bluuebunny/crossref_metadata_2025_split huggingface dataset:
Viral Activity in Two Contrasting Lake Ecosystems
Authors: Bettarel Yvan , Sime-Ngando Télesphore , Amblard Christian , Dolan John 
Published: May 2004

ABSTRACT

For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses. In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat. Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory. Overall, viral infection parameters were much less variable than bacterial production. We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October). FVIC values were significantly higher in Lake Pavin (mean [M] = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium
−1
, respectively. We detected no significant depth-related differences in FVIC or burst size. We found that in both lakes the removal of bacterial production by flagellate grazing (M
Pavin
= 37.7%, M
Aydat
= 18.5%) was nearly always more than the production removed by viral lysis (M
Pavin
= 16.2%, M
Aydat
= 19%) or ciliate grazing (M
Pavin
= 2.7%, M
Aydat
= 8.8%). However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat. In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers. Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system.
Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant,
Arthrobacter chlorophenolicus
A6
Authors: Backman Agneta , Maraha Ninwe , Jansson Janet K. 
Published: May 2004

ABSTRACT

Arthrobacter chlorophenolicus
A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the
gfp
gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [
luc
]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make
A. chlorophenolicus
A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.
Isolation, Characterization, and U(VI)-Reducing Potential of a Facultatively Anaerobic, Acid-Resistant Bacterium from Low-pH, Nitrate- and U(VI)-Contaminated Subsurface Sediment and Description of
Salmonella subterranea
sp. nov
Authors: Shelobolina Evgenya S. , Sullivan Sara A. , O'Neill Kathleen R. , Nevin Kelly P. , Lovley Derek R. 
Published: May 2004

ABSTRACT

A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 μm long and 0.7 to 0.9 μm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H
2
S production, and for gelatin hydrolysis. Strain FRCl was capable of using O
2
, NO
3
−
, S
2
O
3
2−
, fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4% similar to
Salmonella bongori
and 96.3% similar to
Enterobacter cloacae
. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus
Salmonella
and represents a new species,
Salmonella subterranea
sp. nov.
Metal Toxicity Affects Fungal and Bacterial Activities in Soil Differently
Authors: Rajapaksha R. M. C. P. , Tobor-Kapłon M. A , Bååth E. 
Published: May 2004

ABSTRACT
Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load.
Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of
Acinetobacter
sp. Strain ADP1
Authors: Parke Donna , Ornston L. Nicholas 
Published: May 2004

ABSTRACT

Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the
hca
genes in the nutritionally versatile, naturally transformable bacterium
Acinetobacter
sp. strain ADP1. A key step in the
hca
-encoded pathway is activation of the natural substrates caffeate,
p
-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by
hcaC
. As described in this paper,
Acinetobacter
cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates;
Escherichia coli
cells carrying a subclone with the
hcaC
gene are hydroxycinnamate sensitive as well. When the
hcaA
mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated
Acinetobacter
cells to caffeate,
p
-coumarate, or ferulate at 10
−6
M totally inhibited the growth of cells. The toxicity of
p
-coumarate and ferulate to a Δ
hcaA
strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an
Acinetobacter
strain blocked in
hcaA
, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of
hcaC
led to the development of a positive-selection strategy that targets mutations blocking HcaC. An
hcaC
mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.
Novel Polyketide Synthase from
Nectria haematococca
Authors: Graziani Stephane , Vasnier Christelle , Daboussi Marie-Josee 
Published: May 2004

ABSTRACT

We identified a polyketide synthase (PKS) gene,
pksN
, from a strain of
Nectria haematococca
by complementing a mutant unable to synthesize a red perithecial pigment.
pksN
encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: β-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The
pksN
product groups with the
Aspergillus nidulans
WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of
pksN
did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that
pksN
encodes a novel PKS required for the perithecial red pigment biosynthesis.
Survival and Dormancy ofMycobacterium aviumsubsp.paratuberculosisin theEnvironment
Authors: Whittington Richard J. , Marshall D. Jeff , Nicholls Paul J. , Marsh Ian B. , Reddacliff Leslie A. 
Published: May 2004

ABSTRACTThe survival ofMycobacterium aviumsubsp.paratuberculosiswas studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. Adps-like genetic element andrelA, which are involved in dormancy responses in other mycobacteria, are present in theM. aviumsubsp.paratuberculosisgenome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival ofM. aviumsubsp.paratuberculosisin the environment is finite, consistent with its taxonomic description as an obligate parasite of animals.
Studies of Single-Chain Antibody Expression in Quiescent
Escherichia coli
Authors: Mukherjee K. J. , Rowe D. C. D. , Watkins N. A. , Summers D. K. 
Published: May 2004

ABSTRACT

Quiescent
Escherichia coli
cells are generated by overexpressing the Rcd transcript in an
hns
-
205
mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λP
L
under the control of the
c
I857 temperature-sensitive repressor, while the second expresses Rcd from λP
R
. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD
600
] of 3.5) was 37 mg liter
−1
, compared to a maximum of 13 mg liter
−1
in the control culture (final OD
600
of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD
600
of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter
−1
. A control culture with a similar feed reached an OD
600
of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter
−1
. Quiescent cells at an OD
600
of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter
−1
was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent
E. coli
cultures that are capable of efficient synthesis, folding, and export of proteins.
Genomic Diversity of
Erwinia carotovora
subsp.
carotovora
and Its Correlation with Virulence
Authors: Mee-Ngan Yap , Barak Jeri D. , Charkowski Amy O. 
Published: May 2004

ABSTRACT

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen
Erwinia carotovora
subsp.
carotovora
. The results obtained with each method showed that
E. carotovora
subsp.
carotovora
strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial
mdh
sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for
Escherichia coli.
Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven
rrn
operons in all
E. carotovora
subsp.
carotovora
strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant polymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain
E. carotovora
subsp.
carotovora
71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPP17, failed to oxidize cellobiose and was missing several
hrp
and
hrc
genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen.
Particle-Based Multidimensional Multispecies Biofilm Model
Authors: Picioreanu Cristian , Kreft Jan-Ulrich , van Loosdrecht Mark C. M. 
Published: May 2004

ABSTRACT
In this paper we describe a spatially multidimensional (two-dimensional [2-D] and three-dimensional [3-D]) particle-based approach for modeling the dynamics of multispecies biofilms growing on multiple substrates. The model is based on diffusion-reaction mass balances for chemical species coupled with microbial growth and spreading of biomass represented by hard spherical particles. Effectively, this is a scaled-up version of a previously proposed individual-based biofilm model. Predictions of this new particle-based model were quantitatively compared with those obtained with an established one-dimensional (1-D) multispecies model for equivalent problems. A nitrifying biofilm containing aerobic ammonium and nitrite oxidizers, anaerobic ammonium oxidizers, and inert biomass was chosen as an example. The 2-D and 3-D models generally gave the same results. If only the average flux of nutrients needs to be known, 2-D and 1-D models are very similar. However, the behavior of intermediates, which are produced and consumed in different locations within the biofilm, is better described in 2-D and 3-D models because of the multidirectional concentration gradients. The predictions of 2-D or 3-D models are also different from those of 1-D models for slowly growing or minority species in the biofilm. This aspect is related to the mechanism of biomass spreading or advection implemented in the models and should receive more attention in future experimental studies.
Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement
Authors: Garrett James B. , Kretz Keith A. , O'Donoghue Eileen , Kerovuo Janne , Kim William , Barton Nelson R. , Hazlewood Geoffrey P. , Short Jay M. , Robertson Dan E. , Gray Kevin A. 
Published: May 2004

ABSTRACT

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (
myo
-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the
appA
gene from
Escherichia coli
, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the
appA
parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.
Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection
Authors: Vora Gary J. , Meador Carolyn E. , Stenger David A. , Andreadis Joanne D. 
Published: May 2004

ABSTRACT

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, φ29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic
Escherichia coli
O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and φ29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.
Comparative Analysis of the Conventional and Novel
pmo
(Particulate Methane Monooxygenase) Operons from
Methylocystis
Strain SC2
Authors: Ricke Peter , Erkel Christoph , Kube Michael , Reinhardt Richard , Liesack Werner 
Published: May 2004

ABSTRACT

In addition to the conventional
pmoA
gene (
pmoA1
) encoding the active site polypeptide of particulate methane monooxygenase, a novel
pmoA
gene copy (
pmoA2
) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the
pmoA1
and
pmoA2
gene copies in the type II MOB
Methylocystis
strain SC2 are each part of a complete
pmoCAB
gene cluster (
pmoCAB1
,
pmoCAB2
). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either
pmoCAB1
or
pmoCAB2
were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The
amoCAB
gene cluster encodes ammonia monooxygenase and is evolutionarily related to
pmoCAB
. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that
pmoCAB2
encodes a functional equivalent of PmoCAB1. In experiments in which we used 5′ rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of
pmoC1
and
pmoC2
, respectively. Immediately upstream of the transcriptional start sites of both
pmoCAB1
and
pmoCAB2
, sequence motifs similar to
Escherichia coli
σ
70
promoters were identified.