bluuebunny/crossref_metadata_2025_split huggingface dataset:
Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of
Acidianus tengchongensis
Authors: Chen Zhi-Wei , Jiang Cheng-Ying , She Qunxin , Liu Shuang-Jiang , Zhou Pei-Jin 
Published: February 2005

ABSTRACT

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C
31
and C
101
-X-X-C
104
; numbering according to the
Acidianus tengchongensis
numbering system). The thio-modifying reagent
N
-ethylmaleimide and Zn
2+
strongly inhibited the activities of the SORs of
A. tengchongensis
, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C
31
and C
101
-X-X-C
104
, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of
A. tengchongensis
. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in
A. tengchongensis
.
Identification of Cyanophage Ma-LBP and Infection of the Cyanobacterium
Microcystis aeruginosa
from an Australian Subtropical Lake by the Virus
Authors: Tucker Stephen , Pollard Peter 
Published: February 2005

ABSTRACT

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium
Microcystis aeruginosa
(host).
M. aeruginosa
was isolated, cultured, and characterized from a subtropical monomictic lake—Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the
M. aeruginosa
host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h
−1
; generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of
M. aeruginosa
cell lysis (
r
2
= 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the
Podoviridiae
group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 × 10
4
cyanophage · ml
−1
, representing 0.23% of the natural viral population of 2.46 × 10
7
· ml
−1
. Our results showed that this cyanophage could be a major natural control mechanism of
M. aeruginosa
abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.
Fate of Pathogens Present in Livestock Wastes Spread onto Fescue Plots
Authors: Hutchison Mike L. , Walters Lisa D. , Moore Tony , Thomas D. John I. , Avery Sheryl M. 
Published: February 2005

ABSTRACTFecal wastes from a variety of farmed livestock were inoculated with livestock isolates ofEscherichia coliO157,Listeria monocytogenes,Salmonella,Campylobacter jejuni, andCryptosporidium parvumoocysts at levels representative of the levels found in naturally contaminated wastes. The wastes were subsequently spread onto a grass pasture, and the decline of each of the zoonotic agents was monitored over time. There were no significant differences among the decimal reduction times for the bacterial pathogens. The mean bacterial decimal reduction time was 1.94 days. A range of times between 8 and 31 days for a 1-log reduction inC. parvumlevels was obtained, demonstrating that the protozoans were significantly more hardy than the bacteria. Oocyst recovery was more efficient from wastes with lower dry matter contents. The levels of most of the zoonotic agents had declined to below detectable levels by 64 days. However, for some waste types, 128 days was required for the complete decline ofL. monocytogeneslevels. We were unable to find significant differences between the rates of pathogen decline in liquid (slurry) and solid (farmyard manure) wastes, although concerns have been raised that increased slurry generation as a consequence of more intensive farming practices could lead to increased survival of zoonotic agents in the environment.
Relationship of Temporal and Spatial Variabilities of Ammonia-Oxidizing Bacteria to Nitrification Rates in Monterey Bay, California
Authors: O'Mullan G. D. , Ward B. B. 
Published: February 2005

ABSTRACTTemporal and spatial dynamics of ammonia-oxidizing bacteria (AOB) were examined using genes encoding 16S rRNA and ammonia monooxygenase subunit A (AmoA) in Monterey Bay, Calif. Samples were collected from three depths in the water column on four dates at one mid-bay station. Diversity estimators for the two genes showed a strong positive correlation, indicating that overlapping bacterial populations had been sampled by both sets of clone libraries. Some samples that were separated by only 15 m in depth had less genetic similarity than samples that were collected from the same depth months apart. Clone libraries from the Monterey Bay AOB community were dominated byNitrosospira-like sequences and clearly differentiated from the adjacent AOB community in Elkhorn Slough. Many Monterey Bay clones clustered with previously identified 16S rRNA andamoAgroups composed entirely of marine sequences, supporting the hypothesis that these groups are specific to the marine environment and are dominant marine AOB. In addition, novel, phylogenetically distinct groups of AOB sequences were identified and compared to sequences in the database. Only one cluster of gammaproteobacterial AOB was detected using 16S rRNA genes. Although significant genetic variation was detected in AOB populations from both vertical and temporal samples, no significant correlation was detected between diversity and environmental variables or the rate of nitrification.
Accumulation and Fate of Microorganisms and Microspheres in Biofilms Formed in a Pilot-Scale Water Distribution System
Authors: Långmark Jonas , Storey Michael V. , Ashbolt Nicholas J. , Stenström Thor-Axel 
Published: February 2005

ABSTRACT

The accumulation and fate of model microbial “pathogens” within a drinking-water distribution system was investigated in naturally grown biofilms formed in a novel pilot-scale water distribution system provided with chlorinated and UV-treated water. Biofilms were exposed to 1-μm hydrophilic and hydrophobic microspheres,
Salmonella
bacteriophages 28B, and
Legionella pneumophila
bacteria, and their fate was monitored over a 38-day period. The accumulation of model pathogens was generally independent of the biofilm cell density and was shown to be dependent on particle surface properties, where hydrophilic spheres accumulated to a larger extent than hydrophobic ones. A higher accumulation of culturable legionellae was measured in the chlorinated system compared to the UV-treated system with increasing residence time. The fate of spheres and fluorescence in situ hybridization-positive legionellae was similar and independent of the primary disinfectant applied and water residence time. The more rapid loss of culturable legionellae compared to the fluorescence in situ hybridization-positive legionellae was attributed to a loss in culturability rather than physical desorption. Loss of bacteriophage 28B plaque-forming ability together with erosion may have affected their fate within biofilms in the pilot-scale distribution system. The current study has demonstrated that desorption was one of the primary mechanisms affecting the loss of microspheres, legionellae, and bacteriophage from biofilms within a pilot-scale distribution system as well as disinfection and biological grazing. In general, two primary disinfection regimens (chlorination and UV treatment) were not shown to have a measurable impact on the accumulation and fate of model microbial pathogens within a water distribution system.
Kinetic Studies and Biochemical Pathway Analysis of Anaerobic Poly-(
R
)-3-Hydroxybutyric Acid Synthesis in
Escherichia coli
Authors: Carlson Ross , Wlaschin Aaron , Srienc Friedrich 
Published: February 2005

ABSTRACT

Poly-(
R
)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant
Escherichia coli
. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 ± 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study
E. coli
network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in
E. coli
is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.
Effects of Diverse Environmental Conditions on φLC3 Prophage Stability in
Lactococcus lactis
Authors: Lunde Merete , Aastveit Are Halvor , Blatny Janet Martha , Nes Ingolf F. 
Published: February 2005

ABSTRACT

The effects of various growth conditions on spontaneous φLC3 prophage induction in
Lactococcus lactis
subsp.
cremoris
IMN-C1814 was analyzed with a half fraction of a 4
4
factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous φLC3 induction was determined by quantitative analyses of restored DNA attachment sites (
attB
) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected φLC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous φLC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.
Tetracycline-Dependent Conditional Gene Knockout in
Bacillus subtilis
Authors: Kamionka Annette , Bertram Ralph , Hillen Wolfgang 
Published: February 2005

ABSTRACT

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in
Bacillus subtilis
. A chromosomally located gene is controlled by one or two
tet
operators. The precise adjustment of regulatory windows is accomplished by adjusting
tetR
or
revtetR
expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 μM anhydrotetracycline obtained with a
Pr-xylA-tetR
fusion. Reversible 500-fold gene knockouts were obtained in
B. subtilis
after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.
Escherichia coli
Behavior in the Presence of Organic Matter Released by Algae Exposed to Water Treatment Chemicals
Authors: Bouteleux C. , Saby S. , Tozza D. , Cavard J. , Lahoussine V. , Hartemann P. , Mathieu L. 
Published: February 2005

ABSTRACT

When exposed to oxidation, algae release dissolved organic matter with significant carbohydrate (52%) and biodegradable (55 to 74%) fractions. This study examined whether algal organic matter (AOM) added in drinking water can compromise water biological stability by supporting bacterial survival.
Escherichia coli
(1.3 × 10
5
cells ml
−1
) was inoculated in sterile dechlorinated tap water supplemented with various qualities of organic substrate, such as the organic matter coming from chlorinated algae, ozonated algae, and acetate (model molecule) to add 0.2 ± 0.1 mg of biodegradable dissolved organic carbon (BDOC) liter
−1
. Despite equivalent levels of BDOC,
E. coli
behavior depended on the source of the added organic matter. The addition of AOM from chlorinated algae led to an
E. coli
growth equivalent to that in nonsupplemented tap water; the addition of AOM from ozonated algae allowed a 4- to 12-fold increase in
E. coli
proliferation compared to nonsupplemented tap water. Under our experimental conditions, 0.1 mg of algal BDOC was sufficient to support
E. coli
growth, whereas the 0.7 mg of BDOC liter
−1
initially present in drinking water and an additional 0.2 mg of BDOC acetate liter
−1
were not sufficient. Better maintenance of
E. coli
cultivability was also observed when AOM was added; cultivability was even increased after addition of AOM from ozonated algae. AOM, likely to be present in treatment plants during algal blooms, and thus potentially in the treated water may compromise water biological stability.
Monte Carlo Simulation of Pathogen Behavior during the Sprout Production Process
Authors: Montville Rebecca , Schaffner Donald 
Published: February 2005

ABSTRACTFood-borne disease outbreaks linked to the consumption of raw sprouts have become a concern over the past decade. A Monte Carlo simulation model of the sprout production process was created to determine the most-effective points for pathogen control. Published literature was reviewed, and relevant data were compiled. Appropriate statistical distributions were determined and used to create the Monte Carlo model with Analytica software. Factors modeled included initial pathogen concentration and prevalence, seed disinfection effectiveness, and sampling of seeds prior to sprouting, sampling of irrigation water, or sampling of the finished product. Pathogen concentration and uniformity of seed contamination had a large effect on the fraction of contaminated batches predicted by the simulation. The model predicted that sprout sampling and irrigation water sampling at the end of the sprouting process would be more effective in pathogen detection than seed sampling prior to production. Day of sampling and type of sample (sprout or water) taken had a minimal effect on rate of detection. Seed disinfection reduced the proportion of contaminated batches, but in some cases it also reduced the ability to detect the pathogen when it was present, because cell numbers were reduced below the detection limit. Both the amount sampled and the pathogen detection limit were shown to be important variables in determining sampling effectiveness. This simulation can also be used to guide further research and compare the levels of effectiveness of different risk reduction strategies.
Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo
Authors: Mori Koichi , Mukaihara Takafumi , Uesugi Yoshiko , Iwabuchi Masaki , Hatanaka Tadashi 
Published: February 2005

ABSTRACT

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the
Escherichia coli ssb-3
strain and a deletion-directed chimera selection system that uses the
rpsL
+
gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from
Streptomyces septatus
TH-2 and
Streptomyces halstedii
subsp.
scabies
K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the
ssb-3
background than in the
ssb
+
background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.
New Strategy for Identification of Novel
cry
-Type Genes from
Bacillus thuringiensis
Strains
Authors: Berón Corina M. , Curatti Leonardo , Salerno Graciela L. 
Published: February 2005

ABSTRACT

We designed five degenerate primers for detection of novel
cry
genes from
Bacillus thuringiensis
strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known
cry
genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of
cry
-related sequences from novel
B. thuringiensis
isolates.
gerR
, a Novel
ger
Operon Involved in
l
-Alanine- and Inosine-Initiated Germination of
Bacillus cereus
ATCC 14579
Authors: Hornstra Luc M. , de Vries Ynte P. , de Vos Willem M. , Abee Tjakko , Wells-Bennik Marjon H. J. 
Published: February 2005

ABSTRACT

Bacillus cereus
endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the
gerA
family of operons. Analysis of the
Bacillus cereus
ATCC 14579 genome revealed seven
gerA
family homologues. Using a transposon Tn
917
-based insertional mutagenesis approach followed by an enrichment procedure to select for
l
-alanine-induced germination mutants, we isolated a mutant with a defect in the
l
-alanine germination pathway. The transposon disrupted the last gene of a tricistronic
gerA
family operon, designated
gerR
, with the order
gerRA
,
gerRC, gerRB
. A second mutant was created by insertion of pMUTIN4 in
gerRC
. Both mutants showed the same phenotype for nutrient-induced germination. Spores of the
gerR
mutant strains were blocked in their
l
-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. Apparently, germination mediated by
l
-alanine and inosine cannot be compensated for completely by the other germinant receptors, and this points towards an essential role of the
gerR
-encoded receptor in the receptor complex. In food products, spores of the mutant strains showed a reduced germination response compared to spores of the parental strain. High-pressure-initiated germination was not affected by the
gerR
mutations, as experiments with 100 and 550 MPa showed no difference with spores of the parental strain.
Source Bioaerosol Concentration and rRNA Gene-Based Identification of Microorganisms Aerosolized at a Flood Irrigation Wastewater Reuse Site
Authors: Paez-Rubio Tania , Viau Emily , Romero-Hernandez Socorro , Peccia Jordan 
Published: February 2005

ABSTRACT
Reuse of partially treated domestic wastewater for agricultural irrigation is a growing practice in arid regions throughout the world. A field sampling campaign to determine bioaerosol concentration, culturability, and identity at various wind speeds was conducted at a flooded wastewater irrigation site in Mexicali, Baja California, Mexico. Direct fluorescent microscopy measurements for total microorganisms, culture-based assays for heterotrophs and gram-negative enteric bacteria, and small-subunit rRNA gene-based cloning were used for microbial characterizations of aerosols and effluent wastewater samples. Bioaerosol results were divided into two wind speed regimens: (i) below 1.9 m/s, average speed 0.5 m/s, and (ii) above 1.9 m/s, average speed 4.5 m/s. Average air-borne concentration of total microorganisms, culturable heterotrophs, and gram-negative enteric bacteria were, respectively, 1.1, 4.2, and 6.2 orders of magnitude greater during the high-wind-speed regimen. Small-subunit rRNA gene clone libraries processed from samples from air and the irrigation effluent wastewater during a high-wind sampling event indicate that the majority of air clone sequences were more than 98% similar to clone sequences retrieved from the effluent wastewater sample. Overall results indicate that wind is a potential aerosolization mechanism of viable wastewater microorganisms at flood irrigation sites.
Low-Temperature Growth of
Shewanella oneidensis
MR-1
Authors: Abboud Randa , Popa Radu , Souza-Egipsy Virginia , Giometti Carol S. , Tollaksen Sandra , Mosher Jennifer J. , Findlay Robert H. , Nealson Kenneth H. 
Published: February 2005

ABSTRACT

Shewanella oneidensis
MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.