bluuebunny/crossref_metadata_2025 huggingface dataset:
Allelopathy and Exotic Plant Invasion: From Molecules and Genes to Species Interactions
Authors: Bais Harsh P. , Vepachedu Ramarao , Gilroy Simon , Callaway Ragan M. , Vivanco Jorge M.
Published: September 2003

Here we present evidence that
Centaurea maculosa
(spotted knapweed), an invasive species in the western United States, displaces native plant species by exuding the phytotoxin (–)-catechin from its roots. Our results show inhibition of native species' growth and germination in field soils at natural concentrations of (–)-catechin. In susceptible species such as
Arabidopsis thaliana
, the allelochemical triggers a wave of reactive oxygen species (ROS) initiated at the root meristem, which leads to a Ca
2
+
signaling cascade triggering genome-wide changes in gene expression and, ultimately, death of the root system. Our results support a “novel weapons hypothesis” for invasive success.
Neurophilosophy or Philoneuroscience
Authors: Block Ned
Published: September 2003

Brain-Wise
Studies in Neurophilosophy. by Patricia Smith Churchland. MIT Press, Cambridge, MA, 2002. 486 pp., $65, £43.50. ISBN 0-262-03301-1. Paper, $25, £16.95. ISBN 0-262-53200-X.

Churchland explores the implications of our current understanding of the brain for long-debated philosophical questions such as consciousness, free will, and morality.
Constitutive Display of Cryptic Translation Products by MHC Class I Molecules
Authors: Schwab Susan R. , Li Katy C. , Kang Chulho , Shastri Nilabh
Published: September 2003

Major histocompatibility complex (MHC) class I molecules display tens of thousands of peptides on the cell surface, derived from virtually all endogenous proteins, for inspection by cytotoxic T cells (CTLs). We show that, in normal mouse cells, MHC I molecules present a peptide encoded in the 3′ “untranslated” region. Despite its rarity, the peptide elicits CTL responses and induces self-tolerance, establishing that immune surveillance extends well beyond conventional polypeptides. Furthermore, translation of this cryptic peptide occurs by a previously unknown mechanism that decodes the CUG initiation codon as leucine rather than the canonical methionine.
Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR
Authors: Changela Anita , Chen Kui , Xue Yi , Holschen Jackie , Outten Caryn E. , O'Halloran Thomas V. , Mondragón Alfonso
Published: September 2003

The earliest of a series of copper efflux genes in
Escherichia coli
are controlled by CueR, a member of the MerR family of transcriptional activators. Thermodynamic calibration of CueR reveals a zeptomolar (10
–
21
molar) sensitivity to free Cu
+
, which is far less than one atom per cell. Atomic details of this extraordinary sensitivity and selectivity for +1transition-metal ions are revealed by comparing the crystal structures of CueR and a Zn
2
+
-sensing homolog, ZntR. An unusual buried metal-receptor site in CueR restricts the metal to a linear, two-coordinate geometry and uses helix-dipole and hydrogen-bonding interactions to enhance metal binding. This binding mode is rare among metalloproteins but well suited for an ultrasensitive genetic switch.
Indian Ocean Climate and an Absolute Chronology over Dansgaard/Oeschger Events 9 to 13
Authors: Burns Stephen J. , Fleitmann Dominik , Matter Albert , Kramers Jan , Al-Subbary Abdulkarim A.
Published: September 2003

Oxygen-isotope ratios of a stalagmite from Socotra Island in the Indian Ocean provide a record of changes in monsoon precipitation and climate for the time period from 42 to 55 thousand years before the present. The pattern of precipitation bears a striking resemblance to the oxygen-isotope record from Greenland ice cores, with increased tropical precipitation associated with warm periods in the high northern latitudes. The largest change, at the onset of interstadial 12, occurred very rapidly, in about 25 years. The chronology of the events found in our record requires a reevaluation of previously published time scales for climate events during this period.
A Maximum in the Strength of Nanocrystalline Copper
Authors: Schiøtz Jakob , Jacobsen Karsten W.
Published: September 2003

We used molecular dynamics simulations with system sizes up to 100 million atoms to simulate plastic deformation of nanocrystalline copper. By varying the grain size between 5 and 50 nanometers, we show that the flow stress and thus the strength exhibit a maximum at a grain size of 10 to 15 nanometers. This maximum is because of a shift in the microscopic deformation mechanism from dislocation-mediated plasticity in the coarse-grained material to grain boundary sliding in the nanocrystalline region. The simulations allow us to observe the mechanisms behind the grain-size dependence of the strength of polycrystalline metals.
Seasonal Dynamics of Previously Unknown Fungal Lineages in Tundra Soils
Authors: Schadt Christopher W. , Martin Andrew P. , Lipson David A. , Schmidt Steven K.
Published: September 2003

The finding that microbial communities are active under snow has changed the estimated global rates of biogeochemical processes beneath seasonal snow packs. We used microbiological and molecular techniques to elucidate the phylogenetic composition of undersnow microbial communities in Colorado, the United States. Here, we show that tundra soil microbial biomass reaches its annual peak under snow, and that fungi account for most of the biomass. Phylogenetic analysis of tundra soil fungi revealed a high diversity of fungi and three novel clades that constitute major new groups of fungi (divergent at the subphylum or class level). An abundance of previously unknown fungi that are active beneath the snow substantially broadens our understanding of both the diversity and biogeochemical functioning of fungi in cold environments.
Simultaneous Fluorescence and Raman Scattering from Single Carbon Nanotubes
Authors: Hartschuh Achim , Pedrosa Hermeneglido N. , Novotny Lukas , Krauss Todd D.
Published: September 2003

Single-molecule fluorescence spectroscopy was used to determine the electronic properties of individual single-walled carbon nanotubes. Carbon nanotube structure was determined simultaneously from Raman spectroscopy. Fluorescence spectra from individual nanotubes with identical structures have different emission energies and linewidths that likely arise from defects or the local environment. Unlike most other molecules studied to date, the fluorescence intensity or spectrum from a single nanotube unexpectedly did not fluctuate.
Autophagy Genes Are Essential for Dauer Development and Life-Span Extension in
C. elegans
Authors: Meléndez Alicia , Tallóczy Zsolt , Seaman Matthew , Eskelinen Eeva-Liisa , Hall David H. , Levine Beth
Published: September 2003

Both dauer formation (a stage of developmental arrest) and adult life-span in
Caenorhabditis elegans
are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that
bec-1
, the
C. elegans
ortholog of the yeast and mammalian autophagy gene
APG6/VPS30/beclin1
, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires
C. elegans
orthologs of the yeast autophagy genes
APG1, APG7, APG8
, and
AUT10
. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in
C. elegans.
Nuclear Membrane Proteins with Potential Disease Links Found by Subtractive Proteomics
Authors: Schirmer Eric C. , Florens Laurence , Guan Tinglu , Yates John R. , Gerace Larry
Published: September 2003

To comprehensively identify integral membrane proteins of the nuclear envelope (NE), we prepared separately NEs and organelles known to cofractionate with them from liver. Proteins detected by multidimensional protein identification technology in the cofractionating organelles were subtracted from the NE data set. In addition to all 13 known NE integral proteins, 67 uncharacterized open reading frames with predicted membrane-spanning regions were identified. All of the eight proteins tested targeted to the NE, indicating that there are substantially more integral proteins of the NE than previously thought. Furthermore, 23 of these mapped within chromosome regions linked to a variety of dystrophies.
Synchroneity of Tropical and High-Latitude Atlantic Temperatures over the Last Glacial Termination
Authors: Lea David W. , Pak Dorothy K. , Peterson Larry C. , Hughen Konrad A.
Published: September 2003

A high-resolution western tropical Atlantic sea surface temperature (SST) record from the Cariaco Basin on the northern Venezuelan shelf, based on Mg/Ca values in surface-dwelling planktonic foraminifera, reveals that changes in SST over the last glacial termination are synchronous, within ±30 to ±90 years, with the Greenland Ice Sheet Project 2 air temperature proxy record and atmospheric methane record. The most prominent deglacial event in the Cariaco record occurred during the Younger Dryas time interval, when SSTs dropped by 3° to 4°C. A rapid southward shift in the atmospheric intertropical convergence zone could account for the synchroneity of tropical temperature, atmospheric methane, and high-latitude changes during the Younger Dryas.
Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1
Authors: Yin Yili , Manoury Bénédicte , Fåhraeus Robin
Published: September 2003

The glycine-alanine repeat domain (GAr) of Epstein-Barr virus–encoded nuclear antigen 1 (EBNA1) prevents major histocompatibility complex (MHC) class I–restricted presentation of EBNA1 epitopes to cytotoxic T cells. This effect has previously been attributed to the ability of GAr to inhibit its own proteasomal degradation. Here we show, both in vitro and in vivo, that GAr also inhibits messenger RNA translation of EBNA1 in cis and that this effect can be distinguished from its effect on proteasomal degradation. Hence, inhibition of messenger RNA translation, but not protein degradation, is essential to prevent antigen presentation on MHC class I molecules. Thus, by minimizing translation of the EBNA1 transcript, cells expressing EBNA1 avoid cytotoxic T cell recognition. At the same time, blocking degradation maintains the EBNA1 expression level.
Efficient Allelic Exchange and Transposon Mutagenesis in
Mycobacterium avium
by Specialized Transduction
Authors: Otero Joel , Jacobs William R. , Glickman Michael S.
Published: September 2003

Mycobacterium tuberculosis
and
Mycobacterium avium
are pathogenic slow-growing mycobacteria that cause distinct human diseases. In contrast to recent advances in
M. tuberculosis
genetics and pathogenesis investigation,
M. avium
has remained genetically intractable and, consequently, its pathogenic strategies remain poorly understood. Here we report the successful development of efficient allelic exchange and transposon mutagenesis in an opaque clinical strain of
M. avium
by specialized transduction. Efforts to disrupt the
leuD
gene of
M. avium
by specialized transduction were successful but were complicated by inefficient isolation of recombinants secondary to high spontaneous antibiotic resistance. However, by using this leucine auxotroph as a genetic host and the
Streptomyces coelicolor leuD
gene as a selectable marker, we achieved efficient allelic exchange at the
M. avium pcaA
locus. A
leuD
-marked transposon delivered by specialized transduction mutagenized
M. avium
with efficiencies similar to
M. tuberculosis
. These results establish a system for random and directed mutagenesis of
M. avium
. In combination with the forthcoming
M. avium
genome sequence, these tools will allow the distinct physiologic and pathogenic properties of
M. avium
to be dissected in molecular detail.
Biotransformation of Natural and Synthetic Isoflavonoids by Two Recombinant Microbial Enzymes
Authors: Seeger Michael , González Myriam , Cámara Beatriz , Muñoz Liliana , Ponce Emilio , Mejías Lorenzo , Mascayano Carolina , Vásquez Yesseny , Sepúlveda-Boza Silvia
Published: September 2003

Isolation and synthesis of isoflavonoids has become a frequent endeavor, due to their interesting biological activities. The introduction of hydroxyl groups into isoflavonoids by the use of enzymes represents an attractive alternative to conventional chemical synthesis. In this study, the capabilities of biphenyl-2,3-dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) of
Burkholderia
sp. strain LB400 to biotransform 14 isoflavonoids synthesized in the laboratory were investigated by using recombinant
Escherichia coli
strains containing plasmid vectors expressing the
bphA1A2A3A4
or
bphA1A2A3A4B
genes of strain LB400. The use of BphA and BphB allowed us to biotransform 7-hydroxy-8-methylisoflavone and 7-hydroxyisoflavone into 7,2′,3′-trihydroxy-8-methylisoflavone and 7,3′,4′-trihydroxyisoflavone, respectively. The compound 2′-fluoro-7-hydroxy-8-methylisoflavone was dihydroxylated by BphA at
ortho
-fluorinated and
meta
positions of ring B, with concomitant dehalogenation leading to 7,2′,3′,-trihydroxy-8-methylisoflavone. Daidzein (7,4′-dihydroxyisoflavone) was biotransformed by BphA, generating 7,2′,4′-trihydroxyisoflavone after dehydration. Biotransformation products were analyzed by gas chromatography-mass spectrometry and nuclear magnetic resonance techniques.
Aerobic Anoxygenic Photosynthesis in
Roseobacter
Clade Bacteria from Diverse Marine Habitats
Authors: Allgaier Martin , Uphoff Heike , Felske Andreas , Wagner-Döbler Irene
Published: September 2003

The marine
Roseobacter
clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of
Roseobacter
clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the
pufLM
genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl)
a.
A collection of ca. 1,000 marine isolates was screened for members of the marine
Roseobacter
clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42
Roseobacter
clade isolates found tended to form habitat-specific subclusters. The
pufLM
genes were detected in two groups of strains from dinoflagellate cultures but in none of the other
Roseobacter
clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl
a.
Strains within the second group (the DFL-35 cluster) formed a new species of
Roseovarius
and did not produce Bchl
a
under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The
pufL
genes of the dinoflagellate isolates were phylogenetically closely related to
pufL
genes from
Betaproteobacteria
, confirming similar previous observations which have been interpreted as indications of gene transfer events.
Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent
Authors: Ibekwe A. Mark , Grieve Catherine M. , Lyon Stephen R.
Published: September 2003

Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application. The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater. Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms. The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms. There was only moderate removal of total nitrogen and phosphorus. Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the α subunit of the ammonia monooxygenase gene (
amoA
) recovered from soil samples and DGGE bands. DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to
Bacillus
,
Clostridium
,
Mycoplasma
,
Eubacterium
, and
Proteobacteria
originally retrieved from the gastrointestinal tracts of mammals. The population of ammonia-oxidizing bacteria showed a higher percentage of
Nitrosospira
-like sequences from the wetland samples, while a higher percentage of
Nitrosomonas
-like sequences from manure, feces, raw washwater, and facultative pond was found. These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment.
Evidence of Chemolithoautotrophy in the Bacterial Community Associated withAlvinella pompejana, a Hydrothermal Vent Polychaete
Authors: Campbell Barbara J. , Stein Jeffrey L. , Cary S. Craig
Published: September 2003

The deep-sea polychaeteAlvinella pompejanacolonizes tubes on the sides of black smoker chimneys along the East Pacific Rise. A diverse, yet phylogenetically constrained episymbiotic community is obligately associated with its dorsal surface. The morphologically and phylogenetically distinct dominant episymbionts have not yet been cultured, and there are no clearly defined roles for these bacteria in this symbiosis. A large insert fosmid library was screened for the presence of the two dominant phylotypes. Two fosmids, 35.2 and 38 kb, containing phylotype-specific 16S ribosmal DNA sequences were fully sequenced. Each fosmid had a gene encoding ATP citrate lyase, a key enzyme in the reverse tricarboxylic acid (rTCA) cycle, a CO2fixation pathway. A selection of episymbiont communities from various geographic locations and vent sites were screened for the presence, diversity, and expression (via reverse transcription-PCR) of the ATP citrate lyase gene. Our results indicate that the ATP citrate lyase gene is not only a consistent presence in these episymbiont communities but is also expressed. Phylogenetically distinct forms of ATP citrate lyase were also found associated with and expressed by bacteria extracted from the tubes ofA. pompejana. Utilizing PCR with degenerate primers based on a second key enzyme in the rTCA cycle, 2-oxoglutarate:acceptor oxidoreductase, we also demonstrated the persistent presence and expression of this gene in the episymbiont community. Our results suggest that members of both the episymbiont and the surrounding free-living communities display a chemolithoautotrophic form of growth and therefore contribute fixed carbon to other organisms in the vent community.
Environmental Determinants of
Vibrio cholerae
Biofilm Development
Authors: Kierek Katharine , Watnick Paula I.
Published: September 2003

Vibrio cholerae
is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the
vps
genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for
V. cholerae
biofilm development in Luria-Bertani broth. In this work, we showed that
V. cholerae
forms
vps
-dependent biofilms and
vps-
independent biofilms. The
vps
-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of
vps
-dependent biofilm development are present in freshwater, while environmental activators of
vps
-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that
vps-
dependent biofilm development and
vps-
independent biofilm development may play distinct roles in the natural environment.
Characterization of an Inducible Chlorophenol
O
-Methyltransferase from
Trichoderma longibrachiatum
Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines
Authors: Coque Juan-José R. , Álvarez-Rodríguez María Luisa , Larriba Germán
Published: September 2003

A novel
S
-adenosyl-
l
-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of
Trichoderma longibrachiatum
CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [
methyl
-
3
H]SAM, and this reaction was prevented by previous incubation with
S
-adenosylhomocysteine. Gel filtration indicated that the
M
r
was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The

K
m

values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively.
S
-Adenosylhomocysteine acted as a competitive inhibitor, with a

K
i

of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu
2+
, Hg
2+
, Zn
2+
, and Ag
+
, and to a lesser extent by
p
-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.
Purification and Functional Characterization of a Novel α-
l
-Arabinofuranosidase from
Bifidobacterium longum
B667
Authors: Margolles Abelardo , de los Reyes-Gavilán Clara G.
Published: September 2003

The gene encoding a novel α-
l
-arabinofuranosidase from
Bifidobacterium longum
B667,
abfB
, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-
l
-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in
Lactococcus lactis
under control of the tightly regulated, nisin-inducible
nisA
promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70°C), and it had an optimum pH of 6.0 and an optimum temperature of 45°C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55°C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu
2+
, Hg
2+
, and Zn
2+
markedly reduced enzymatic activity. The protein exhibited a high level of activity with
p
-nitrophenyl α-
l
-arabinofuranoside, with apparent
K

m

and
V
max
values of 0.295 mM and 417 U/mg, respectively. AbfB released
l
-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of
l
-arabinose-containing polysaccharides.
Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages
Authors: Rakonjac Jasna , Ward Lawrence J. H. , Schiemann Anja H. , Gardner Paul P. , Lubbers Mark W. , O'Toole Paul W.
Published: September 2003

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium
Lactococcus lactis
. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P
E
1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P
E
1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P
E
1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.
Heat Adaptation Alters
Escherichia coli
O157:H7 Membrane Lipid Composition and Verotoxin Production
Authors: Yuk Hyun-Gyun , Marshall Douglas L.
Published: September 2003

The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of
Escherichia coli
O157:H7 (ATCC 43895), an
rpoS
mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant
E. coli
O157:H7 (B6-914), and nonpathogenic
E. coli
(ATCC 25922) was investigated.
D
values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and
cis
-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the
rpoS
mutant, whereas there was a reduction and no change in the amount of
cis
-vaccenic acid in nonpathogenic and verotoxin mutant
E. coli
, respectively. The ratio of palmitic to
cis
-vaccenic acids decreased in ATCC 43895 and in the
rpoS
mutant, whereas the ratio increased in nonpathogenic
E. coli
and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and
rpoS
mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The
rpoS
gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.
Genetic Engineering of a Highly Solvent-Tolerant
Pseudomonas putida
Strain for Biotransformation of Toluene to
p-
Hydroxybenzoate
Authors: Ramos-González María-Isabel , Ben-Bassat Arie , Campos María-Jesús , Ramos Juan L.
Published: September 2003

The solvent-tolerant strain
Pseudomonas putida
DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA).
P. putida
DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The
todC1C2
genes encode the α and β subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EΔ
todC
mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The
P. putida pobA
gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a
Pseudomonas mendocina pobA1 pobA2
double mutant. This
pobA
gene was knocked out in vitro and used to generate a double mutant, DOT-T1EΔ
todCpobA
, that was unable to use either toluene or 4-HBA as a carbon source. The
tmo
and
pcu
genes from
P. mendocina
KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of
p-
cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn
5Tc
and stably recruited in the chromosome of DOT-T1EΔ
todCpobA
. Expression of the
tmo
and
pcu
genes took place in a DOT-T1E background due to cross-activation of the
tmo
promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.
Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of
Bacillus
and Related Genera
Authors: Daffonchio Daniele , Cherif Ameur , Brusetti Lorenzo , Rizzi Aurora , Mora Diego , Boudabous Abdellatif , Borin Sara
Published: September 2003

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera
Bacillus
,
Brevibacillus
,
Geobacillus
, and
Paenibacillus
. We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.
Predictive Thermal Inactivation Model for Effects of Temperature, Sodium Lactate, NaCl, and Sodium Pyrophosphate on
Salmonella
Serotypes in Ground Beef
Authors: Juneja Vijay K. , Marks Harry M. , Mohr Tim
Published: September 2003

Analyses
of survival data of a mixture of
Salmonella
spp. at fixed
temperatures between 55°C (131°F) and 71.1°C
(160°F) in ground beef matrices containing concentrations of
salt between 0 and 4.5%, concentrations of sodium pyrophosphate
(SPP) between 0 and 0.5%, and concentrations of sodium lactate
(NaL) between 0 and 4.5% indicated that heat resistance of
Salmonella
increases with increasing levels of SPP and salt,
except that, for salt, for larger lethalities close to 6.5, the effect
of salt was evident only at low temperatures (<64°C).
NaL did not seem to affect the heat resistance of
Salmonella
as much as the effects induced by the other variables studied. An
omnibus model for predicting the lethality for given times and
temperatures for ground beef matrices within the range studied was
developed that reflects the convex survival curves that were observed.
However, the standard errors of the predicted lethalities from this
models are large, so consequently, a model, specific for predicting the
times needed to obtained a lethality of 6.5 log
10
, was
developed, using estimated results of times derived from the individual
survival curves. For the latter model, the coefficient of variation
(CV) of predicted times range from about 6 to 25%. For example,
at 60°C, when increasing the concentration of salt from 0 to
4.5%, and assuming that the concentration of SPP is 0%,
the time to reach a 6.5-log
10
relative reduction is
predicted to increase from 20 min (CV = 11%) to 48 min
(CV = 15%), a 2.4 factor (CV = 19%). At
71.1°C (160°F) the model predicts that more than 0.5
min is needed to achieve a 6.5-log
10
relative
reduction.
Cyanobacterial Diversity in Natural and Artificial Microbial Mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a Morphological and Molecular Approach
Authors: Taton Arnaud , Grubisic Stana , Brambilla Evelyne , De Wit Rutger , Wilmotte Annick
Published: September 2003

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from
Nodularia
and
Hydrocoryne
, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.
Impact of Seasonal Variations and Nutrient Inputs on Nitrogen Cycling and Degradation of Hexadecane by Replicated River Biofilms
Authors: Chénier Martin R. , Beaumier Danielle , Roy Réal , Driscoll Brian T. , Lawrence John R. , Greer Charles W.
Published: September 2003

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both
alkB
and
nirK
were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for
nirK
from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.
Real-Time PCR for Quantification of
Giardia
and
Cryptosporidium
in Environmental Water Samples and Sewage
Authors: Guy Rebecca A. , Payment Pierre , Krull Ulrich J. , Horgen Paul A.
Published: September 2003

The protozoan pathogens
Giardia lamblia
and
Cryptosporidium parvum
are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of
Giardia
and
Cryptosporidium
are very low. Primers and TaqMan probes based on the β-giardin gene of
G. lamblia
and the COWP gene of
C. parvum
were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of
G. lamblia
and one oocyst of
C. parvum
. A multiplex real-time PCR (qPCR) assay for simultaneous detection of
G. lamblia
and
C. parvum
resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify
G. lamblia
in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of
G. lamblia
in the sewage. No
Cryptosporidium
was detected in these samples by either method.
Biogeography of the Purple Nonsulfur Bacterium
Rhodopseudomonas palustris
Authors: Oda Yasuhiro , Star Bastiaan , Huisman Louis A. , Gottschal Jan C. , Forney Larry J.
Published: September 2003

The biogeography of the purple nonsulfur bacterium
Rhodopseudomonas palustris
on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as
R. palustris
based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (
C
MH
), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (
C
MH
, ≥0.95) but there was almost no similarity between locations that were ≥9 m apart (
C
MH
, ≤0.25). These calculations showed there was a gradual decrease in similarity among the five locations as a function of distance and that clones of
R. palustris
were lognormally distributed along the linear 10-m transect. These data indicate that natural populations of
R. palustris
are assemblages of genetically distinct ecotypes and that the distribution of each ecotype is patchy.
Spatial Distribution of
Rhodopseudomonas palustris
Ecotypes on a Local Scale
Authors: Bent S. J. , Gucker C. L. , Oda Y. , Forney L. J.
Published: September 2003

The number, spatial distribution, and significance of genetically distinguishable ecotypes of prokaryotes in the environment are poorly understood. Oda et al. (Y. Oda, B. Star, L. A. Huisman, J. C. Gottschal, and L. J. Forney, Appl. Environ. Microbiol. 69:xxx-xxx, 2003) have shown that
Rhodopseudomonas palustris
ecotypes were lognormally distributed along a 10-m transect and that multiple strains of the species could coexist in 0.5-g sediment samples. To extend these observations, we investigated the clonal diversity of
R. palustris
in 0.5-g samples taken from the corners and center of a 1-m square. A total of 35 or 36 clones were recovered by direct plating from each sample and were characterized by BOX A1R repetitive element-PCR genomic DNA fingerprinting. Isolates with fingerprint images that were ≥80% similar to each other were defined as the same genotype. Among the 178 isolates studied, 32 genotypes were identified, and each genotype contained between 1 and 40 isolates. These clusters were consistent with minor variations found in 16S rRNA gene sequences. The Shannon indices of the genotypic diversity within each location ranged from 1.08 (5 genotypes) to 2.18 (13 genotypes). Comparison of the rank abundance of genotypes found in pairs of locations showed that strains from three locations were similar to each other, with Morisita-Horn similarity coefficients ranging from 0.59 to 0.71. All comparisons involving the remaining two locations resulted in coefficients between 0 and 0.12. From these results we inferred that the patterns of ecotype diversity at the sampling site are patchy at a 1-m scale and postulated that factors such as mixing, competitive interactions, and microhabitat variability are likely to be responsible for the maintenance of the similarities between some locations and the differences between others.
Effects of Soil pH on the Biodegradation of Chlorpyrifos and Isolation of a Chlorpyrifos-Degrading Bacterium
Authors: Singh Brajesh K. , Walker Allan , Morgan J. Alun W. , Wright Denis J.
Published: September 2003

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of ≥6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of
Pseudomonas
. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (
opd
) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.
Identification of
cry1I-
Type Genes from
Bacillus thuringiensis
Strains and Characterization of a Novel
cry1I-
Type Gene
Authors: Song Fuping , Zhang Jie , Gu Aixing , Wu Yue , Han Lanlan , He Kanglai , Chen Zhongyi , Yao Jiang , Hu Yuqin , Li Guoxun , Huang Dafang
Published: September 2003

A PCR-restriction fragment length polymorphism method for identification of
cry1I
-type genes from
Bacillus thuringiensis
was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp
cry1I-
type gene fragments. Amplification products were digested with the
Bsp
119I and
Ban
I enzymes, and four kinds of known
cry1I
-type genes were successfully identified. The results showed that
cry1I
-type genes appeared in 95 of 115
B. thuringiensis
isolates and 7 of 13 standard strains. A novel
cry1I
-type gene was found in one standard strain and six isolates. The novel
cry1I
gene was cloned from
B. thuringiensis
isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in
Escherichia coli
BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (
Plutella xylostella
), Asian corn borer (
Ostrinia furnacalis
), and soybean pod borer (
Leguminivora glycinivorella
). However, it was not toxic to the cotton bollworm (
Helicoverpa armigera
), beet armyworm (
Spodoptera exigua
), or elm leaf beetle (
Pyrrhalta aenescens
) in bioassays. Subsequently, the Cry protein encoded by this novel
cry
gene was designated Cry1Ie1 by the
B. thuringiensis
δ-endotoxin nomenclature committee.
Detection of Staphylococcal Enterotoxin B via Biomolecular Interaction Analysis Mass Spectrometry
Authors: Nedelkov Dobrin , Nelson Randall W.
Published: September 2003

Detection of
Staphylococcus
enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work. The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation. Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin. Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of ∼500 amol of SEB, was readily achieved from both the standard and mushroom samples. A certain amount of SEB degradation was indicated by the signals in the mass spectra. The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms.