bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in
Saccharomyces cerevisiae
Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation
Authors: Mensonides Femke I. C. , Brul Stanley , Klis Frans M. , Hellingwerf Klaas J. , Teixeira de Mattos M. Joost 
Published: August 2005

ABSTRACT

This paper reports on physiological and molecular responses of
Saccharomyces cerevisiae
to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a
tps1
mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.
Involvement of a Branched-Chain Aminotransferase in Production of Volatile Sulfur Compounds in
Yarrowia lipolytica
Authors: Cernat Bondar Daniela , Beckerich Jean-Marie , Bonnarme Pascal 
Published: August 2005

ABSTRACT

The enzymatic degradation of
l
-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast
Yarrowia lipolytica
, the degradation of
l
-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (Yl
BCA1
) of
Y. lipolytica
was amplified, and the
l
-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that
l
-methionine degradation via transamination is involved in formation of VSCs in
Y. lipolytica
.
Characterization of an Inducible Phenylserine Aldolase from
Pseudomonas putida
24-1
Authors: Misono Haruo , Maeda Hiroshi , Tuda Kouiti , Ueshima Sakuko , Miyazaki Naoto , Nagata Shinji 
Published: August 2005

ABSTRACT

An inducible phenylserine aldolase (
l
-
threo
-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of
l
-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of
Pseudomonas putida
24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5′ phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on
l
-
threo
-3-phenylserine (

K
m

, 1.3 mM),
l
-
erythro
-3-phenylserine (

K
m

, 4.6 mM),
l
-threonine (

K
m

, 29 mM), and
l
-
allo
-threonine (

K
m

, 22 mM). In the reverse reaction,
threo
- and
erythro
- forms of
l
-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from
Pseudomonas putida
24-1 was cloned and overexpressed in
Escherichia coli
cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5′-phosphate.
Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria
Authors: Naz Naghma , Young Hilary K. , Ahmed Nuzhat , Gadd Geoffrey M. 
Published: August 2005

ABSTRACT

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains
Desulfovibrio desulfuricans
DSM 1926 and
Desulfococcus multivorans
DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the
smtAB
,
cadAC
, and
cadD
genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known
smtAB
genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of
Desulfomicrobium norvegicum
I1 and
Desulfovibrio desulfuricans
DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.
Mobility of Protozoa through Narrow Channels
Authors: Wang Wei , Shor Leslie M. , LeBoeuf Eugene J. , Wikswo John P. , Kosson David S. 
Published: August 2005

ABSTRACT

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 μm s
−1
(or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 μm s
−1
. Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.
Critical Factors Influencing the Occurrence of
Vibrio cholerae
in the Environment of Bangladesh
Authors: Huq Anwar , Sack R. Bradley , Nizam Azhar , Longini Ira M. , Nair G. Balakrish , Ali Afsar , Morris J. Glenn , Khan M. N. Huda , Siddique A. Kasem , Yunus Mohammed , Albert M. John , Sack David A. , Colwell Rita R. 
Published: August 2005

ABSTRACT

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype
Vibrio cholerae
O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic
Vibrio cholerae
serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a “reemerging” disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis.
187:
96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably
V. cholerae
). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of
V. cholerae
is proving useful in developing environmental models for the prediction of cholera epidemics.
Molecular Characterization of Korean
Bacillus anthracis
Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis
Authors: Ryu Chunsun , Lee Kyunghee , Hawng Han-Jun , Yoo Cheon-Kwon , Seong Won-Keun , Oh Hee-Bok 
Published: August 2005

ABSTRACT

We analyzed the genetic relationships and molecular characteristics of 34
Bacillus anthracis
isolates from soil and clinical samples in various regions of Korea and 17 related
Bacillus
species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of
B. anthracis
isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate
B. anthracis
strains from other
Bacillus
species. The
B. cereus
group was separated from other
Bacillus
species at a level of similarity of 0.68. Among them, some
B. cereus
strains showed genetic interspersion with
B. thuringiensis
strains. The evolutionary pattern of nucleotide differences among
B. anthracis
strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic
B. anthracis
isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.
Expression of 17 Genes in
Clostridium thermocellum
ATCC 27405 during Fermentation of Cellulose or Cellobiose in Continuous Culture
Authors: Stevenson David M. , Weimer Paul J. 
Published: August 2005

ABSTRACT

Clostridium thermocellum
is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in
C. thermocellum
ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a ∼10-fold range of dilution rates (0.013 to 0.16 h
−1
). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (
recA
and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type:
sdbA
(cellulosomal scaffoldin-dockerin binding protein),
cdp
(cellodextrin phosphorylase),
cbp
(cellobiose phosphorylase),
hydA
(hydrogenase),
ldh
(lactate dehydrogenase),
ack
(acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h
−1
:
cipA
(cellulosomal scaffolding protein),
celS
(exoglucanase),
manA
(mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.
Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis
Authors: Ben-Amor Kaouther , Heilig Hans , Smidt Hauke , Vaughan Elaine E. , Abee Tjakko , de Vos Willem M. 
Published: August 2005

ABSTRACT

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to
Clostridium coccoides
,
Clostridium leptum
, and
Bacteroides
. We found that some of the butyrate-producing related bacteria, such as
Eubacterium rectale
and
Eubacterium hallii
, were obviously viable at the time of sampling. However, amplicons affiliated with
Bacteroides
and
Ruminococcus obeum
- and
Eubacterium biforme
-like bacteria, as well as
Butyrivibrio crossotus
, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the
Clostridium leptum
cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to
Bifidobacterium adolescentis
,
Bifidobacterium longum
,
Bifidobacterium infantis
,
Bifidobacterium pseudocatenulatum
, and
Bifidobacterium bifidum
. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations;
B. longum
and
B. infantis
were retrieved from all sorted fractions, while
B. adolescentis
was recovered mostly from the sorted dead fraction.
Study of Spanish Grape Mycobiota and Ochratoxin A Production by Isolates of
Aspergillus tubingensis
and Other Members of
Aspergillus
Section
Nigri
Authors: Medina Angel , Mateo Rufino , López-Ocaña Laura , Valle-Algarra Francisco Manuel , Jiménez Misericordia 
Published: August 2005

ABSTRACT

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were
Alternaria
,
Cladosporium
, and
Aspergillus
. The isolation frequency of
Aspergillus
spp. section
Nigri
in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of
Aspergillus carbonarius
and 14.3% of
Aspergillus tubingensis
isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No
Aspergillus niger
isolate had the ability to produce this toxin under the conditions assayed. Identification of the
A. niger
aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of
A. niger
CECT 2807 and
A. tubingensis
CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of
A. tubingensis
matched 99 to 100% with the nucleotide sequence of strain
A. tubingensis
CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of
A. tubingensis
to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.
Proteins Encoded bySphingomonas elodeaATCC 31461rmlAandugpGGenes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities
Authors: Silva Elisabete , Marques Ana Rita , Fialho Arsénio Mendes , Granja Ana Teresa , Sá-Correia Isabel 
Published: August 2005

ABSTRACTThe commercial gelling agent gellan is a heteropolysaccharide produced bySphingomonas elodeaATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of therml4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gelcluster). Based on sequence homology, the putativermloperon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared fromEscherichia coliIPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Kmof 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Kmof 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. TheS. elodeaATCC 31461 UgpG protein, encoded by theugpGgene which maps outside thegelcluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing ofugpGgene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).
Engineering of Phenylacetaldehyde Reductase for Efficient Substrate Conversion in Concentrated 2-Propanol
Authors: Makino Yoshihide , Inoue Kousuke , Dairi Tohru , Itoh Nobuya 
Published: August 2005

ABSTRACT
Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.
Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates
Authors: Green Stefan J. , Minz Dror 
Published: August 2005

ABSTRACT

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from
Streptomyces
spp., and selective restriction of the
Streptomyces
16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.
Biological Control of Hog Waste Odor through Stimulated Microbial Fe(III) Reduction
Authors: Coates John D. , Cole Kimberly A. , Michaelidou Urania , Patrick Jennifer , McInerney Michael J. , Achenbach Laurie A. 
Published: August 2005

ABSTRACT

Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family
Geobacteraceae
as the dominant species. These organisms were present at concentrations as high as 2 × 10
5
cells g
−1
. Several pure cultures of Fe(III) reducers, including
Geobacter metallireducens
,
Geobacter humireducens
,
Geobacter sulfurreducens
,
Geobacter grbiciae
,
Geothrix fermentans
, and
Geovibrio ferrireducens
, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast,
Shewanella algae
did not degrade any of these compounds. We isolated an Fe(III) reducer,
Geobacter
strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.