zealand:
Kyphosus sydneyanus
(family Kyphosidae),
Odax pullus
(family Odacidae), and
Aplodactylus arctidens
(family Aplodactylidae). Ex vivo preparations of freshly caught fish were maintained with their respiratory and circulatory systems intact, radiolabeled acetate was injected into ligated hindgut sections, and gut fluid was sampled at 20-min intervals for 2 h. Ranges for acetate turnover in the hindguts of the studied species were determined from the slope of plots as the log of the specific radioactivity of acetate versus time and pool size, expressed on a nanomole per milliliter per minute basis. Values were 450 to 570 (
K. sydneyanus
), 373 to 551 (
O. pullus
), and 130 to 312 (
A. arctidens
). These rates are comparable to those found in the guts of herbivorous reptiles and mammals. To determine the contribution of metabolic pathways to the fate of acetate, rates of sulfate reduction and methanogenesis were measured in the fore-, mid-, and hindgut sections of the three fish species. Both rates increased from the distal to proximal end of the hindgut, where sulfate reduction accounted for only a small proportion (<5%) of acetate methyl group transformed to CO
2
, and exceeded methanogenesis from acetate by >50-fold. When gut size was taken into account, acetate uptake from the hindgut of the fish species, determined on a millimole per day per kilogram of body weight basis, was 70 (
K. sydneyanus
), 18 (
O. pullus
), and 10 (
A. arctidens
).
Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer
Authors: Kleikemper Jutta , Schroth Martin H. , Sigler William V. , Schmucki Martina , Bernasconi Stefano M. , Zeyer Josef
Published: July 2002

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO
4
2−
reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day
−1
for sulfate reduction and from 0.13 to 0.60 day
−1
for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.
EglC, a New Endoglucanase from
Aspergillus niger
with Major Activity towards Xyloglucan
Authors: Hasper Alinda A. , Dekkers Ester , van Mil Marc , van de Vondervoort Peter J. I. , de Graaff Leo H.
Published: July 2002

A novel gene,
eglC
, encoding an endoglucanase, was cloned from
Aspergillus niger
. Transcription of
eglC
is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from
Trichoderma reesei
was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from
A. niger
, EglA and EglB, which exhibit the greatest activity towards β-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by
A. niger
.
Identification ofListeria monocytogenesGenes Expressed in Response to Growth at Low Temperature
Authors: Liu Siqing , Graham James E. , Bigelow Lance , Morse Philip D. , Wilkinson Brian J.
Published: July 2002

Listeria monocytogenesis a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in theL. monocytogenesstrain EGD-e genome were obtained by SCOTS. These included cDNAs forL. monocytogenesgenes involved in previously described cold-adaptive responses (flaAandflp), regulatory adaptive responses (rpoN,lhkA,yycJ,bglG,adaB, andpsr), general microbial stress responses (groEL,clpP,clpB,flp, andtrxB), amino acid metabolism (hisJ,trpG,cysS, andaroA), cell surface alterations (fbp,psr, andflaA), and degradative metabolism (eutB,celD, andmleA). Four additional cDNAs were obtained corresponding to genes potentially unique toL. monocytogenesand showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated thatL. monocytogenesacclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.
Formation of Protoplasts from Cultured Tobacco Cells and
Arabidopsis thaliana
by the Action of Cellulosomes and Pectate Lyase from
Clostridium cellulovorans
Authors: Tamaru Yutaka , Ui Sadaharu , Murashima Koichiro , Kosugi Akihiko , Chan Helen , Doi Roy H. , Liu Bo
Published: July 2002

The crude culture supernatants from
Clostridium cellulovorans
were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and
Arabidopsis thaliana
. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from
C. cellulovorans
was more effective than those of commercial enzymes based on protein content.
Empirical and Theoretical Bacterial Diversity in Four Arizona Soils
Authors: Dunbar John , Barns Susan M. , Ticknor Lawrence O. , Kuske Cheryl R.
Published: July 2002

Understanding patterns of biodiversity in microbial communities is severely constrained by the difficulty of adequately sampling these complex systems. We illustrate the problem with empirical data from small surveys (200-member 16S rRNA gene clone libraries) of four bacterial soil communities from two locations in Arizona. Among the four surveys, nearly 500 species-level groups (

Dunbar et al.,
Appl. Environ. Microbiol.
65
:
662
-1669,
1999

) and 21 bacterial divisions were documented, including four new candidate divisions provisionally designated SC1, SC2, SC3, and SC4. We devised a simple approach to constructing theoretical null models of bacterial species abundance. These null models provide, for the first time, detailed descriptions of soil bacterial community structure that can be used to guide experimental design. Models based on a lognormal distribution were consistent with the observed sizes of the four communities and the richness of the clone surveys. Predictions from the models showed that the species richness of small surveys from complex communities is reproducible, whereas the species composition is not. By using the models, we can now estimate the required survey scale to document specified fractions of community diversity. For example, documentation of half the species in each model community would require surveys of 16,284 to 44,000 individuals. However, quantitative comparisons of half the species in two communities would require surveys at least 10-fold larger for each community.
Antigenic and Genetic Characterization of Lipoprotein LppQ fromMycoplasma mycoidessubsp.mycoidesSC
Authors: Abdo El-Mostafa , Nicolet Jacques , Frey Joachim
Published: July 2002

Lipoprotein LppQ, a predominant 48-kDa antigen, and its corresponding gene,lppQ, were characterized inMycoplasma mycoidessubsp.mycoidesSC, the etiological agent of contagious bovine pleuropneumonia. ThelppQgene is specific toM. mycoidessubsp.mycoidesSC and was found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccinal strains. LppQ is encoded as a precursor with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site. The leader sequence shows significant prominent transmembrane helix structure with a predicted outside-to-inside helix formation capacity. The N-terminal domain of the mature LppQ was shown to be surface exposed. It induced a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. The C-terminal domain of LppQ possesses an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, which have a pore formation potential. A recombinant peptide representing the N-terminal domain of LppQ was obtained by site-directed mutagenesis of nineMycoplasma-specific TGA (Trp) codons into universal TGG (Trp) codons and expression inEscherichia colihosts. It was used for serodetection of cattle infected withM. mycoidessubsp.mycoidesSC, in which it was detected postinfection for significantly longer than conventional serological test reactions.
Helicobacter pylori
Intrafamilial Infections: Change in Source of Infection of a Child from Father to Mother after Eradication Therapy
Authors: Taneike Ikue , Tamura Yukiko , Shimizu Toshiaki , Yamashiro Yuichiro , Yamamoto Tatsuo
Published: July 2002

Biopsy specimens of the antrum and corpus were obtained from four
Helicobacter pylori
-infected members of a family and from the same boy (son 1) in whom the infection reappeared after simultaneous successful eradication treatment of three family members, excluding the mother. A total of 18 to 60
H. pylori
isolates were obtained from each specimen and subjected to rRNA gene restriction pattern analysis. The father's isolates and the initial isolates from son 1 showed the same
Hin
dIII type, which was divided into three
Hae
III subtypes. Isolates from the mother and a brother (son 2) and posttreatment isolates from son 1 showed a distinct
Hin
dIII type (with one minor subtype), which was divided into six
Hae
III subtypes. All subtypes of the initial isolates from son 1 were present in the father's isolates, and all subtypes of the posttreatment isolates from son 1 were present in the mother's isolates but not in son 2's. Electron microscopic analysis of the biopsy specimens demonstrated extremely high levels of
H. pylori
colonization in the father's gastric mucosa.
H. pylori
adherence with a ruffle formation was also demonstrated. The findings suggest that son 1 was infected initially with the
H. pylori
strain of the father and son 2 was infected with the
H. pylori
strain of the mother and that after eradication therapy son 1 was reinfected with the
H. pylori
strain of the mother, who did not undergo eradication therapy.
Comparison of Chemicon SimulFluor Direct Fluorescent Antibody Staining with Cell Culture and Shell Vial Direct Immunoperoxidase Staining for Detection of Herpes Simplex Virus and with Cytospin Direct Immunofluorescence Staining for Detection of Varicella-Zoster Virus
Authors: Chan Edward L. , Brandt Ken , Horsman Greg B.
Published: July 2002

A new rapid direct immunofluorescence assay, the SimulFluor direct fluorescent-antibody (DFA) assay, which can simultaneously detect herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), was evaluated in comparison with our current standard procedures of (i) shell vial direct immunoperoxidase (shell vial IP) staining and cell culture for detection of HSV and (ii) cytospin DFA staining for VZV detection. A total of 517 vesicular, oral, genital, and skin lesion specimens were tested by all three procedures. For HSV detection, the SimulFluor DFA assay had an overall sensitivity, specificity, positive predictive value, and negative predictive value of 80.0, 98.3, 92.3, and 95.1%, respectively, when compared to culture. Shell vial IP staining had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.6, 100, 100, and 96.9%, respectively, when compared with cell culture. The SimulFluor DFA assay, however, offers same-day, 1.5-hours results versus a 1- to 2-day wait for shell vial IP staining results and a 1- to 6-day wait for culture results for HSV. For VZV detection SimulFluor DFA staining detected 27 positive specimens as compared to 31 by our standard cytospin DFA technique—a correlation of 87.1%. A positive SimulFluor reaction for VZV is indicated by yellow-gold fluorescence compared to the bright apple-green fluorescence observed by cytospin DFA staining. There is no difference in turnaround time between the two assays. The SimulFluor DFA assay is a rapid immunofluorescence assay that can detect 80% of the HSV-positive specimens and 87% of the VZV-positive specimens with a 1.5-h turnaround time.
Selective In Vivo Depletion of CD4+T Lymphocytes with Anti-CD4 Monoclonal Antibody during Acute Infection of Calves withAnaplasma marginale
Authors: Valdez Reginald A. , McGuire Travis C. , Brown Wendy C. , Davis William C. , Jordan Jeffrey M. , Knowles Donald P.
Published: July 2002

To investigate the in vivo role of CD4+T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4+T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain ofAnaplasma marginalein two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3+CD4+and CD4+CD45R+(naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection withA. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginaleantibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginalemajor surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4+-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4+T lymphocytes would abrogate resistance to acute infection.
Development, Evaluation, and Application of Lateral-Flow Immunoassay (Immunochromatography) for Detection of Rotavirus in Bovine Fecal Samples
Authors: Al-Yousif Yousif , Anderson Joe , Chard-Bergstrom Cindy , Kapil Sanjay
Published: July 2002

A lateral-flow immunoassay (LFT) was developed to detect bovine rotavirus in fecal samples. Using samples (n= 74) from diarrheic calves, a comparison of the LFT with a commercial latex agglutination test (LAT) and transmission electron microscopy (EM) was conducted. When EM was used as the reference method, initial studies of 29 samples indicated 70 and 80% sensitivities of the LFT and LAT, respectively, with both being 100% specific. When the LAT was the reference test, the LFT was 75% sensitive and 91% specific. Additional specimens (n= 45) were tested by the LFT and LAT alone, and results were identical for both methods.
Lung Infections Associated with Cystic Fibrosis
Authors: Lyczak Jeffrey B. , Cannon Carolyn L. , Pier Gerald B.
Published: July 2002

While originally characterized as a collection of related syndromes, cystic fibrosis (CF) is now recognized as a single disease whose diverse symptoms stem from the wide tissue distribution of the gene product that is defective in CF, the ion channel and regulator, cystic fibrosis transmembrane conductance regulator (CFTR). Defective CFTR protein impacts the function of the pancreas and alters the consistency of mucosal secretions. The latter of these effects probably plays an important role in the defective resistance of CF patients to many pathogens. As the modalities of CF research have changed over the decades from empirical histological studies to include biophysical measurements of CFTR function, the clinical management of this disease has similarly evolved to effectively address the ever-changing spectrum of CF-related infectious diseases. These factors have led to the successful management of many CF-related infections with the notable exception of chronic lung infection with the gram-negative bacterium Pseudomonas aeruginosa. The virulence of P. aeruginosa stems from multiple bacterial attributes, including antibiotic resistance, the ability to utilize quorum-sensing signals to form biofilms, the destructive potential of a multitude of its microbial toxins, and the ability to acquire a mucoid phenotype, which renders this microbe resistant to both the innate and acquired immunologic defenses of the host.
Interleukin-4 (IL-4) and IL-13 Signaling Pathways Do Not RegulateBorrelia burgdorferi-Induced Arthritis in Mice: IgG1 Is Not Required for Host Control of Tissue Spirochetes
Authors: Potter Melissa R. , Noben-Trauth Nancy , Weis John H. , Teuscher Cory , Weis Janis J.
Published: July 2002

Previous studies have suggested that interleukin-4 (IL-4) has a protective effect in host defense toBorrelia burgdorferiinfection, both in limiting the severity of arthritis and in controlling spirochete numbers in tissues, and a mapping study revealed suggestive linkage to a cluster of genes on mouse chromosome 11, including the genes for IL-4 and IL-13. In contrast, other studies have questioned the importance of IL-4. In this study the involvement of IL-4 in murine Lyme disease was examined in C57BL/6J and BALB/cJ mice with targeted disruptions in the IL-4 gene, the IL-4Rα chain gene, or both. A spectrum of arthritis severity was seen in BALB/cJ mice, and ablation of IL-4, IL-4Rα, or both had no effect on the overall severity of arthritis as determined by joint swelling and histopathology. Wild-type C57BL/6J mice exhibited mild to moderate arthritis, and ablation of IL-4 again had no effect on arthritis severity. IL-4- and IL-4Rα-deficient mice produced extremely low levels of immunoglobulin G1 (IgG1) and showed increased production of IgG2b. This shift in immunoglobulin isotype had no effect on the host's ability to control spirochete growth in either strain of mouse, as determined by PCR detection ofB. burgdorferiDNA from heart and ankle tissues. In summary, the IL-4-IL-4Rα pathway, including IL-13 signaling, neither limits arthritis severity nor is required for control of spirochete growth duringB. burgdorferiinfection of mice. Furthermore, the IgG1 isotype is not required to controlB. burgdorfericell numbers in tissues. These findings suggest the host defense againstB. burgdorferiinfection is not dependent on the Th1-Th2 paradigm of T-cell responses.